The Scripps Research Institute Scientific Report

T HE S CRIPPS R ESEARCH I NSTITUTE

Scientific Report 2006 ON THE COVER: “Click” chemistry refers to the development and use of highly reliable chemical reactions to make molecules that perform a desired function. The most successful click reaction is the copper-catalyzed azide-alkyne cycloaddition (CuAAC) process, an abstract representation of which is shown here. Blue (azide) and red (alkyne) components are efficiently joined by spherical copper ions to give pentagonal 1,4-triazoles. Surrounding the large triazole motif in the foreground are energy diagrams highlighting favorable aspects of the reaction, and its biological applications are represented by icosahedral virus particles. The brick-like mosaic tying the entire image together suggests both the highly modular nature of the CuAAC reaction and its use- fullness in the creation of functional small molecules, polymers, and materials. The overall theme celebrates the importance of connections: in chemistry, as in life, while individuals may be unique and pleasing, it is when they “click” together that true art is created. Painting by Yeon-Hee Lim, Ph.D., research associate in the laboratory of M.G. Finn, Ph.D., Department of Chemistry. VOLUME 32 THE SCRIPPS RESEARCH INSTITUTE SCIENTIFIC REPORT

PRESIDENT’S INTRODUCTION 5 INFECTOLOGY Staff and Fellows 355

CALIFORNIA Chairman’s Overview 355 Investigators’ Reports 356 THE SKAGGS INSTITUTE FOR CHEMICAL TRANSLATIONAL RESEARCH INSTITUTE BIOLOGY Staff and Fellows 363 Staff and Fellows 11 Chairman’s Overview 364 Director’s Overview 13 Investigators’ Reports 365 Investigator’s Reports 15

CELL BIOLOGY Staff and Fellows 19 A WARDS, EDUCATION, CENTERS AND Chairman’s Overview 22 INSTITUTES, AND ORGANIZATIONS Investigators’ Reports 24 Staff Awards and Activities 373

CHEMISTRY Kellogg School of Science and Technology 377 Staff and Fellows 63 Chairman’s Overview 67 Center for Integrative Molecular Biosciences 382 Investigators’ Reports 69 The Harold L. Dorris Neurological Research Center 383

IMMUNOLOGY Helen L. Dorris Child and Adolescent 384 Staff and Fellows 99 Neuro-Psychiatric Disorder Institute Chairman’s Overview 102 The Institute for Childhood and Neglected Diseases 384 Investigators’ Reports 104 Society of Fellows 386 MOLECULAR BIOLOGY Staff and Fellows 153 Author Index 387 Chairman’s Overview 158 Subject Index 395 Investigators’ Reports 160

MOLECULAR AND EXPERIMENTAL MEDICINE Staff and Fellows 239 Chairman’s Overview 242 Investigators’ Reports 245

MOLECULAR AND INTEGRATIVE NEUROSCIENCES Staff and Fellows 287 Chairman’s Overview 289 Investigators’ Reports 291

NEUROBIOLOGY Staff and Fellows 327 Chairman’s Overview 328 Investigators’ Reports 330

FLORIDA

BIOCHEMISTRY Staff and Fellows 339 Chairman’s Overview 340

Investigators’ Reports 340 Vessels of a human retina (macula). The image shows the avascular zone of the fovea. Image provided by Edith CANCER BIOLOGY Aguilar de Diaz, M.D., Scientific Associate, Matthew Ritter, Staff and Fellows 350 Ph.D., Research Associate, and Martin Friedlander, M.D., Ph.D., Chairman’s Overview 350 Professor. Work done in the Friedlander laboratory in the Investigators’ Reports 351 Department of Cell Biology.

BOARD OF TRUSTEES

John J. Moores Alexander W. Dreyfoos Chair of the Board, Scripps Research Private Investor Former Chairman, Board of Regents of the University of California Chairman, Raymond F. Kravis Center for the Performing Arts Chairman, JMI and San Diego Padres Charles C. Edwards, M.D. Chair, Board of Trustees, The Carter Center Former Director, Food and Drug Administration Warren Beatty Former President, Scripps Clinic and Research Foundation President, Mulholland Productions Incorporated Richard J. Elkus, Jr. Vincent E. Benstead Director, KLA-Tencor, Lam Research, Virage Logic Former Partner, PricewaterhouseCoopers Member, Board of Trustees, Palo Alto Medical Foundation

Mrs. William McCormick Blair, Jr. Marjorie Fink Vice President, Albert and Mary Lasker Foundation Philanthropist

J. Gary Burkhead Phillip Frost, M.D. Retired, Vice-Chairman, Fidelity Investments Chairman and Chief Executive Officer, IVAX Corporation

Gary N. Coburn Louis L. Gonda Retired Senior Managing Director, Putnam Investments Chairman and Chief Executive Officer, Lexington Commercial Holdings Chairman, Lexington Ventures, L.L.C. Gerald Cohn Chairman, Lexington Realty, L.L.C. Retired Executive, Private Investor Paul L. Herrling, Ph.D. George H. Conrades Head, Corporate Research, Novartis International AG Chairman and Chief Executive Officer, Akamai Technologies, Inc. Lawrence C. Horowitz, M.D. J. Michael Cook President and Managing General Partner Retired Chairman and Chief Executive Officer, Deloitte + Touche Selby Lane Enterprises II, L.L.C. Rod Dammeyer Thomas H. Insley President, CAC, L.L.C. Vice President and Chief Financial Officer, SkinMedica, Inc. John G. Davies, Esq. Richard A. Lerner, M.D. Of Counsel, Allen Matkins President, The Scripps Research Institute Judicial Appointments Advisor for Governor Arnold Schwarzenegger Claudia S. Luttrell Thomas E. Dewey, Jr. President, The Skaggs Institute for Research Member, McFarland Dewey & Co., L.L.C. James R. Mellor Former Chairman and Chief Executive Officer General Dynamics Corporation

The Hon. Lynn Schenk Former Congresswoman, California

Ralph J. Shapiro Chair, Avondale Investment Company

Mark S. Skaggs Board Member, The ALSAM Foundation

The Hon. Alice D. Sullivan (Ret.) California Superior Court Judge, Retired

Chris D. Van Gorder President and Chief Executive Officer, ScrippsHealth

Andrew Viterbi, Ph.D. President, Viterbi Group, L.L.C.

OFFICERS

Richard A. Lerner, M.D. President Human keratinocyte polarising in response to Douglas A. Bingham EGF-1. Microtubules are shown in red, Clip170 a microtu- Executive Vice President and Chief Operating Officer and Secretary bule + end binding protein in yellow, and EB1 different micro- Donna J. Weston tubule +end binding protein in blue. Work done by Ann Wheeler, Senior Vice President and Chief Financial Officer and Treasurer Ph.D., Research Associate, in the laboratory of Clare Waterman- Storer, Ph.D., Department of Cell Biology. Thomas E. Northrup, Esq., Ph.D. Chief Business Counsel and Assistant Secretary THE SCRIPPS RESEARCH INSTITUTE 2006 3

BOARD OF SCIENTIFIC GOVERNORS

Dr. Gunter Blobel* Dr. Inder Verma The Rockefeller University The Salk Institute New York, New York La Jolla, California

Professor Piet Borst Dr. Semir Zeki The Netherlands Cancer Institute University College Amsterdam, the Netherlands London, England Dr. Sydney Brenner* The Salk Institute * Nobel Laureate La Jolla, California

Dr. Michael S. Brown* The University of Texas Southwestern Medical Center Dallas, Texas

Professor Jean-Pierre Changeux Institut Pasteur Paris, France

Dr. Samuel Danishefsky Memorial Sloan-Kettering Cancer Center and Columbia University New York, New York

Professor Raymond A. Dwek, F.R.S. University of Oxford Oxford, United Kingdom

Professor Mitchell Feigenbaum The Rockefeller University New York, New York

Dr. Edmond Fischer* University of Washington Seattle, Washington

Dr.Walter Gilbert* Harvard University Cambridge, Massachusetts

Dr. Joseph L. Goldstein* The University of Texas Southwestern Medical Center Dallas, Texas

Dr. Paul Greengard* The Rockefeller University New York, New York The synapse revealed. The artist created a pencil sketch Dr. Har Gobind Khorana* based on micrograph data, but opted to cut the number of neu- Massachusetts Institute of Technology ronal interactions to ~ 30% in order to clarify the physiology. He Cambridge, Massachusetts constructed 3-dimensional models of two cells to render on top Professor Aaron Klug* of the approved sketch. He designed a texture map to skin the Medical Research Council Laboratory of Molecular Biology two primary neurons that would insinuate a proteinated bilayer Cambridge, England and simultaneously simulate organelles deep to the surface. Light- ing sweeps attention over the entire image before settling it on Professor Sir Harold Kroto, F.R.S.* the synapse by reflecting hints of color from the focus into periph- University of Sussex Falmer, Brighton, United Kingdom eral regions of the drawing. Cool desaturated colors in the background allow the warm synapse region to command attention as Dr. Phillip A. Sharp* it glows with a hint of anticipatory presynaptic transmission. This Massachusetts Institute of Technology image received a Certificate of Merit award from the Association Cambridge, Massachusetts of Medical Illustrators and won first place in the National Science Dr. Susumu Tonegawa* Foundation’s Scientific Visualization Challenge of 2005 compe- Massachusetts Institute of Technology tition. Illustration by Graham Johnson, courtesy of the Howard Cambridge, Massachusetts Hughes Medical Institute ©2004. 4 THE SCRIPPS RESEARCH INSTITUTE 2006

ADMINISTRATION

Richard A. Lerner, M.D. Judith T. Muñoz, Ph.D. President Vice President, Human Resources

Douglas A. Bingham Polly A. Murphy, DVM, Ph.D. Executive Vice President and Chief Operating Officer Senior Vice President, Business and Scientific Services

Donna J. Weston Harry Orf, Ph.D. Senior Vice President and Chief Financial Officer Vice President, Scientific Operations, Scripps Florida

Emily M. Holmes, Ph.D. William R. Roush, Ph.D. Vice President, Research Services Associate Dean, Graduate Studies, Scripps Florida

Gerald F. Joyce, M.D., Ph.D. Denise M. Scalzo Dean, Faculty Vice President, Development

Jeffery W. Kelly, Ph.D. James R. Williamson, Ph.D. Dean, Graduate and Postgraduate Studies Associate Dean, Graduate Studies

Stephen Mayfield, Ph.D. Kaye I. Wynne Associate Dean, Graduate Studies Vice President, Office of Sponsored Programs

Keith McKeown Vice President, Communications and Public Relations

Ben F. Morris, Jr. SCIENTIFIC DEPARTMENT CHAIRMEN Vice President, Facilities Services

Ernest Beutler, M.D. Department of Molecular and Experimental Medicine

Tamas Bartfai, Ph.D. Department of Neuropharmacology

John Cleveland, M.D. Department of Cancer Biology

Gerald M. Edelman, M.D., Ph.D. Department of Neurobiology

Steve A. Kay, Ph.D. Department of Biochemistry

K.C. Nicolaou, Ph.D. Department of Chemistry

Sandra L. Schmid, Ph.D. Department of Cell Biology

Richard J. Ulevitch, Ph.D. Department of Immunology

Charles Weissmann, M.D., Ph.D. Department of Infectology The figure shows the variable domain of antibody 4-4-20, which binds the antigen fluorescein, and its free-energy sur- Peter E. Wright, Ph.D. faces at various stages of evolution. Work carried out in the Department of Molecular Biology laboratory of Floyd Romesberg showed that 4-4-20’s binding site has evolved from a flexible progenitor that populates many conformations into a rigid binding site with only one conformation, thus confirming a longstanding hypothesis in immunology. The image depicts collaborative research between Floyd Romesberg, Ph.D., Charles Brooks, Ph.D., and their colleagues. Graphics by Joerg Zimmermann, Ph.D., and Floyd E. Romesberg, Ph.D., Department of Chemistry. THE SCRIPPS RESEARCH INSTITUTE 2006 5

which relies on automated robots to analyze a large number of compounds at once, is available to Scripps Research faculty on both coasts. In January, the Access to Technologies Program also opened the system to scientists from universities and research institutions throughout Florida, enhancing our other collaborations in the state. The State of Florida awarded its first research grant to 1 of our faculty members this year. Awarded on the basis of scientific merit, the Florida Department of Health’s James & Esther King Biomedical Research grant will provide support for Layton Smith, associate director of Pharmacology at Scripps Florida, who is conducting research in the field of metabolism. In another Florida development this year, we welcomed the first entering classman to our graduate program in Jupiter, where he joins several students who transferred from other institutions. A new 2-way, web- based conferencing technology is enabling Florida students to participate in California lectures in real time, as well as open future Florida classes to interested Cali- Richard A. Lerner, M.D. fornia students.

NEW RESEARCH ALLIANCES President’s Introduction In 2006, we forged a number of new alliances that will advance science at the institute in the years ahead. ne of the pleasures of being associated with In February, we announced a collaborative initiative Scripps Research is that we so often have good with IBM, called “Project Check-mate,” that will con- O news to report. So it is this year, when we can duct research on pandemic viruses to develop ways to share progress on the Florida campus; new contributions anticipate, manage, and contain infectious diseases. of our faculty, staff, and trustees in both Florida and Check-mate capitalizes on Scripps Research’s world- California; and groundbreaking research in our under- class research in biochemical modeling and drug discov- standing of health and disease. ery and on IBM’s expertise in computational biology FLORIDA ADVANCES biopatterning and supercomputing. The joint research With the Palm Beach County commissioners’ selec- team will harness both IBM’s Blue Gene supercomputer tion in February of a new site for Scripps Florida—on and Scripps Florida’s screening technology. the north campus of Florida Atlantic University in In March, we joined forces with 3 preeminent San Jupiter—we have moved forward with plans for a per- Diego research institutions—the Burnham Institute for manent facility. Scheduled to open in 2009, the facility Medical Research, the Salk Institute for Biological Stud- will be a world-class, 350,000-square-foot biomedical ies, and the University of California, San Diego—to research operation focusing on basic biomedical science, establish an independent, nonprofit consortium dedi- drug discovery, and technology development. cated to stem cell research. The alliance, called the In the meantime, Scripps Florida opened a second San Diego Consortium for Regenerative Medicine, will temporary building this fall on the Florida Atlantic Uni- explore the tremendous therapeutic potential of stem versity site. The structure will provide 33,000 square cells to repair and replace damaged tissue. feet of space to house our growing faculty and staff while In April, we became part of Microsoft’s new BioIT the permanent campus is under construction. Alliance, a cross-industry group working to integrate The state-of-the-art screening technologies at Scripps science and technology to speed the pace of drug dis- Florida have begun to make contributions to science covery and development. The alliance’s first project, as evidenced by published papers this year. The system, Collaborative Molecular Environment, strives to make 6 THE SCRIPPS RESEARCH INSTITUTE 2006

research more efficient through a data management solu- immune system and cause persistent and recurrent tion targeting common technology problems faced in gonorrhea infections. the life sciences. • Professor Hugh Rosen and colleagues developed In May, a new robotic crystallization facility opened a chemical tool that allows manipulation of the on the California campus, thanks to support from the passage of substances through the barriers Joint Center for Structural Genomics (funded through between blood and organ tissues, findings that the National Institutes of Health’s Protein Structure have therapeutic implications for organ trans- Initiative) and global nonprofit group International AIDS plants, autoimmune disease, multiple sclerosis, Vaccine Initiative. One of the largest machines of its and adult respiratory distress syndrome. kind, the integrated robotics system will enhance sci- • Immunology Department Chair Richard Ulevitch entists’ ability to solve molecular structures, increas- and colleagues uncovered a new and potentially ing our understanding of basic biology and strategies important function for the protein Nod1, inhibit- for combating a variety of diseases. ing the growth of estrogen-sensitive human breast GROUNDBREAKING RESEARCH cancer cells. The institute’s science stands at the forefront of • Associate Professor Elizabeth Winzeler and col- basic biomedical research, a vital endeavor that seeks leagues discovered hundreds of novel genes that to comprehend the most fundamental processes of life. may help the malaria parasite evade destruction by In addition to well-publicized research on an anti- the human immune system and antimalarial drugs. obesity vaccine, reactivation of the gene responsible for The findings could lead to the development of Friedreich’s ataxia, heart damage from prion disease, new therapies or vaccines for the deadly disease. and the threat of the avian flu virus, Scripps Research OTHER NOTEWORTHY DEVELOPMENTS scientists made many significant contributions in 2006. New agreements with Novartis and the Genomics A few key studies are highlighted below. Institute of the Novartis Research Foundation (GNF) • Scientists demonstrated an innovative combina- will provide approximately $50 million over the next 5 tion of immunotherapy and small-molecule drug years to fund the Scripps Research laboratories of 20 design for producing anticancer targeting antibod- investigators, including Professor Peter Schultz, 5 sci- ies. One study, led by Professor Carlos Barbas III, entists moving to Scripps Research from GNF, and 14 highlighted the potential of such an approach assistant professors. Terms also facilitate the future against melanoma. In another study, Associate funding of Scripps Research faculty by Novartis. Professor Subhash Sinha and I developed a The Consortium for Functional Glycomics, led by compound against metastatic breast cancer. Scripps Research Professor James Paulson, received a • Professor Chi-Huey Wong and colleagues discov- $40.7 million “glue” grant for the international group ered a class of compounds that block the SARS of some 300 participating scientists to continue col- virus from replicating, a finding that may open the laborative study of the complex dynamics of protein- door to new drug targets against the deadly disease. carbohydrate interactions. The 5-year grant from the • Professor Dale Boger and Kellogg School Ph.D. National Institute of General Medical Science of the candidate Brendan Crowley re-engineered a well- National Institutes of Health follows a grant of $34 known antibiotic to ensure its effectiveness against million awarded in 2001. both sensitive and resistant enterococci, a com- The Integrative Neuroscience Initiative on Alcoholism, mon strain of bacteria responsible for widespread led by Scripps Research Professor George Koob, won hospital infections. renewal of support from the National Institutes of • Professor John Tainer and colleagues determined Health’s National Institute on Alcohol Abuse and Alco- the crystal structure and molecular mechanisms holism. The grant, which is expected to total $38 of a key part of WRN, a protein that protects million over 5 years, supports the efforts of a multi- humans from premature aging and cancer. They institutional consortium of investigators to identify the also uncovered the structural chemistry behind molecular basis of alcoholism. the bacterial GC Type IV pilus filament, which Scripps Research launched a research and edu- plays an essential role in allowing antibiotic- cational initiative with McDonald’s to drive progress resistant strains of N. gonorrhoeae to escape the toward a solution to childhood obesity and type 2 dia- THE SCRIPPS RESEARCH INSTITUTE 2006 7 betes. McDonald’s will contribute $2 million to the his elucidation of multiple interferon genes and institute to address these critical health issues. the pharmaceutical development of Intron A PEOPLE NEWS (interferon alpha2b). In 2006, Scripps Research continued to be served • Chair of the Department of Chemistry, K.C. Nico- by an outstanding group of trustees and administrators. laou, won both the 2006 American Chemical At our commencement ceremony in May that grad- Society Auburn G.M. Kosolapoff Award and Ger- uated 31 students from the Kellogg School of Science many’s Burkhardt-Helferich Prize. He is also an and Technology, we conferred 2 honorary degrees in author of 1 of Chemical Abstracts Service’s 10 recognition of Hon. Alice Sullivan (Ret.), retiring chair most requested papers (second quarter), “Palla- of the Scripps Research Board of Trustees who will dium-catalyzed cross-coupling reactions in total continue as a trustee, and Alexander Dreyfoos, also a synthesis,” in Angewandte Chemie. member of the Board of Trustees. • Two patents on “click chemistry” by Professor The business leader and philanthropist John Moores K. Barry Sharpless, Associate Professor Valery was unanimously elected new chair of the board—he Fokin, and Associate Professor M.G. Finn were will bring enormous skill and energy to the position. among the Chemical Abstracts Service’s 10 most We also have the pleasure of welcoming back Ralph J. requested patent families (second quarter). Shapiro of Beverly Hills, California, chair of Avondale • Associate Professor Clare Waterman-Storer won Investment Company, and of welcoming new member the 2006 R.R. Bensley Award in Cell Biology from Marjorie Fink of Palm Beach County, Florida. the American Association of Anatomists, which With the appointment of Professor Gerald Joyce as recognized her for innovation in molecular micros- dean of the faculty and Professor Jeffery W. Kelly as copy and contributions to the understanding of dean of graduate and postgraduate studies, in July we cytoskeletal dynamics in cell motility. formalized a new distribution of administrative responsi- •Professor Argyrios Theofilopoulos was honored bilities. This change will enhance efficiency and commu- several times this year for lifetime contributions nication in our academic programs. to medicine and autoimmune research; he received Barbara Suflas Noble, who has been part of our honorary doctoral degrees from the Aristotle administrative team in Florida, will assume the position University of Thessaloniki Medical School and of director of external affairs for Scripps Florida, reach- the Democritos Medical School of Alexandroupo- ing out to our generous and enthusiastic base of donors lis and was elected a corresponding member of in the state. Peter Policastro joins our team as senior the Academy of Athens. director of business development for Scripps Florida. •Professor Bruce Beutler won the Cancer Research We also welcome investigator John Cleveland, who Institute’s 2006 William B. Coley Award for Dis- will head the new Cancer Biology department on the tinguished Research in Basic Immunology for his Scripps Florida campus. contribution to our understanding of the events AWARDS AND HONORS leading to the initiation of innate immunity. Many awards and honors lauded our faculty, post- • Associate Professor Phil Baran received the Sloan doctoral fellows, and graduate students in 2006. Research Fellowship for “outstanding researchers Among the faculty recognitions: early in their academic careers.” He also received •Professor Dale Boger was elected to the Ameri- the Bristol-Myers Squibb Unrestricted Freedom can Academy of Arts and Sciences. Fellows are to Discover Grant (2006–2010) and a National selected through a highly competitive process Science Foundation CAREER award (2006–2010). that recognizes individuals who have made pre- • Norman Klinman, who became professor emeritus eminent contributions to their disciplines and to this year, received the 2006 Excellence in Mentor- society at large. ing Award from the American Association of Immu- • Chair of the Scripps Florida Department of Infec- nologists for exemplary career contributions to a tology, Charles Weissmann, received the presti- future generation of scientists. gious DART/NYU Biotechnology Achievement Our hardworking postdoctoral fellows were also Award from the Biotechnology Study Center of recognized by numerous grants and awards. As a few the New York University School of Medicine for examples, Ian Schneider of the Waterman-Storer lab 8 THE SCRIPPS RESEARCH INSTITUTE 2006

won a Damon Runyon Fellowship Award; Adam Mullick of the Curtiss-Tobias lab, a fellowship from the Ameri- can Heart Association; Terry Meehan of the Havran lab, a Crohn’s & Colitis Foundation of America Research Fellowship Award; Jeff Lee of the Ollmann Saphire lab, the Canadian Governor General’s Gold Medal; and David Edmonds of the Nicolaou lab, a European Merck Post- doctoral Fellowship. As for our Ph.D. candidates in the Kellogg School of Science and Technology, an unprecedented 5 students— Dan Bachovchin, Christine Fang, Graham Johnson, Costas Lyssiotis, and Adrian Ortiz—were awarded National Science Foundation Fellowships this year. In addition, students garnered prestigious awards from private donors, the National Institutes of Health, Novar- tis, and many other organizations including the Hertz Foundation, the American Heart Association, the Cali- fornia Breast Cancer Research Program, and the Ameri- can Chemical Society. This year’s achievements make me proud to be part of The Scripps Research Institute. My congratula- tions go out to faculty, staff, postdoctoral fellows, students, trustees, and loyal supporters for another year well done. Skaggs Institute for Chemical Biology

Wide-angle perspective: Looking down the barrel of the cylindrical capsule, we find n-tetradecane in a coiled, helical conformation. At first glance, this imposition of host on guest appears unfavor- able, but in the end, adaptation maximizes contact between the two and results in an induced-fit match. Work for the image done by

Michael P. Schramm, Ph.D., The Skaggs Institute for Chemical Biology. Trevor Dale

Graduate Student, Chemistry

The Skaggs Institute for

Chemical Biology THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 11

THE SKAGGS INSTITUTE M.G. Finn, Ph.D.* Peter Schultz, Ph.D.* Lionel Moisan, Ph.D. Associate Professor Professor FOR CHEMICAL BIOLOGY Andrew Myles, Ph.D. Scripps Family Chair Elizabeth D. Getzoff, Ph.D.†† Severin Odermatt, Ph.D. STAFF Professor K. Barry Sharpless, Ph.D.* Riccardo Salvio, Ph.D. Julius Rebek, Jr., Ph.D.* W.M. Keck Professor of M. Reza Ghadiri, Ph.D.* Professor and Director Chemistry Michael Schramm, Ph.D. Professor Siddhartha Shenoy, Ph.D. Carlos F. Barbas III, Ph.D.** Subhash C. Sinha, Ph.D.** Professor Kim D. Janda, Ph.D.* Associate Professor Alessandro Volonterio, Ph.D. Professor Janet and W. Keith Kellogg II Felix Zelder, Ph.D. Chair in Molecular Biology Ely R. Callaway, Jr., Chair John A. Tainer, Ph.D.** in Chemistry Professor Tamas Bartfai, Ph.D. Director, The Worm Institute James R. Williamson, Ph.D.††† Professor for Research and Medicine * Joint appointment in the Chairman, Molecular and Professor Department of Chemistry Integrative Neurosciences Gerald F. Joyce, M.D., Associate Dean, Kellogg ††† ** Joint appointment in the Department, Scripps Ph.D. School of Science and Department of Molecular Biology Professor Technology Research *** Joint appointment in the Director, Harold L. Dorris Dean, Faculty Department of Molecular and Ian A. Wilson, D.Phil.** Neurological Research Experimental Medicine Ehud Keinan, Ph.D.** Professor Institute **** Joint appointments in the Adjunct Professor Departments of Cell Biology and Chemistry Ernest Beutler, M.D.*** Chi-Huey Wong, Ph.D.* Jeffery W. Kelly, Ph.D.* Professor Ernest W. Hahn Professor ***** Joint appointment in the Lita Annenberg Hazen Department of Cell Biology Chairman, Department of and Chair in Chemistry Molecular and Experimental Professor of Chemistry † Joint appointment in the Peter E. Wright, Ph.D.** Medicine, Scripps Research Dean, Graduate and Department of Neurobiology Postgraduate Studies Professor †† Joint appointments in the Dale L. Boger, Ph.D.* Cecil H. and Ida M. Green Departments of Molecular ††† Richard and Alice Cramer Richard A. Lerner, M.D. Investigator in Biomedical Biology and Immunology Professor of Chemistry President, Scripps Research Research ††† Joint appointments in the Lita Annenberg Hazen Chairman, Department of Departments of Chemistry and Molecular Biology Geoffrey Chang, Ph.D.** Professor of Molecular Biology, Scripps †††† Associate Professor Immunochemistry Research Research associates in the labo- Cecil H. and Ida M. Green ratories of staff other than Dr. Rebek are included in the lists Benjamin F. Cravatt, ††† Chair in Chemistry Kurt Wüthrich, Ph.D. of the respective departments in Ph.D.**** Cecil H. and Ida M. Green which the associates hold joint Professor Stephen P. Mayfield, Professor of Structural appointments. Director, Helen L. Dorris Ph.D.***** Biology Child & Adolescent Neuro- Professor Psychiatric Disorder Associate Dean, Kellogg RESEARCH Institute School of Science and ASSOCIATES†††† Technology Philip Dawson, Ph.D.***** Associate Professor Dariush Ajami, Ph.D. K.C. Nicolaou, Ph.D.* Aline W. and L.S. Skaggs Elizabeth Barrett, Ph.D. Gerald M. Edelman, M.D., Ph.D.† Professor of Chemical Biology Sara Butterfield, Ph.D. Darlene Shiley Chair in Professor Alexandre Carella, Ph.D. Chairman, Department of Chemistry Neurobiology, Scripps Chairman, Department of Naran Gombosuren, Ph.D. Research Chemistry, Scripps Research Clemens Haas, Ph.D. ††† Albert Eschenmoser, Ph.D.* Paul R. Schimmel, Ph.D. Junli Hou, Ph.D. Professor Ernest and Jean Hahn Richard J. Hooley, Ph.D. Professor and Chair in Martha J. Fedor, Ph.D.** Molecular Biology and Tetsuo Iwasawa, Ph.D. Associate Professor Chemistry Enrique Mann, Ph.D. THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY

In 1996, The Scripps Research Institute established The Skaggs Institute for Chemical Biology, made possible by a gift of more than $100 million to The Skaggs Institute for Research from Aline W. and L.S. Skaggs. Scientific members of the Skaggs Institute hold dual appointments in various departments at Scripps Research. These scientists have broad expertise in areas including the structure of biological macromolecules, chemical and antibody catalysis, synthetic and combinatorial chemistry, molecular recognition, and molecular modeling methods. With the achievements of its staff, the Skaggs Institute has assumed its research identity in the United States and throughout the world at the interface of biology and chemistry. THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 13

cinated against anthrax. Other antibodies are targeted as anticancer agents, particularly those related to childhood lymphomas. Chi-Huey Wong and his group are using enzymes as reagents for organic synthesis; an ultimate target is to use these enzymes to modify proteins with sugars on their surfaces, leading to the development of vaccines against HIV, influenza, and breast cancer. This approach is complemented by a new ligation method that uses a sugar derivative to accelerate the coupling of protein fragments. Paul Schimmel’s research continues to be centered on the apparatus that converts genetic information into proteins. The enzymes that attach amino acids to tRNA were discovered to have an additional activity as cell- signaling proteins. These cytokines are needed for control of blood vessel growth and inflammation; controlling these signaling activities may eventually lead to therapies. Peter Wright, chairman of the Department of Molecular Biology, is studying protein-protein and protein–nucleic acid interactions in solution. Specific targets include the transcriptional coactivators that have Julius Rebek, Jr., Ph.D. been implicated in human diseases such as leukemia, cancer, and mental retardation. Dale Boger and his group Director’s Overview are studying small-molecule nucleic acid binding agents; they have synthesized naturally occurring antitumor he Skaggs Institute for Chemical Biology, estab- agents and have showed that the natural compound lished in 1996, is now completing its 1st decade. and its mirror image are equally effective at covalent T During this time, the generous endowment of the binding to nucleic acids. Dr. Boger’s group is also working Skaggs family has supported the research of more than on redesigning the vancomycin antibiotic to overcome 30 principal investigators and some 500 postdoctoral resistance. This exercise in molecular recognition has fellows and graduate students. These researchers have led to a new structure that is 100-fold more effective at produced more than 2,000 publications in the areas of binding to resistance peptides. Carlos Barbas and mem- chemistry, chemical biology, molecular biology, and bers of his laboratory are developing strategies to produce immunology gaining the Skaggs Institute a worldwide antibodies that officially form or break carbon-carbon reputation for excellence. Highlights of some of the bonds for use in organic synthesis. These antibodies research done this past year are discussed briefly below, are developed by using novel recombinant strategies. and the individual reports of the principal investigators Dr. Barbas is also spearheading an effort to use small are presented elsewhere in this volume. organic molecules that show enzyme-like activities for Researchers in the laboratories of Geoffrey Chang organic synthesis. and M.G. Finn have taken multidisciplinary measures to Ernest Beutler, chairman of the Department of Molec- design inhibitors to biological molecules that cause can- ular and Experimental Medicine, is studying mechanisms cer drug resistance. These measures take advantage of involved in the checkpoints that maintain genome sta- x-ray structures of drug “antiporters” and electron cry- bility. These are principle defense mechanisms against omicroscopy to study changes in the shapes of the mol- malignant phenotypes and forces for tumor growth. ecules as they perform. Elizabeth Getzoff characterizes the mechanisms of light- Stephen Mayfield’s group is using algae as a sys- induced protein activities, particularly in the cryptochrome tem for production of therapeutic proteins. Specifically, flavoproteins that are components of the circadian clocks they have developed a human anti-anthrax antibody by in animals and humans. Jamie Williamson’s group is using samples obtained from soldiers who had been vac- using nuclear magnetic resonance to study very large 14 THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE molecules such as viral capsid proteins in solution. The opportunities provided by the Skaggs family’s support. nuclear magnetic resonance spectra reveal protein seg- We look forward to our second decade. ments that are sufficiently mobile so as not to be observ- able by using x-ray or electron microscopy. Ian Wilson also pursues the crystallographic characterization of influenza virus glycoproteins. Dr. Wilson and his group intend to improve the potency of antiviral drugs by solving the structure of the neuraminidase and the viral coat protein involved in the 1918 influenza pandemic. K.C. Nicolaou, chairman of the Department of Chem- istry, has made great progress in the synthesis of several biologically active molecules. These include a number of antitumor agents from the cytoskyrin family, murine- derived antibiotics, and marinomycins. K. Barry Sharpless and Valery Fokin continue to develop “click chemistry,” extremely versatile reactions that drive spontaneous, selective, and irreversible linkages between molecular building blocks. They and their researchers are using these reactions as a means for rapid exploration of chemical space. President Richard Lerner and Subhash Sinha are developing antibodies for selective chemotherapy. These involve drug conjugates and prodrugs that target cell-surface receptors and prostate-specific membrane antigen. The antibodies convert prodrugs into active molecules at the sites where they are most needed. Kim Janda’s group is exploring applications of botox in the treatment of multiple sclerosis, stroke, and migraine. Molecules that activate the toxin could ultimately allow lower doses of the agent to be effective and could reduce the immune response in these applications. Tamas Bartfai, chairman of the Molecular and Integrative Neuroscience Department, continues his studies on the temperature regulation of mammals. Dr. Bartfai and his group have used transgenic methods to develop an animal model, “the cool mouse,” whose thermo set has been effectively lowered, resulting in reduced energy expenditure and a longer lifetime for the animal. Jeffery Kelly, dean of the Graduate Program, is studying the protein folding and misfolding involved in Alzheimer’s, Parkinson’s, and Gaucher’s diseases. They have found that oxidative cholesterol metabolites can modify amyloid peptide and accelerate the precipitation associated with Alzheimer’s disease. My own group continues the study of molecules in extremely small spaces, and how they interact when confined to close range. This has led to newly discovered stereochemical properties and a spring-loaded device for nanomachinery. All of us are thrilled to be associated with The Skaggs Institute for Research and are grateful for the research THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 15

INVESTIGATOR’S REPORT resembles that of an α-helical protein. We have built small libraries of these compounds that target a number of protein-protein interactions implicated in, for Convex and Concave example, prolonged cancer states, chronic neuropathic pain, and epilepsy. Recognition Surfaces CAVITANDS AS RECEPTORS Cavitands are concave hosts that bind small mole- J. Rebek, Jr., D. Ajami, E. Barrett, S. Biros, S. Butterfield, cules of complementary size, shape, and chemical sur- A. Carella, T.J. Dale, N. Gombosuren, C. Haas, R.J. Hooley, face. Deepened cavitands enclose most of a small guest, T. Iwasawa, E. Mann, L. Moisan, A. Myles, B. Purse, but the open end reduces the selectivity and exposes R. Salvio, M. Schramm, H. Van Anda, A. Volonterio, F. Zelder part of the guest to the external medium. Exquisite MIMETICS OF α -HELICES selectivities can be achieved by using capsules that rotein-protein interactions are involved in the reg- completely surround the guest. We recently prepared a ulation of a wide variety of biological processes. water-soluble cavitand (Fig. 2) that coaxes hydropho- P These recognition events often occur between a large protein containing a well-defined binding site and a smaller protein with features complementary to the site. This relationship has been compared with that of a lock and its key: only a key with the correct grooves and notches will fit and elicit a response. Regulation of these events by small, synthetic molecules is a challenging but desirable goal in medicinal chemistry. Structures that can selectively enhance or antagonize protein-protein interactions have much promise as pharmaceuticals. Fig. 2. A water-soluble synthetic receptor extracts normal alka- We have constructed a series of molecules that tar- nes and other insoluble species into aqueous solution. Inside the get protein-protein interactions in which the smaller cavity, the alkanes coil into a helix to maximize hydrophobic con- protein adopts an α-helical conformation (Fig. 1). The tacts with the receptor and tumble rapidly on the nuclear magnetic resonance timescale.

bic guests into the cavity, where they are more or less shielded from the aqueous environment. These complexes are kinetically stable; that is, exchange of guests is slow on the nuclear magnetic resonance timescale. The guests are surrounded by surfaces made of aromatic subunits, allowing van der Waals interactions between host and guest. This attraction leads to conformational changes for normal hydrocarbons such as octane; the hydrocarbons coil to make better contacts with the inner lining of the receptor and reduce the surfaces exposed to the aqueous environment. A cavitand with doors that can be rotated over the Fig. 1. Left, Line structure and skeletal model of a synthetic, open end has been synthesized and characterized. The scaffold-based α-helix mimetic. Right, Overlay of the scaffold (light doors shield guests from water and limit the size of gray) with an α-helical peptide (black tube); the side-chain functional groups used to recognize other proteins are shown as spheres. guests that fit the space. The increase in selectivity for small guests allows cycloalkanes inside but excludes synthesis of these molecules is modular and readily longer linear counterparts of cycloalkanes. Cyclopentane amenable to combinatorial techniques. These struc- inside the cavitand is shown in Figure 3. The binding tures act as scaffolds to project functional groups (the of n-hexane causes the doors to fully open and expose “grooves” and “notches”) in a manner that closely the guest to the aqueous surroundings. The closed doors 16 THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Menozzi, E., Onagi, H., Rheingold, A.L., Rebek, J., Jr. Extended cavitands of nanoscale dimensions. Eur. J. Org. Chem. 3633, 2005, Issue 17.

Menozzi, E., Rebek, J., Jr. Metal directed assembly of ditopic containers and their complexes with alkylammonium salts. Chem. Commun. (Camb.) 5530, 2005, Issue 44.

Purse, B.W., Gissot, A., Rebek, J., Jr. A deep cavitand provides a structured environment for the Menschutkin reaction. J. Am. Chem. Soc. 127:11222, 2005.

Purse, B.W., Rebek, J., Jr. Functional cavitands: chemical reactivity in structured environments. Proc. Natl. Acad. Sci. U. S. A. 102:10777, 2005.

Fig. 3. Two views of a water-soluble cavitand with rotating doors Zelder, F.H., Rebek, J., Jr. Cavitand templated catalysis of acetylcholine. Chem. Commun. (Camb.) 753, 2006, Issue 7. on its upper rim. Two doors close access to the cavity and slow the uptake and release of guests. The guest shown is cyclopentane. Zelder, F.H., Salvio, R., Rebek, J., Jr. A synthetic receptor for phosphocholine esters. Chem. Commun. (Camb.) 1280, 2006, Issue 12. also reduce the rate of motion as various small guests go in and out of the cavitand. Cavitands have also been outfitted for catalysis by introducing a metal complex fused to the upper rim. This complex features a deep cavity for driving guest recognition and a metal ion at the top of the cavity that is positioned to coordinate a phosphate group of the guest. These binding forces act simultaneously on smaller molecules that bear phosphocholine subunits as shown in Figure 4. Reactions that have been catalyzed involv-

Fig. 4. Left, Line drawing of a cavitand outfitted with a salen ligand with a zinc ion. Right, Energy-minimized structure of the complex of the cavitand and a simplified phosphocholine model (a wall of the cavitand has been removed for viewing clarity).

ing a choline substrate include acylation, aminolysis, and ester cleavage.

PUBLICATIONS Haas, C.H., Biros, S.M., Rebek, J., Jr. Binding properties of cavitands in aqueous solution: the influence of charge on guest selectivity. Chem. Commun. (Camb.) 6044, 2005, Issue 48.

Hooley, R.J., Biros, S.M., Rebek, J., Jr. A deep, water-soluble cavitand acts as a phase-transfer catalyst for hydrophobic species. Angew. Chem. Int. Ed. 45:3517, 2006.

Hooley, R.J., Biros, S.M., Rebek, J., Jr. Normal hydrocarbons writhe and tumble rapidly in a deep, water-soluble cavitand. Chem. Commun. (Camb.) 509, 2006, Issue 5.

Hooley, R.J., Rebek, J., Jr. Deep cavitands provide organized solvation of reactions. J. Am. Chem. Soc. 27:11904, 2005.

Hooley, R.J., Van Anda, H.J., Rebek, J., Jr. Cavitands with revolving doors regulate binding selectivities and rates in water. J. Am. Chem. Soc. 128:3894, 2006. Cell Biology

Electron cryomicroscopy and single-particle analysis were used to determine the 30-Å structure of the self-assembling coat protein complex-II cage nanoparticle. Shown are the

2-fold (bottom), 3-fold (middle), and 4-fold (top) fold symmetry axes of the biologically unprecedented cuboctahedron structure responsible for directing cargo selection and membrane curvature during endoplasmic reticulum vesicle budding. Reprinted from Stagg, S.M., Gurkan, C., Fowler, D.M.,

LaPointe, P., Foss, T.R., Potter, C.S., Carragher, B., Balch,

W.E. Structure of the Sec13/31 COPII coat cage. Nature

439:234, 2006. This work is a collaboration between the laboratories of William Balch, Ph.D., Bridget Carragher, Ph.D, and Clinton Potter, B.S., at the National Resource for

Automated Molecular Microscopy. Gaudenz Danuser, Ph.D., Associate Professor

Dinah Leorke, Ph.D., Research Associate

James Lim, Graduate Student

Department of Cell Biology CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 19

DEPARTMENT OF Stephen P. Mayfield, A DJUNCT APPOINTMENTS Robert Fischer, Ph.D. CELL BIOLOGY Ph.D.***** Professor Alan Bell, B.S.C.S. Elizabeth Wilson, Ph.D. Xerox Palo Alto Research STAFF Associate Dean of Graduate Studies Center Sandra L. Schmid, Ph.D.* Palo Alto, California SENIOR RESEARCH Professor and Chairman Mark Mayford, Ph.D.*** ASSOCIATES Richard Bruce, Ph.D. Associate Professor Francisco Asturias, Ph.D.** Xerox Palo Alto Research Brian Adair, Ph.D. Center Associate Professor Lindsey Miles, Ph.D. Palo Alto, California Barbara Calabrese, Ph.D. Associate Professor William E. Balch, Ph.D.* Professor Douglas Curry, B.S. (E.E.C.S.) Mark Daniels, Ph.D. Ronald A. Milligan, Ph.D.** Xerox Palo Alto Research Professor Kristin Baldwin, Ph.D.*** Center Jeremiah Joseph, Ph.D. Director, Center for Assistant Professor Palo Alto, California Integrative Biosciences Edward Korzus, Ph.D.††† Bridget Carragher, Ph.D.** Bertil Daneholt, M.D. University of California Associate Professor Ulrich Müller*** Karolinska Institutet Riverside, California Professor Stockholm, Sweden Benjamin Cravatt, Ph.D.**** Matthew Ritter, Ph.D. † Professor Ardem Patapoutian, Ph.D. Scott Elrod, Ph.D. Director, Helen L. Dorris Associate Professor Xerox Palo Alto Research Martin Schwander, Ph.D. Child & Adolescent Neuro- Center Psychiatric Disorder Clinton Potter , B.S.** Palo Alto, California Gina Story, Ph.D.††† Institute Associate Professor Washington University Mark Ginsberg, M.D. St. Louis, Missouri Gaudenz Danuser , Ph.D.** James Quigley, Ph.D. University of California Associate Professor Professor San Diego, California Defne Yarar, Ph.D.

Philip E. Dawson, Ph.D.***** Lisa Stowers, Ph.D.†† David Goldberg, Ph.D. Andries Zijlstra, Ph.D. Xerox Palo Alto Research Associate Professor Assistant Professor Center Velia Fowler, Ph.D.** Palo Alto, California Heidi Stuhlmann, Ph.D. RESEARCH ASSOCIATES Professor Associate Professor Xiaohua Gong, Ph.D. Jessica Alexander, Ph.D. Martin Friedlander, M.D., University of California Kevin F. Sullivan, Ph.D.††† Ph.D. Berkeley, California University of Ireland Geza Ambrus-Aikelin, Ph.D. Professor Galway, Ireland Klaus Hahn, Ph.D. Veronica Ardi, Ph.D. Larry R. Gerace, Ph.D.* University of North Carolina Peter N.T. Unwin, Ph.D.** Professor Chapel Hill, North Carolina Angelique Aschrafi, Ph.D.†††† Professor Shelley Halpain, Ph.D.*** Eric Peeters, Ph.D. Andrea Bacconi, Ph.D. Associate Professor Clare Waterman-Storer, Xerox Palo Alto Research Ph.D.** Center Hongdong Bai, Ph.D. Natasha Kralli, Ph.D. Associate Professor Palo Alto, California Associate Professor Kent Baker, Ph.D. Elizabeth Winzeler, Ph.D.†

Peter Kuhn, Ph.D.** Associate Professor STAFF SCIENTISTS Claudia Barros, Ph.D. Associate Professor John R. Yates III, Ph.D. Michael Bracey, Ph.D. Maria Beligni, Ph.D. David Loskutoff, Ph.D. Professor Professor Emeritus Anchi Cheng, Ph.D. Richard Belvindrah, Ph.D. Mari Manchester, Ph.D.** Mark J. Yeager, M.D., Ph.D. Associate Professor Professor Elena Deryugina, Ph.D. Edward Brignole III, Ph.D. 20 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Florence Brunel, Ph.D. Anna Durrans, Ph.D. Paul LaPointe, Ph.D. Jacobus Neels, Ph.D.

Anja Bubeck, Ph.D.††† Samer Eid, Ph.D.††† Nicole Lazarus, Ph.D. Sherry Niessen, Ph.D. Gen-Probe, Inc. Merck Research San Diego, California Laboratories, Neuroscience Donmienne Leung, Ph.D.††† Silvia Ortega-Gutierrez, Drug Discovery Applied Molecular Evolution Ph.D. Gang Cai, Ph.D. West Point, Pennsylvania San Diego, California Lesley Page, Ph.D. Gregory Cantin, Ph.D. Michael Fitch, Ph.D.††† John Lewis, Ph.D.††† Tanabe Research Dalhousie University Ana Maria Pasaperi-Limon, Eric Carlson, Ph.D. Laboratories U.S.A. Halifax, Nova Scotia, Canada Ph.D. San Diego, California Aurelia Cassany, Ph.D. Lujian Liao, Ph.D. Olivier Pertz, Ph.D.††† Santos Franco, Ph.D. University of California Yuriy Chaban, Ph.D. Maria Lillo, Ph.D.††† San Diego, California Margaret Gardel, Ph.D. University of Salamanca Pablo Chamero, Ph.D. Salamanca, Spain Barbie Pornillos, Ph.D. Maria Gonzalez, Ph.D.††† Emily Chen, Ph.D. Rincon Pharmaceuticals Jennifer Lin, Ph.D. Anita Pottekat, Ph.D. La Jolla, California Ihsiung Chen, Ph.D.††† Ryan Littlefield, Ph.D.††† Judith Prieto, Ph.D. CODA Genomics University of Washington Jorg Grandl, Ph.D. Laguna Hills, California Seattle, Washington Natalie Prigozhina, Ph.D.††† Vala Sciences, Inc. Nicolas Grillet, Ph.D. Yei Hua Chen, Ph.D. Dinah Loerke, Ph.D. La Jolla, California Cemal Gurkan, Ph.D.†††† Charmian Cher, Ph.D. Darren Logan, Ph.D. Thomas Pucadyil, Ph.D.

Johannes Hewel, Ph.D. Smita Chitnis, Ph.D. Bingen Lu, Ph.D. Rajesh Ramachandran, Ph.D. Michael Hock, Ph.D. Esther Choi, Ph.D. Matthias Machacek, Ph.D. Vandana Ramachandran, Ke Hu, Ph.D. Parag Chowdhury, Ph.D. Kalotina Machini, Ph.D. Ph.D.

Jill Chrencik, Ph.D. Michael Huber, Ph.D. Mark Madsen, Ph.D. Abbas Razvi, Ph.D.

Michael Churchill, Ph.D. Darren Hutt, Ph.D. Valentina Marchetti, Ph.D. Leon Reijmers, Ph.D.

Francesco Conti, Ph.D. Eric Hwang, Ph.D. Julia Marin-Navarro, Ph.D. Anna Reynolds, Ph.D.

Judith Coppinger, Ph.D. Khuloud Jaqaman, Ph.D. Michael Matho, Ph.D. Edwin Romijn, Ph.D.††† Philips Scientific Equipment †††† Kaustuv Datta, Ph.D. Anass Jawhari, Ph.D. Naoki Matsuo, Ph.D. Division Eindhoven, the Netherlands Leif Dehmelt, Ph.D. Lin Ji, Ph.D. Daniel McClatchy, Ph.D. Cristian Ruse, Ph.D. Ajay Dhaka, Ph.D. Nobutaka Kato, Ph.D. Caroline McKeown, Ph.D. Mohsen Sabouri-Ghomi, Anouk Dirksen, Ph.D. Claire Kidgell, Ph.D.†††† Marcel Mettlen, Ph.D. Ph.D.

Meng-Qui Dong, Ph.D. Katsuhiro Kita, Ph.D. Helena Mira, Ph.D. Alan Saghatelian, Ph.D.††† Harvard University Michael Dorrell, Ph.D. Kevin Koehntop, Ph.D. Jennifer Mitchell, Ph.D. Cambridge, Massachusetts

Kelly A. Dryden, Ph.D. Jenny Kohler, Ph.D. Machiko Muto, Ph.D. Kumar Saikatendu, Ph.D.

Jerome Dupuy, Ph.D. Atanas Koulov, Ph.D. Andromeda Nauli, Ph.D. Tomoyo Sakata, Ph.D. CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 21

Cleo Salisbury, Ph.D. Ge Yang, Ph.D. * Joint appointment in the Department of Molecular Biology Ian Schneider, Ph.D. Masahiro Yasuda, Ph.D.††† ** Joint appointment in the Center University of Michigan for Integrative Molecular Christina Schroeder, Ph.D. Ann Arbor, Michigan Biosciences *** Joint appointment in the Stephan Sieber, Ph.D.††† Rie Yasuda, Ph.D.††† Institute for Childhood and Ludwig-Maximilians University Osaka University Neglected Diseases Munich, Germany Osaka, Japan **** Joint appointments in the Department of Chemistry, the Pratik Singh, Ph.D. Zhongmin Zou, Ph.D.††† Skaggs Institute for Chemical Institute of Combined Injury Biology, and the Helen L. Dorris Scott Stagg, Ph.D. of PLA, Third Military Child and Adolescent Neuro- Psychiatric Disorder Institute Medical University Mark Surka, Ph.D. Chongqing, P.R. China ***** Joint appointment in the Skaggs Institute for Chemical Biology

† Patricia Szainer, Ph.D. Joint appointments in the Institute for Childhood and SCIENTIFIC ASSOCIATES Neglected Diseases and the Claire Tiraby Nguyen, Ph.D. Genomics Institute of the Novartis Hilda Edith Aguilar de Diaz, Research Foundation Tuija Uusitalo, Ph.D.††† M.D. †† Joint appointments in the Helen University of Helsinki L. Dorris Child and Adolescent Helsinki, Finland Alexei Brooun, Ph.D. Neuro-Psychiatric Disorder Institute Valerie Uzzell, Ph.D. Claire Delahunty, Ph.D. ††† Appointment completed; new location shown John Venable, Ph.D. Mohammed El-Kalay, Ph.D. †††† Appointment completed

Josep Villena, Ph.D. Tinglu Guan, Ph.D.

Xiaodong Wang, Ph.D.††† Anand Kolatkar, Ph.D. Medical University of Ohio Toledo, Ohio

Kari Bradtke Weber, Ph.D.

Eranthie Weerapana, Ph.D.

BinQing Wei, Ph.D.

Scott Westenberger, Ph.D.

Ann Wheeler, Ph.D.

Torsten Wittmann, Ph.D.††† University of San Francisco San Francisco, California

James Wohlschlegel, Ph.D.

Catherine Wong, Ph.D.

Aaron Wright, Ph.D.

Lihua Wu, Ph.D.††† Department of Immunology Scripps Research 22 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

A unique attribute of Scripps Research that distinguishes it from academic institutions whose faculty must meet diverse educational obligations is its ability to build research efforts around areas of strength, ensuring critical mass and leadership in important areas of biomedical research. Although the department is proud of the success of its individual scientists, we believe that this success reflects in part the strong synergies that have developed as we have built on areas of strength within cell biology. Synergy is defined as 2 or more groups working together in such a way that the result is greater than the sum of their individual capabilities. Although there are numerous foci of synergy driving innovation and research in the department, I highlight here the 3 areas that represent the strengths on which we will continue to build. An early and unique strength of our department, which synergizes with the structural efforts in the Department of Molecular Biology, is the use of electron cryomicroscopy to provide structural insights into the workings of complex, multisubunit cellular machines. Francisco Sandra Schmid, Ph.D. Asturias, Bridget Carragher, Clint Potter, Ron Milligan, Nigel Unwin, Mark Yeager, and their colleagues have Cell Biology Overview built an internationally preeminent center for electron cryomicroscopy. Together, these groups are developing embers of the Department of Cell Biology con- new methodologies, solving important structures, and tinue to excel in the rich environment of Scripps training the next generation of electron cryomicroscopy M Research, and their successes are being rec- structural biologists, not only among students and fellows ognized by others. Clare Waterman-Storer, who launched at Scripps Research, but through popular intensive sum- her independent career at Scripps Research, received the mer courses offered to students from around the world. National Institutes of Health Director’s Pioneer Award These synergistic interactions have driven unprecedented this year, along with a 5-year grant to support her research productivity. Many important structures have been solved program. Dr. Waterman-Storer was 1 of 13 scientists in the past year, including (1) the chloroplast ribosome chosen from more than 800 applicants for this extremely to reveal functionally important differences between it prestigious award; the Director’s Pioneer Award recog- and its bacterial progenitor (Dr. Milligan in collaboration nizes leading scientists in the United States and invests with members of Dr. Mayfield’s laboratory), (2) the intact in their potential to make important advances in bio- infectious P22 bacteriophage to reveal the mechanisms medical research. Also this year John Yates received of viral DNA transfer into the host cell (Drs. Carragher the prestigious Christian B. Anfinsen Award from the and Potter in collaboration with Jack Johnson in the Protein Society, which recognizes significant technical Department of Molecular Biology), (3) the coat protein achievements in the field of protein science. Gaudenz complex II cage to reveal a unique architecture for deform- Danuser, Martin Friedlander, Dr. Waterman-Storer, and I ing a membrane and collecting cargo molecules into trans- have given plenary addresses recognizing notable scien- port vesicles (Drs. Carragher and Potter in collaboration tific achievements and leadership in our respective fields. with members of Bill Balch’s laboratory), (4) the struc- Finally, Dr. Danuser and Natasha Kralli were promoted ture of DNA polymerase epsilon, providing new insight to associate professor, and Stephen Mayfield, who also into its interaction with its DNA template (Dr. Asturias), serves as associate dean of Graduate Studies, was pro- (5) the structure of a minus-end directed kinesin to reveal moted to full professor—positions befitting their acade- the mechanism of its unusual directionality (Dr. Milligan mic and scientific accomplishments. in collaboration with Ron Vale at the University of Cali- CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 23 fornia, San Francisco), and (6) the structure of an inte- the cell. The work of Drs. Danuser and Waterman-Storer grin complexed to its substrate revealing the activated is revealing the complex molecular and physical inter- form of this cell adhesion molecule (Dr. Yeager). These actions between multiple moving parts of the cell that remarkable accomplishments reveal the accelerated pace are required for directed cell locomotion. Dynamic inter- of electron cryomicroscopy structural biology made pos- actions and interdependencies between the actin cyto- sible through the relatively recent expansion of these skeleton and the endocytic machinery are being revealed efforts within the Center for Integrative Molecular Bio- in collaboration with scientists in my laboratory. Spatial sciences, and the resulting innovations in technology and temporal regulation of signaling events that direct and efforts to automate this historically slow and tedious cellular behavior are being analyzed in collaboration with technology. The importance of these efforts, under the Gary Bokoch in the Department of Immunology. The leadership of Drs. Carragher and Potter, has been recog- sophisticated microscopy, image analysis, and mathe- nized with funding from the National Center for Research matical modeling techniques being developed in the Resources and designated as a National Resource for Center for Molecular Biosciences, combined with innova- Automated Molecular Microscopy. tions in molecular biology, are enabling cell biologists to A second area of synergy, which has been built in manipulate and study the complex behavior of whole cells, association with the Institute for Childhood Diseases, is rather than the traditional and more limited “divide and in cellular and molecular neurobiology. Kristen Baldwin conquer” approaches of the past. recently joined these efforts from Columbia University Although the Department of Cell Biology at Scripps after completing her postdoctoral training with Richard Research is clearly leading in these important areas of Axel, the recipient of the 2004 Nobel Prize in Physiol- research and innovation, in this fast-paced and compet- ogy and Medicine for his work on olfaction. Using the itive arena if you’re not moving forward, you’ll quickly olfactory system as a model, Dr. Baldwin plans to dis- slip backward. Therefore, we hope to continue to build sect the mechanisms governing neuronal diversity and on these areas of strength with the recruitment of tal- the establishment of neural connectivities that enable us ented and creative faculty who bring new and comple- to process and respond to complex sensory input. She mentary expertise to our efforts and who will contribute joins a group of investigators at the Institute for Child- to and benefit from the synergistic environments we hood Diseases that includes Shelley Halpain, Mark May- have established. ford, Uli Mueller, Ardem Patapoutian, and Lisa Stowers, who work on diverse but complementary aspects of neuronal development, sensory perception (especially touch, smell, and hearing), the establishment of neuronal circuitry, and higher-order functions of learning and memory. Their combined expertise allows them to tackle the complexities of how the brain is wired for higher-order functioning. The outcomes of their studies have important implications for childhood diseases such as autism, as well as for mental retardation, schizophrenia, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. A third area of synergy on which we plan to build is in the quantitative spatial and temporal analyses of higher- order cellular processes, such as cell migration, signal transduction, the establishment of polarity, and intracellular trafficking. These efforts, also being carried out within the Center for Molecular Biosciences, are spear- headed by Dr. Danuser, Velia Fowler, and Dr. Waterman- Storer. Traditionally, cell biologists have focused their efforts on dissecting a single cellular process or a single piece of the cellular machinery, often in isolation from 24 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

INVESTIGATORS’REPORTS 2 of the 4 subunit components of Pol ε and to docu- ment and measure changes in the relative orientation of the 2 large domains that constitute the structure. Structural Characterization of Although no atomic resolution structures of the component subunits were available to help in interpreting Macromolecular Machines the electron microscopy map, we used the structural information to design template elongation assays that F.J. Asturias, Y. Chaban, J. Brown, E. Brignole, G. Cai resulted in a model for interaction of Pol ε with DNA e use state-of-the-art electron microscopy and that explains the intrinsic processivity and capacity of image analysis techniques to determine the Pol ε to interact with a variety of templates (Fig. 1). W 3-dimensional structures of macromolecular complexes involved in a variety of cellular processes, including DNA transcription, DNA replication, chromatin modification and remodeling, and fatty acid synthesis. Macromolecular electron microscopy is an ideal technique for these studies because it requires only a small amount of material and the conditions for preparing samples are physiologically relevant. Images of individual macromolecules are recorded and then computationally combined to obtain structures of low to moderate (25–10 Å) resolution. These structures are often interpreted by docking atomic resolution structures of component subunits in the lower resolution map of an entire complex. Our ultimate goal is to use a combination of biochemical and structural information to reveal the mechanism by which a macromolecular complex carries out its function. In our current research on DNA transcription and its regulation, we are analyzing the basal machinery and assembly of the RNA polymerase II preinitiation complex. We are also studying complexes involved in the regulation of transcription during initiation and in previous steps in which the structure of chromatin is altered to control access to DNA. We are particularly Fig. 1. Structure of Pol ε and a model for its interaction with DNA. interested in the structure and function of Mediator, a Electron microscopy and single-particle image analysis were used to calculate the structure of the polymerase at a resolution of 16 Å. complex that plays a central role in regulating tran- The structure of the catalytic Pol2 subunit was calculated indepen- scription in eukaryotes at the time transcription begins. dently, as was the structure of the Pol2–Dpb2 subunit complex. We have developed a reproducible protocol for purify- Image analysis also revealed changes in the relative orientation of the ing Mediator that will enable us to pursue biochemical Pol2 and Dpb2-Dpb3-Dpb4 domains made possible by a flexible and structural studies to get to the heart of the mech- connection. These changes in relative orientation might result in a anism of regulation by Mediator.We are also gearing conformation that would allow access of the DNA to the active-site cleft (top). A return of the tail to its normal conformation after entry up to use fluorescence microscopy to validate the in of double-stranded DNA into the active-site cleft would result in close vivo relevance of our in vitro studies. interaction of the nucleic acid with the extended tail domain (bottom). In the past year we also made progress in analyz- This mode of interaction with DNA would explain the intrinsic proces- ing the structure and mechanism of DNA polymerase ε sivity of Pol ε and the involvement of the Dpb3–Dpb4 subunit com- (Pol ε). We used electron microscopy and single-parti- plex in double-stranded DNA binding. The processivity dependence cle image analysis to calculate a 16-Å resolution of the of the length of the double-stranded primer region revealed by elongation assays would be explained by the requirement for a minimal polymerase, the first of a multisubunit eukaryotic DNA length of double-stranded DNA to ensure proper interaction with the polymerase. We were able to determine the location of full length of the extended tail domain in the Pol ε structure. CELL BIOLOGY 2006 25

Finally, we continue to investigate the role that con- protein folding and how that defect affects the ability formational changes play in the function of mammalian of the protein to function normally within the context fatty acid synthase (FAS), the enzyme responsible for of the cell’s intracellular transport machinery or in the the synthesis of long-chain fatty acids. In this true extracellular environment of the host. macromolecular assembly line, the different enzymes Our broad objective is to define the molecular involved in the synthesis of fatty acids have fused into basis for the trafficking of normal and misfolded pro- a single polypeptide chain that includes 6 catalytic and teins through the secretory pathway of eukaryotic cells. 1 acyl carrier protein domains. Using a novel approach We use chemical, structural, biological, and bioinfor- in which FAS point mutants were imaged in the pres- matics approaches. ence of substrates (effectively pausing the enzyme at a Eukaryotic cells are highly compartmentalized; each given catalytic step), we were able to determine an compartment of the exocytic and endocytic pathways FAS structure that led to a revised model for FAS provides a unique chemical landscape in which protein organization. That model has now been confirmed by function and folding may be modulated. Movement a recently published partial x-ray structure of FAS. As between these compartments involves the activity of a result of the molecular flexibility that appears to be both anterograde and retrograde transport tubules and essential for the function of FAS, 2 of the FAS domains vesicles. Many conformational diseases are a conse- were not observed in the x-ray structure. Further elec- quence of dysfunction at different stages of this trans- tron microscopy analysis of FAS will reveal the location port pathway or outside the cell. of all domains and provide information about the vari- Transport through the secretory pathway involves a ety of conformational states that make possible the selective mechanism in which cargo molecules are con- multitude of interdomain interactions required for the centrated into carrier vesicles. Vesicle-mediated trans- function of the enzyme. port is regulated by a diverse group of small GTPases belonging to the Ras superfamily. Each of these mole- PUBLICATIONS cules acts as a “molecular sensor” to regulate different Asturias, F.J., Cheung, I., Sabouri, N., Chilkova, O., Wepplo, D., Johansson, E. Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron steps in the reversible assembly of vesicle coats and microscopy. Nat. Struct. Mol. Biol. 13:35, 2006. targeting-fusion complexes. During export from the first

Takagi, Y., Chadick, J.Z., Davis, J.A., Asturias, F.J. Preponderance of free Mediator compartment of the secretory pathway, the endoplasmic in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 280:31200, 2005. reticulum, coat recruitment to budding sites involves activation of the GTPase Sar1. After activation, the cytosolic coat components Sec23/24 and Sec13/31 Chemical Biology of form the coatomer complex II coat (COPII) that polymerizes to promote budding from the surface of the Conformational Disease endoplasmic reticulum. This machinery directs exit and Membrane Traffic from the endoplasmic reticulum of proteins encoded by nearly one third of the genome in eukaryotes. Recently, in collaboration with C. Potter and B. Car- W.E. Balch, Y. An, C. Chen, J. Conkright-Johnson, D. Fowler, ragher, Department of Cell Biology, we solved the C. Gurkan, D. Hutt, A. Koulov, P. LaPointe, J. Matteson, 2-dimensional electron cryomicroscopy structure of the A. Nauli, L. Page, H. Plutner, A. Pottekat, A. Razvi, S. Stagg, Sec13/31 cage (Fig. 1). This cage is a self-assembling P. Szajner, I. Yonemoto nanoparticle that collects cargo by assembling into a major challenge is to understand and treat the polymer scaffold that interacts with an adaptor protein many protein-misfolding diseases that affect complex bound to “exit codes” found on the cytoplas- A human health, including cystic fibrosis, emphy- mic domains of cargo and cargo receptors. These exit sema, type 2 diabetes, and amyloidosis. These codes bind to a multivalent adaptor platform found on abnormalities are classified as membrane-trafficking the surface of Sec24 facing the lipid layer. With J.R. conformational diseases because a defect in protein Yates, Department of Cell Biology, we are using state-of- folding at some stage of the eukaryotic secretory path- the-art proteomics (multidimensional protein identification way results in loss of activity or protein aggregation. A technology or MudPIT) to identify unknown components key concern is to determine the underlying defect in involved in cargo selection. 26 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

architecture of eukaryotic cells found in different tissues. This systems biology approach provides for the first time a global view of membrane traffic from the top down, integrating form with function. Of particular importance is our characterization of the structure of the Rab1 tether p115, done in collaboration with I.A. Wilson, Department of Molecular Biology. The structure reveals a superhelical coiled coil multivalent assembly platform that facilitates Rab-dependent maturation of tethering-fusion complexes. In addition, Rab proteins are recycled for use in multiple rounds of tether assembly. We recently showed the surprising importance of the Hsp90 chaperone system in Rab recycling after vesicle fusion. Many mutation disrupt cargo traffic from the endoplasmic reticulum by preventing proper protein folding during synthesis, resulting in loss of recognition by the COPII selection machinery. Other protein conformational diseases have mutations that disrupt function at later steps of the secretory pathway and outside the cell in new chemical environments that can alter the protein fold. In collaboration with J. Kelly, Department of Chemistry, we are studying the link between trafficking defects and the protein-folding energetics of a number of conformational diseases, including cystic Fig. 1. Structure of the self-assembling COPII cage. Illustrated are fibrosis, hereditary childhood emphysema, Gaucher the 3 different symmetry-related views of the Sec13/31 complex that disease, familial amyloidosis of Finnish type, Parkin- self-assembles to form unprecedented cuboctahedron geometry on the surface of the endoplasmic reticulum to form a molecular scaf- son’s disease, and transthyretin amyloidosis. These fold (cage) that collects cargo for export. By coordinating cargo con- analyses have led to a new understanding of the func- centration with membrane curvature and fission, the cage can tion of the endoplasmic reticulum in normal physiol- generate a transit vesicle that mobilizes cargo to the cell surface. ogy, suggesting that this compartment functions as a Reprinted from Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, capacitor for protein folding and human evolution. Our P., Foss, T.R., Potter, C.S., Carragher, B., Balch, W.E. Structure of analysis of cystic fibrosis has revealed that system-wide the Sec13/31 COPII coat cage. Nature 439:234, 2006. modification of the chaperone folding pathways (the After budding and fusion of COPII transport vesicles chaperone) can alter the steady-state energetic pools from the endoplasmic reticulum, targeting and fusion of of unfolded and folded macrostates (conformational the vesicles to generate the next compartment of the populations) that allow for rescue of the trafficking secretory pathway, the Golgi apparatus, require a differ- defect and restore the function of chloride channels at ent class of Ras-like GTPases that belong to the Rab the cell surface. family. Members of the large Rab family (>70 mem- Through a multidisciplinary approach that com- bers) act as molecular switches that assemble combines the tools of chemistry, biology, systems biology, plexes involved in vesicle tethering and fusion. Using bioinformatics, and structure, we hope to gain critical a bioinformatics approach involving hierarchial cluster- insight into the fundamental principles of cargo traf- ing and mRNA expression profiling (microarray), we ficking and the basis for a variety of inherited transport found that each Rab GTPase executes targeting and diseases. Knowledge of the function of these cargo fusion decisions at a distinct step in the exocytic or selection pathways will enable the development of endocytic pathway. By integrating the interactions of small-molecule chemical chaperones to encourage multiple distinct effectors at each step, Rab GTPases export and stability of misfolded proteins, leading to act as hubs to define the highly distinctive membrane restoration of normal cellular function. CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 27

PUBLICATIONS cessing. Our goal is to genetically label the neurons that Bannykh, S.I., Plutner, H., Matteson, J., Balch, W.E. The role of ARF1 and Rab GTPases in polarization of the Golgi stack. Traffic 6:803, 2005. respond to specific odors in the nose and the olfactory bulb, to trace the projections of the neurons into the cor- Chen, C.Y., Balch, W.E. The Hsp90 chaperone complex regulates GDI-dependent Rab recycling. Mol. Biol. Cell 17:3494, 2006. tical regions where inputs converge, and to identify the molecular mechanisms that govern the formation of spe- Chen, C.Y., Sakisaka, T., Balch, W.E. Use of Hsp90 inhibitors to disrupt GDI- dependent Rab recycling. Methods Enzymol. 403:339, 2005. cific neural circuits. We anticipate that our findings will

Fowler, D.M., Koulov, A.V., Alory-Jost, C., Marks, M.S., Balch, W.E., Kelly, J.W. reveal mechanisms common to neural circuit formation Functional amyloid formation within mammalian tissue. PLoS Biol. 4:e6, 2006. throughout the brain and provide insight into genetic

Gurkan, C., Balch, W.E. Recombinant production in baculovirus-infected insect bases of human cognitive and behavioral disorders. cells and purification of the mammalian Sec13/Sec31 complex. Methods Enzymol. CLONING MICE FROM NEURONS 404:58, 2005. In contrast to gene activation in other sensory sys- Gurkan, C., Lapp, H., Alory, C., Su, A.I., Hogenesch, J.B., Balch, W.E. Large-scale tems, odorant receptor genes are activated by stochastic profiling of Rab GTPase trafficking networks: the membrome. Mol. Biol. Cell 16:3847, 2005. mechanisms. Stochastic gene activation in the immune system is due to irreversible DNA rearrangements. We Gurkan, C., Lapp, H., Hogenesch, J.B., Balch, W.E. Exploring trafficking GTPase function by mRNA expression profiling: use of the SymAtlas Web-application and searched for chromosomal rearrangements by cloning the Membrome datasets. Methods Enzymol. 403:1, 2005. mice from the nuclei of olfactory sensory neurons. We Kelly, J.W., Balch, W.E. The integration of cell and chemical biology in protein found that odorant receptor choice is reversible, as is folding. Nat. Chem. Biol. 2:224, 2006. neuronal differentiation. We will now clone mice from Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.J., Venable, J., Yates, J., Kelly, J.W., other types of neurons to determine whether irrevers- Balch, W.E. Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. Embo J. 24:4124, 2005. ible chromosomal alterations accompany neuronal diversification in the brain. Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, P., Foss, T.R., Potter, C.S., Carragher, B., Balch, W.E. Structure of the Sec13/31 COPII coat cage. Nature VISUALIZING OLFACTORY INPUTS TO THE BRAIN 439:234, 2006. A major challenge is to understand how olfactory Suk, J.Y., Zhang, F., Balch, W.E., Linhardt, R.J., Kelly, J.W. Heparin accelerates information is integrated in the olfactory cortex. An gelsolin amyloidogenesis. Biochemistry 45:2234, 2006. important first step is to describe the anatomy of the Wang, X., Venable, J., LaPointe, P., Hutt, D.M., Koulov, A.V., Coppinger, J., Gurkan, second-order circuit. This endeavor has been hindered C., Kellner, W., Matteson, J., Plutner, H., Riordan, J.R., Kellly, J.W., Yates, J.R. III, Balch, W.E. Hsp90 cochaperone rescue of misfolding disease. Cell, in press. by the lack of specific promoters for the output neurons (mitral cells) of the olfactory bulb. We identified a gene Wiseman, R.L., Balch, W.E. A new pharmacology: drugging stressed folding pathways. Trends Mol. Med. 11:347, 2005. that is expressed specifically in mitral cells and have produced mice in which subsets of these cells express fluorescent proteins. We use confocal and 2-photon Molecular Mechanisms of microscopy to map the projections of individual mitral cells into the brain. By visualizing the second-order Olfactory Perception and Neural olfactory circuit, we can begin to understand how the brain recognizes odors. Circuit Formation NEURAL DIVERSITY AND CIRCUIT FORMATION Although genes that regulate axon guidance and K.K. Baldwin, S. Tate, B. Fields, S. Ghosh large-scale brain patterning have been identified, the n mammals, the sense of smell is critical for sur- genes that endow neurons with the precise patterns of vival. Scents trigger suckling at birth, distinguish neuronal synaptic connectivity remain enigmatic. We I food from poison, provide warning of predators, and reasoned that genes expressed in subsets of mitral cells identify attractive mates. A primary goal of neurobiol- would be good candidates to direct the formation of ogy is to discover how neural circuits link these types specific neuronal circuits. Using bioinformatics and of sensory inputs to appropriate behavioral outputs. single-cell gene profiling, we have identified about 70 Surprisingly little is known about how neural circuits genes that can be used to subdivide mitral cells into specific to one set of inputs are organized or built. different classes. We will use gene targeting to test the We take advantage of the unique architecture and role of these genes in neural circuit formation. genetic tractability of the mouse olfactory system to One class of genes that diversifies mitral cells is the study specific olfactory circuits at the first 2 levels of pro- large family of approximately 60 clustered genes for pro- 28 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE tocadherins. Protocadherins are expressed throughout use the infrastructure developed to open up the some- the nervous system. Intriguingly, each neuron seems to times esoteric practices of electron cryomicroscopy to express a distinct combination of several protocadherin a much wider group of researchers, including investi- proteins. We have produced mice that do not express one gators in cell biology, x-ray crystallography, and mate- subfamily of protocadherins. These mice have behavioral rials science. abnormalities consistent with defects in neuronal func- During the past 3 years, the new techniques and tion. We are investigating the cellular and physiologic technologies that we developed included a new grid consequences of loss of protocadherin diversity. substrate designed to improve quality and throughput for vitreous ice specimens; a prototype of a robotic grid-handling system used for screening; Leginon, an Automated Molecular Imaging automated system for microscope control and image acquisition; a relational database that tracks and man- B. Carragher, C.S. Potter, A. Cheng, D. Fellmann, G. Lander, ages data acquired by Leginon and tools for viewing S. Mallick, P. Mercurio, J. Pulokas, J. Quispe , S. Stagg, and delivering the data via Web browsers; and ACE, a program for the automated measurement and correction C. Yoshioka of contrast transfer function. These technologies all uring the past decade, electron cryomicroscopy contributed in demonstrating the potential for automated has emerged as a powerful method for deter- high-throughput data acquisition and analysis in an D mining the structure of large macromolecular experiment in which images of more than 280,000 complexes. Elucidating the structure and mechanism particles of GroEL, a molecule involved in protein fold- of action of these “molecular machines” is an emerging ing, were acquired in a single 25-hour session at the frontier in understanding how the information in the microscope and subsequently subjected to completely genome is transformed into cellular activities. Examples automated procedures to reconstruct a 3-dimensional of the machines include ribosomes, transcription com- map to a resolution better than 8 Å (Fig. 1). plexes, track-motor complexes, and membrane-embedded pumps and channels. In electron cryomicroscopy, the macromolecular specimen is preserved in a thin layer of vitreous (glassy) ice and imaged in an electron microscope by using low doses of electrons. The low signal-to-noise ratio of the resulting images means that averaging is required to recover the signal and reconstruct a 3-dimensional map of the structure. In 2002, we established the National Resource for Automated Molecular Microscopy (NRAMM) to develop, test, and apply technology for automating the processes involved in using electron cryomicroscopy to solve macromolecular structures. The goal of automation is Fig. 1. More than 280,000 particles of GroEL were acquired not only to facilitate the process of molecular micros- from a single grid by using Leginon in a period of 25 hours. These copy, although this facilitation is a welcome benefit, particles were sorted by ice thickness and used to reconstruct a but also to expand the scope of accessible problems 3-dimensional density map to a resolution of approximately 8 Å. Visualization by Scott Stagg and Mike Pique. and push experimental frontiers by making possible investigations deemed too difficult or high risk because These technological developments have been of the considerable effort involved in using manual designed for and used in a number of collaborative methods. An additional goal of automation is to enable research projects, including reconstruction of a mini- much higher throughput of data and thus improve res- mal coatomer complex II cage and reconstruction of olution for single-particle reconstructions by increasing an intact infectious P22 virion. The infrastructure has the numbers of particles that contribute to the average also, in accordance with our mission, made electron 3-dimensional map. Another mission of NRAMM is to cryomicroscopy accessible to a much wider commu- CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 29 nity, leading to publications from research groups in Stagg, S.M., Lander, G.C., Pulokas, J., Fellmann, D., Cheng, A., Quispe, J.D., Mallick, S.P., Avila, R.M., Carragher, B., Potter, C.S. Automated cryoEM data chemistry, x-ray crystallography, materials science, acquisition and analysis of 284,742 particles of GroEL. J. Struct. Biol., in press. and industry. NRAMM currently provides support for Suloway, C., Pulokas, J., Fellmann, D., Cheng, A., Guerra, F., Quispe, J., Stagg, more than 35 collaborative and service projects, and S., Potter, C.S., Carragher, B. Automated molecular microscopy: the new Leginon the Leginon software, including the database, has been system. J. Struct. Biol. 151:41, 2005. distributed to about 30 laboratories outside Scripps Research. We are also distributing ACE, a variety of other software packages, and the novel grid substrates. Regulation of These efforts are complemented by training activities that include small-group training, a biennial large Cytomechanochemical Systems training course in electron cryomicroscopy, and small G. Danuser, A. Bacconi, J. Dorn, K. Jaqaman, L. Ji, workshops focused on various aspects of automation. J. Kunken, D. Loerke, M. Machacek, A. Matov, M. Sabouri, An additional project, sponsored by the National K. Thompson, G. Yang Science Foundation, is the development of automated data collection techniques for imaging serial sections by e study how force-generating molecular using an electron microscope. Understanding the fine machines are spatially and temporally regu- structure of cells and cellular components contributes W lated to mediate complex cell functions, to a more profound understanding of cellular function including migration, division, and intracellular transport and intracellular or intercellular interactions. In order of organelles and vesicles. Specifically, we investigate to visualize these large, complex structures in 3 dimen- the relationships between assembly and contraction of sions at resolutions sufficient to observe structure on the actin cytoskeleton and the dynamic coupling of actin the nanoscale, the cells must be cut into sections and filaments with other components of the cytoskeleton then examined by using a transmission electron micro- during cell migration. We also study how assembly scope. Acquiring high-magnification images of a long and disassembly of microtubules and motor-driven slid- series of sections is difficult and extremely labor inten- ing of microtubule bundles are orchestrated to symmet- sive. The region of interest in each section must be rically segregate replicated DNA from the dividing tracked across sections and across grids, a process mother cell into 2 daughter cells. that requires examining the sections at a variety of In the past year, we expanded our research pro- scales before acquiring high-magnification images of gram with 2 new, collaborative projects. In the first interesting areas. Multiscale imaging of this sort is project, we aim to establish the requirements for local not straightforward because the image formed by an regulation of cortical actin mechanics during endocy- electron microscope shifts and rotates as the magnifi- tosis. In the second, we are analyzing the modes of cation is changed. The overall task of reconstructing a interaction between microtubule plus end– and minus 3-dimensional volume from a set of serial sections is end–directed motor families in vesicle transport along challenging and time consuming, and the number of neuronal axons. large-scale reconstructions has been limited to a few To examine molecular systems, we develop com- spectacular examples. Our objectives are to design, putational models to predict the relationship between develop, and implement a software application to the dynamics of molecular-level component processes and cellular-level outputs. Subsequently, we validate automate the task of acquiring high-magnification the models and estimate unknown parameters by fit- images of specific regions of the cell across tens to ting the parameters to measurements of cell dynam- hundreds of serial sections. ics. The challenges in such data-driven, multiscale

PUBLICATIONS modeling are 2-fold: the precise and complete charac- Cheng, A., Fellmann, D., Pulokas, J., Potter, C.S., Carragher, B. Does contamination terization of cell dynamics in space and time and the buildup limit throughput for automated cryoEM? J. Struct. Biol. 154:303, 2006. implementation of numerical tools for fitting cellular- Fellmann, D., Banez, R., Carragher, B., Potter, C.S. Temperature monitoring of an level data to models with molecular resolution. EM environment. Microsc. Today 14:24, January 2006. In our studies of cell migration, we made 2 major Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, P., Foss, T.R., Potter, C.S., Car- advancements. First, in collaboration with C. Waterman- ragher, B., Balch, W.E. Structure of the Sec13/31 COPII coat cage. Nature 439:234, 2006. Storer, Department of Cell Biology, we extended fluores- 30 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE cent speckle microscopy to accomplish an integrated, Danuser, G. Coupling the dynamics of two actin networks: new views on the mechanics of cell protrusion. Biochem. Soc. Trans. 33:1250, 2005. correlative multiparameter analysis of cytoskeleton dynamics. We can now determine accurately how cell Danuser, G., Waterman-Storer, C.M. Quantitative fluorescent speckle microscopy of cytoskeleton dynamics. Ann. Rev. Biophys. Biomol. Struct. 35:361, 2006. movements depend on molecular processes such as deRooij, J., Kerstens, A., Danuser, G., Schwartz, M.A., Waterman-Storer, C.M. the assembly, disassembly, and transport of cytoskele- Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J. ton components or adhesions, and we can use biosen- Cell Biol. 171:153, 2005. sor probes to visualize activation of signals. We recently Dorn, J.F., Jaqaman, K., Rines, D.R., Jelson, G.S., Sorger, P.K., Danuser, G. Yeast used our analysis framework in studies in which we dis- kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy. Biophys. J. 89:2834, 2005. sected the roles of several signaling cascades in the Ji, L., Danuser, G. Tracking quasi-stationary flow of weak fluorescent features by regulation of cell motility and the involvement of adhe- adaptive multi-frame correlation. J. Microsc. 220:150, 2005. sion molecules in the transient, integrin-mediated cou- Lussi, J.W., Tang, C., Kuenzi, P.A., Staufer, U., Csucs, G., Voros, J., Danuser, G., pling of the actin cytoskeleton to the extracellular matrix. Hubbell, J.A., Textor, M. Selective molecular assembly patterning at the nanoscale: A second breakthrough was achieved in our effort a novel platform for producing protein patterns by electron-beam lithography on SiO2/indium tin oxide-coated glass substrates. Nanotechnology 16:1781, 2005. to reconstruct intracellular force distributions from light Machacek, M., Danuser, G. Morphodynamic profiling of protrusion phenotypes. microscopic measurements of cytoskeleton deformation. Biophys. J. 90:1439, 2006. We established a unique method to probe the relation- Meijering, E., Smal, I., Danuser, G. Tracking in molecular bioimaging. IEEE Signal ship between spatially distributed force generation and Process. Mag. 23:46, May 2006. the resulting cell morphologic outputs, for example, Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman-Storer, C.M., Danuser, G. during cell migration. We will use this tool to dissect Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithe- the mechanism of force regulation by signals and iden- lial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J. 89:3456, 2005. tify feedback interactions between force transduction Shah, S., Yang, G., Danuser, G., Goldstein, L.S.B. Axonal transport: imaging and and signal activation, which are a central element in modeling of a neuronal process. Springer Lecture Notes in Physics. In: The Nobel molecular systems control, not only in cell motility but Symposium. Springer, New York, in press. also in a broad set of other cell functions. Yang, G., Matov, A., Danuser, G. Reliable tracking of large scale dense antiparallel To study chromosome segregation, we use fluores- particle motion for fluorescence live cell imaging. In: Proceedings of the 2005 IEEE Computer Society Conference on Computer Vision and Pattern Recognition (CVPR’05) cent speckle microscopy to analyze the dynamics of Workshops. IEEE Computer Society,Washington, DC, 2005, Vol. 3, p. 138. microtubule scaffolds associated with the spindle apparatus in animal cells and 3-dimensional, high-resolution light microscopy to analyze the dynamics of single chro- Synthetic Protein Chemistry mosomes in yeast. The first approach should reveal how microtubule assembly and disassembly across the spin- P.E. Dawson, A. Dirksen, F. Brunel, M. Churchill, F. Hansen, dle are coregulated with motor-mediated generation of E. Lempens, N. Metanis, T. Shekhter, T. Tiefenbrunn force. The second approach should allow us to identify e use chemical synthesis to design and engi- the functions of proteins in the kinetochore, a molecu- neer proteins with novel structures and func- lar complex that regulates the attachment of chromo- W tions. We continue to develop methods to somes to spindle microtubules. link fully unprotected peptides and carbohydrates via We have developed fully automated, image-based native and nonnative linkages. During the past year, approaches of unprecedented sensitivity for investiga- we focused on synthesizing mimics of the HIV envelope tions of phenotype microtubule and chromosome dynam- protein gp41, the oxidoreductase enzyme glutaredoxin, ics. In collaboration with E.D. Salmon, University of a glycosylated form of monocyte chemoattractant pro- North Carolina, T. Kapoor, Rockefeller University, and tein-3, and carbohydrate-binding proteins. We are also P. Sorger, Massachusetts Institute of Technology, we are using these chemoselective ligation reactions to label using the data obtained to systematically characterize proteins such as thrombin and nanoparticle quantum the involvement of spindle- and kinetochore-associated dots. Overall, our goal is to use synthetic chemistry to proteins in the regulation of chromosome motion understand the molecular basis of protein structure throughout the cell cycle. and function.

PUBLICATIONS SELENOGLUTAREDOXIN Cameron, L.A., Yang, G., Cimini, D., Canman, J.C., Kisurina-Evgenieva, O., Selenoenzymes have a central role in maintaining Khodjakov, A., Danuser, G., Salmon, E.D.S. Kinesin 5-independent poleward flux of kinetochore microtubules in Ptk1 cells. J. Cell Biol. 173:173, 2006. cellular redox potential. These enzymes have selenylsul- CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 31 fide bonds in their active sites that catalyze the reduction in the rate of thioredoxin reduction by the seleno-Grx3 of peroxides, sulfoxides, and disulfides. The preparation analogs indicates that compared with their sulfide coun- of enzymes containing selenocysteine is experimentally terparts, oxidoreductases containing either selenylsulfide challenging. As a result, little is known about the kinetic or diselenide bonds can have physiologically compatible role of selenols in enzyme active sites, and the redox redox potentials and enhanced reduction kinetics. potential of a selenylsulfide or diselenide bond in a pro- HIV VACCINE DESIGN tein has not been experimentally determined. The transmembrane protein gp41 is an attractive To fully evaluate the effects of selenocysteine on target for the development of an HIV vaccine. We are oxidoreductase redox potential and kinetics, we synthe- collaborating with M.B. Zwick and D.R. Burton, Depart- sized glutaredoxin 3 (Grx3; Fig. 1) and all 3 seleno- ment of Immunology, and I.A. Wilson, Department of Molecular Biology, to design peptides that mimic the gp41 epitopes of known neutralizing antibodies. The membrane-proximal external region of gp41 contains several neutralizing epitopes, including 4E10 and Z13e1. On the basis of our previous work on 4E10, we designed and synthesized peptides to map the Z13 epitope and performed an alanine scan to identify key elements within the sequence. Structural constraints are being introduced into the peptides to obtain an antigen capable of eliciting both 4E10- and Z13e1-like antibodies. To better mimic the molecular environment of native gp41, we plan to introduce steric constraints such as polyethylene glycol and carbohydrates. To mimic the viral membrane, we have appended a transmembrane helix and have incorporated the peptide into soluble lipid bilayers. Recently, neutralizing antibodies to the N-heptad repeat of gp41 have been discovered. We designed and synthesized 3-helix bundles to mimic this region of gp41, and we are using them to identify these epitopes and map the key binding interactions.

PUBLICATIONS Brunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R., Fig. 1. Chemical synthesis of Grx3 by using folding to accelerate Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutraliz- the ligation reaction. Selective oxidation of the active-site disulfide ing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006. allowed alkylation of the cysteine residue at the ligation site. Cremeens, M.E., Fujisaki, H., Zhang, Y., Zimmermann, J., Sagle, L.B., Matsuda, S., Dawson, P.E., Straub, J.E., Romesberg, F.E. Efforts toward developing direct cysteine variants of the enzyme’s conserved 11CXX14C probes of protein dynamics. J. Am. Chem. Soc. 128:6028, 2006. active site. Grx3, Grx3(C11U), and Grx3(C14U) had Delehanty, J.B., Medintz, I.L., Pons, T., Brunel, F.M., Dawson, P.E., Mattoussi, H. redox potentials of –193, –259 and –273 mV, respec- Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery. Bioconjug. Chem. 17:920, 2006. tively. The position of redox equilibrium between Grx3(C11U-C14U) (–308 mV) and thioredoxin (–270 Medintz, I.L., Clapp, A.R., Brunel, F.M., Tiefenbrunn, T., Uyeda, H.T., Chang, E.L., Deschamps, J.R., Dawson, P.E., Mattoussi, H. Proteolytic activity monitored mV) suggests a possible role for diselenide bonds in by fluorescence resonance energy transfer through quantum-dot-peptide conju- biological systems. Kinetic analysis indicated that the gates. Nat. Mater. 5:581, 2006. lower redox potentials of the selenocysteine variants is Sagle, L.B., Zimmermann, J., Matsuda, S., Dawson, P.E., Romesberg, F.E. Redox- due primarily to the greater nucleophilicity of the active- coupled dynamics and folding in cytochrome c. J. Am. Chem. Soc. 128:7909. 2006. site selenium rather than to the role of the selenium Yamamoto, N., Takayanagi, A., Sakakibara, T., Dawson, P.E., Kajihara, Y. Highly as either a leaving group or a “central atom” in the efficient synthesis of sialylglycopeptides overcoming unexpected aspartimide formation during activation of Fmoc-Asn(undecadisialyloligosaccharide)-OH. Tetrahedron exchange reaction. The 100- to 10,000-fold increase Lett. 47:1341, 2006. 32 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE Regulation of Actin Dynamics in Despite the high level of sequence conservation (~70%) among vertebrate tropomodulins, comparisons of Morphogenesis and Development their actin-binding activities reveals that tropomodulin 3, but not tropomodulin 1, binds actin monomers and V.M. Fowler, T. Fath, R.S. Fischer, C. McKeown, J. Moyer, nucleates actin filament assembly in addition to capping R. Nowak, J. Palomique, K. Weber pointed ends. Tropomodulin 3 can be chemically cross- linked to actin in a 1:1 complex, providing a tool to egulation of actin dynamics at the ends of fila- identify the amino acids at the tropomodulin 3–actin ments determines the organization and turnover binding interface. Initial results from tryptic digestion R of actin cytoskeletal structures and is critical and mass spectrometry indicate that tropomodulin 3 for cell motility and architecture and actin-based mor- interacts with actin monomers via a unique interface on phogenetic processes in development. We focus on the the actin and on the tropomodulin 3. Site-directed muta- tropomodulin family of proteins that cap the pointed genesis plus structural and functional interaction studies ends of actin filaments. Tropomodulins are a conserved are in progress to further define the tropomodulin family of proteins of about 40 kD that bind to tropo- 3–actin binding interface and to develop tropomodulin myosin and actin. The tropomodulins are expressed in mutants for studies of cellular functions in vivo. a tissue-specific and developmentally regulated fash- To investigate the in vivo function of tropomodulin 1 ion in vertebrates, flies, and worms. in myofibril assembly and cardiac development, we are In vertebrates, the tropomodulin 1 isoform is asso- using mice that lack the gene for this tropomodulin. ciated with stable architectural arrays of actin filaments We showed previously that myofibril assembly in the such as thin filaments in striated muscle myofibrils and heart is grossly aberrant in the embryos of these actin filaments in the membrane skeleton of red blood mutants, leading to aborted cardiac development and cells (RBCs) and on the lateral membranes of the fiber the death of embryos between days 9 and 10 of devel- cells of the eye lens. Previous research indicated that opment. To investigate the primary defect in myofibril tropomodulin 1 regulates the dynamics of actin pointed assembly, we examined nascent myofibrils on myocyte ends and thus the length and stability of thin filaments membranes in embryos at 4–5 days of development, in myofibrils of cultured cardiac muscle cells. Tropo- before the appearance of gross cardiac abnormalities. modulin 3, the isoform in the cytoplasm, is associated In wild-type embryos, the earliest myofibrils con- with dynamic actin filaments in the lamellipodia of tain 1–3 sarcomeres in tandem with regularly spaced crawling endothelial cells, where it is a negative regu- Z bodies and continuous F-actin, indicative of unregu- lator of cell migration. lated filament lengths. Such sarcomere structures are Our goal is to tie the molecular and cellular regula- never observed in the absence of tropomodulin 1; tion of the dynamics of actin pointed ends by tropo- instead, α-actinin and F-actin are present in rodlike, modulins to the in vivo functions of the proteins in aberrant Z disc structures on myocyte membranes. This actin-based morphogenetic processes in development. finding suggests that tropomodulin 1 has a novel early We use mouse genetic models to study the function of function in the organization of Z discs into sarcomeres. tropomodulins in myofibril assembly and cardiac develop- More recently, we found that cardiac development fails ment, the biogenesis and stability of the RBC membrane specifically at the stage of looping morphogenesis, at skeleton, and the morphogenesis and transparency of an earlier stage than that observed in all other mice fiber cells in the eye lens. that lack genes for contractile proteins. We are testing The structure and function of tropomodulins is best the hypotheses that defective myofibril assembly in understood for tropomodulin 1, which consists of 2 absence of tropomodulin 1 may lead directly or indi- domains: an unstructured, flexible N-terminal domain rectly to aberrant cell-cell contacts or polarity, and/or and a compact, folded C-terminal domain composed to defective cell proliferation, leading to failure of loop- of 5 leucine-rich repeats. The N-terminal domain binds ing morphogenesis. tropomyosin and is regulated by tropomyosin to cap To investigate the consequences in RBCs of delet- tropomyosin-actin pointed ends with nanomolar affinity. ing the gene for tropomodulin 1, we prevented death The C-terminal domain caps actin pointed ends with in the embryos of the mutant mice by expressing a submicromolar affinity and is unaffected by tropomyosin. tropomodulin 1 transgene solely in the heart. The result CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 33 was viable mice with no tropomodulin 1 in their RBCs. We use a neonatal mouse retina model to identify Hematologic analyses revealed that these mice had a regulators of developmental angiogenesis and under- compensated mild hemolytic anemia, with increased stand endothelial guidance mechanisms. In addition, reticulocytosis and RBCs that were abnormally variable in a long-standing collaboration with D.A. Cheresh, in size in blood smears. Measurements of mechanical University of California, San Diego, we are using this stability and deformability indicated that tropomodulin system to evaluate the role of integrins in this process. 1–deficient RBCs were less deformable and more fragile In collaboration with P.R. Schimmel, Department of than normal RBCs. Western blotting indicated increased Molecular Biology, we found that fragments of tryptophan levels of tropomodulin 3 in the tropomodulin 1–defi- tRNA synthetase are potent angiostatics that signifi- cient RBCs. cantly reduce retinal neovascularization. The synthetase Using these tropomodulin 1–deficient RBCs, we fragments are also angiostatic in vivo when delivered can test the effects of tropomodulin 3 on the length by a cell-based method. and dynamics of actin filaments and the consequences Most recently, we used combination therapy to show for stability of the membrane skeleton, RBC survival, that targeting multiple, distinct angiogenic pathways and function in vivo. Mice with the transgene also pro- with fragments of tryptophan tRNA synthetase and vide an opportunity to examine the function of tropo- antagonists of integrins and vascular endothelial cell modulin 1 in vivo in other tropomodulin 1–expressing growth factor provides highly synergistic, potent angio- cells and tissues such as the eye lens, neurons, and static activity. Although this therapeutic approach should kidney. We are also producing mice that lack the gene be useful in the treatment of diseases in which com- for tropomodulin 3 to obtain mice with RBCs deficient plete inhibition of angiogenesis is desirable, it may not in both tropomodulin 1 and tropomodulin 3 to assess be efficacious in the treatment of ischemic retinal dis- the consequences of complete lack of tropomodulin on ease. In ischemic retinal disease, relief of hypoxia by RBC structure and function. vascular reconstruction, rather than destruction, may be the desired outcome. PUBLICATIONS Fowler, V.M., McKeown, C.R., Fischer, R.S. Nebulin: does it measure up as a To examine possible therapies for diseases of reti- ruler? Curr. Biol. 16:R18, 2006. nal ischemia, we explored the potential usefulness of stem cells derived from the bone marrow of adult mice for cell-based delivery of angiostatic and neurotrophic Angiogenesis-Dependent substances and for the trophic actions of the cells themselves in vascular and neuronal degenerative dis- Disease and Membrane eases. We found that both lineage-negative hematopoietic progenitors and CD44hi-expressing myeloid Protein Topogenesis progenitors specifically target activated retinal astrocytes, incorporate into and around new vessels, and, M. Friedlander, E. Aguilar, E. Banin, F. Barnett, R. Bautchek, in a mouse model of retinal degeneration, rescue and M. Dorrell, M. El-Kalay, S.F. Friedlander, S. Hanekamp, stabilize a degenerating retinal vasculature. R. Jacobson, A. Johnson, V. Machetti, M. Ritter, L. Scheppke, We also showed that both types of stem cells have J. Trombley, H. Uusitalo-Jarvinen, V. Marchetti, W. Ruf a profound neurotrophic effect when injected into eyes ANGIOGENESIS-DEPENDENT DISEASE of mice with inherited retinal degeneration; not only is ost diseases that cause catastrophic loss of the vasculature rescued in these mice but photorecep- vision do so as a result of abnormal growth tors and visual function are also preserved. The stem M of blood vessels. Similarly, tumors depend on cells also rescue retinal vasculature subject to hypoxic a blood supply for their growth and use these new ves- stress and may be useful in the treatment of ischemic sels as an avenue for metastasis. Blood vessels them- retinal abnormalities such as diabetic retinopathy and selves can generate tumors (e.g., hemangiomas) when retinopathy of prematurity. The mechanism of rescue the growth and organization of vascular endothelial is not clear, but it is related to high levels of heat-shock cells is not properly controlled. Our goal is to under- proteins found in these populations of cells. We also stand the mechanisms of ocular neovascularization in defined a role for CD44hi-derived microglia in facilitat- normal and pathologic situations. ing vascular recovery in models of retinal ischemia. 34 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Glioblastoma multiforme is an incurable brain tumor PUBLICATIONS Banin, E., Dorrell, M.I., Aguilar, E., Ritter, M.R., Aderman, C.M., Smith, A.C.H., that is usually fatal within 1 year after diagnosis. We Friedlander, J., Friedlander, M. T2-TrpRS inhibits preretinal neovascularization and enhances physiological vascular regrowth in OIR as assessed by a new method of are using gene therapy and a rat model of this disease quantification. Invest. Ophthalmol. Vis. Sci. 47:2125, 2006. to study the efficacy of an antiangiogenic approach in Dorrell, M., Uusitalo-Jarvinen, H., Aguilar, E., Friedlander, M. Ocular angiogene- treating these tumors. Hemangiomas are endothelial sis; basic mechanisms and therapeutic advances. Surv. Ophthalmol., in press. tumors that proliferate rapidly and later involute spon- Dorrell, M.I., Friedlander, M. Mechanisms of endothelial cell guidance and vascular taneously. We are using DNA microarrays to study patterning in the developing mouse retina. Prog. Retin. Eye Res. 25:277, 2006. changes in gene expression as hemangiomas progress. Friedlander, M. Stem cells and retinal disease. In: Retina, 4th ed. Ryan, S.J. Our goal is to identify (1) new targets for therapy for (Editor-in-Chief). St. Louis, Mosby, 2006, Vol. 1, p 23.* these tumors and (2) novel regulators of angiogenesis. Friedlander, S.F., Ritter, M.R., Friedlander, M. Recent progress in our understanding of the pathogenesis of infantile hemangiomas. Lymphat. Res. Biol. 3:219, 2005. In a collaboration with G.R. Nemerow, Department of Jin, H., Aiyer, A., Su, J., Borgstrom, P., Stupack, D., Friedlander, M., Varner, J. A Immunology, we used pseudotyped adenovirus to selec- homing mechanism for bone marrow-derived progenitor cell recruitment to the neo- tively target specific cell types in the retina. By using vasculature. J. Clin. Invest. 116:652, 2006. the appropriate fiber type, we can deliver transgenes Ritter, M., Aguilar, E., Banin, E., Scheppke, L., Uusitalo-Jarvinen, H., Friedlander, M. Three-dimensional in vivo imaging of the mouse ocular vasculature during develop- to cells, such as photoreceptors, that ordinarily are not ment and disease. Invest. Ophthalmol. Vis. Sci. 46:3021, 2005. targeted by adenovirus. Ritter, M., Banin, E., Aguilar, E.A., Dorrell, M.I., Moreno, S.K., Friedlander, M. MEMBRANE PROTEIN TOPOGENESIS Myeloid progenitors differentiate into microglia and promote vascular repair in a We are also studying the mechanism whereby pro- model of ischemic retinopathy. J. Clin. Invest., in press. teins are asymmetrically integrated into cell membranes. Ritter, M., Friedlander, M. Integrins in ocular angiogenesis. In: Ocular Angiogenesis: Diseases, Mechanisms, and Therapeutics. Tobran-Tink, J., Barnstable, C. (Eds.). In addition to studies of membrane protein topogene- Humana Press. Totowa, NJ, 2006, p. 279. sis at the molecular level, we are studying defects in Ritter, M., Reinisch, J., Friedlander, S.F., Friedlander, M. Myeloid cells in infantile protein processing and insertion that occur in several hemangioma. Am. J. Pathol. 168:621, 2006. degenerative diseases of the eye. In collaboration with K. Philipson, University of California, Los Angeles, we are investigating the topology of the cardiac sodium-cal- Nucleocytoplasmic Transport cium exchanger. On the basis of hydropathy analysis of and Role of the Nuclear the amino acid sequence, the exchanger is proposed to contain 12 hydrophobic segments, the first of which Lamina in Higher Level is a cleaved signal sequence. Using a variety of reporter Nuclear Organization domains (glycosylation sites, epitopes, and proteolytic cleavage sites), we analyzed the topology of the L. Gerace, G. Ambrus-Aikelin, A. Aschrafi, J. Bednenko, exchanger both in vitro and in oocyte expression systems. A. Bubeck, A. Cassany, B. Chen, E. Choi, K. Datta, T. Guan, Because nearly all other polytopic eukaryotic membrane M. Huber, K. Kanelakis proteins do not have cleaved signal sequences, we are he nuclear envelope is a specialized domain of investigating the putative role of such a sequence in the endoplasmic reticulum that forms the bound- the insertion and targeting of these exchangers. T ary of the nucleus in eukaryotic cells. The enve- Our results indicate that the native, cleaved N-ter- lope consists of inner and outer nuclear membranes, the minal signal sequence is not necessary for insertion of nuclear lamina, and nuclear pore complexes (NPCs). a functional exchanger into the cell membrane. In con- The nuclear lamina, a protein meshwork lining the trast, the photoreceptor exchanger does not have a inner nuclear membrane, provides a structural scaffold cleaved N-terminal signal sequence. If the N-terminal for the nuclear envelope and an anchoring site at the 65 amino acids are deleted, translocation of the N ter- nuclear periphery for chromatin. NPCs are large supra- minus of the protein is disrupted, but the remainder of molecular assemblies that span the nuclear envelope the exchanger is integrated into the membrane. We are and serve as channels for molecular transport between also using large-scale genomic analysis to study trans- the nucleus and the cytoplasm. We are using a combina- genic mice in which mutated exchanger is expressed tion of biochemical, structural, and functional approaches and mice that lack the gene for the exchanger. to investigate NPCs and the lamina. CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 35

NUCLEOCYTOPLASMIC TRANSPORT MECHANISMS tify small-molecule inhibitors of Rev transport and func- Transport of protein and RNA through NPCs is an tion; the goal is to find compounds for developing new energy-dependent process mediated by nucleocyto- drugs to inhibit HIV replication in humans. plasmic shuttling receptors of the karyopherin β fam- NUCLEAR LAMINA AND HIGHER LEVEL NUCLEAR ily. Karyopherins bind to transport signals on protein ORGANIZATION or RNA cargo molecules, and the receptor-cargo com- The nuclear lamina in vertebrates contains a poly- plexes are translocated through the NPC by receptor mer of 2–4 related intermediate filament proteins called binding to a group of NPC proteins (nucleoporins) that lamins, which are associated with a number of trans- contain phenylalanine-glycine amino acid motifs. The membrane proteins of the inner nuclear membrane. directionality of nuclear transport is determined largely The lamina plays essential roles in nuclear structure by the small GTPase Ran, which directly interacts with and functions, as indicated by the recent findings that karyopherins and thereby regulates cargo binding. more than 15 inherited diseases in humans, including Conformational flexibility of karyopherins is thought to several muscular dystrophies, are caused by mutations be fundamental to their dynamic interactions with in lamins or lamina-associated transmembrane proteins. cargo, Ran, and nucleoporins. The involvement of the lamina in disease is thought to We are using in vitro assays with digitonin-perme- be linked to its roles in nuclear integrity, cell signaling, abilized cells to analyze the molecular events that specify and gene expression. Until recently, only about 12 translocation of cargo-receptor complexes through NPCs. transmembrane proteins specific to the nuclear enve- Recently, using site-directed mutagenesis of importin β, lope had been identified. the prototypical nuclear import receptor, we character- To determine the full complement of proteins in the nuclear envelope, we carried out a proteomics analysis ized 2 distinct binding sites in importin β for nucleo- of the nuclear envelope of rodent liver cells in collabo- porins containing the phenylalanine-glycine motif and ration with J.R. Yates, Department of Cell Biology. We defined mutational hot spots for cargo binding. A major identified 67 novel putative nuclear envelope trans- goal is to determine how the conformational dynamics membrane proteins. Almost all members of this group of importin β are linked to discrete transport steps. To that we have examined are authentic components of this end, we are complementing structure-function stud- the nuclear envelope. ies with analysis involving small-molecule inhibitors. Currently, we are analyzing nuclear envelope trans- In a related project, we are analyzing nuclear import membrane proteins in muscle, because this is the tis- of the adenovirus genome, which consists of a 36-kb sue most sensitive to disruption of lamina function by double-stranded DNA molecule. Results from our in disease-causing mutations. Using transcriptional pro- vitro transport studies indicate that adenovirus DNA filing of cultured myoblasts, we found that the genes transport is driven by import signals on DNA-associated for 6 of the nuclear envelope transmembrane proteins proteins. Our characterization of multiple import signals are strongly upregulated in myoblast differentiation. The in adenovirus protein VII and the tight association of genes also are highly expressed in muscle in adults, the protein with the genome suggest that this viral pro- consistent with a role of the genes in muscle differen- tein may be the protein adaptor involved in the DNA tiation and/or maintenance. We have confirmed that import. Nuclear import of protein VII involves several these nuclear envelope transmembrane proteins are of the major cellular importins, suggesting that adeno- authentic nuclear envelope proteins; we are using gene virus has evolved to use redundant import pathways silencing approaches to analyze their requirement in to ensure efficient nuclear delivery of its genome. muscle cell function. Our goals are to identify novel We also are analyzing nuclear export of HIV type 1 genes that may have a role in human muscular dystro- mRNA mediated by the viral regulatory protein Rev. Rev phies and to further elucidate how the protein network polymerizes on a cis-acting sequence of viral mRNAs, consisting of lamins and associated transmembrane providing a platform for assembly of nuclear export fac- proteins directs nuclear structure and functions. tors. We are using proteomics combined with a permeabilized cell assay for Rev-dependent HIV mRNA export PUBLICATIONS Ospina, J.K., Gonsalvez, G.B., Bednenko, J., Darzynkiewicz, E., Gerace, L., Matera, to functionally characterize the proteins assembled on A.G. Cross-talk between snurportin1 subdomains. Mol. Biol. Cell 16:4660, 2005. the Rev platform. This project is part of a larger collabo- Schirmer, E.C., Gerace, L. The nuclear membrane proteome: extending the enve- ration with a research team at Scripps Research to iden- lope. Trends Biochem. Sci. 30:551, 2005. 36 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Wodrich, H., Cassany, A., D’Angelo, M.A., Guan, T., Nemerow, G., Gerace, L. the receptive, postsynaptic element at glutamate syn- Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways. J. Virol. 80:9608, 2006. apses. Spines become lost or dysmorphic in many types of mental retardation and in psychiatric conditions such as chronic depression and schizophrenia. Furthermore, Organization and Function of spines are vulnerable to injury in diseases such as stroke and epilepsy, in which excessive release of glutamate the Neuronal Cytoskeleton can induce neuronal injury and subsequent cell death (a condition termed excitotoxicity). Understanding how S. Halpain, J. Braga, B. Calabrese, L. Dehmelt, E. Hwang, spines form, what regulates their stability, and how they J. Koehler, K. Spencer recover from injury is therefore of therapeutic interest uring the past year, we made significant progress for several neurologic conditions. in research on the development and regeneration Our most recent results suggest a neuroprotective D of neurons. In 2 main projects, we focused on role for spines, because preventing the collapse of den- cytoskeletal proteins of nerve cells, key proteins that dritic spines attenuates neuronal cell death induced by a underlie the structure and morphologic flexibility required subsequent lethal stimulus. The spine cytoskeleton is by neurons for transmitting, storing, and processing composed mainly of actin filaments. We discovered that synaptic signals. We used biochemical, molecular bio- actin filaments in spines are rapidly broken down within logical, and microscopy-based approaches to understand minutes of an injury-inducing stimulus. However, this the function of these molecules. Fluorescence time-lapse damage to the spine can be rapidly reversed within imaging of living neurons is an important tool that we minutes under appropriate conditions, indicating for used to uncover structure-function relationships for cyto- the first time that spines can regrow after they collapse. skeletal proteins and the consequences of the dysfunc- We also discovered that the membrane-associated tion of the proteins. The results of these projects have protein myristoylated alanine-rich C kinase substrate contributed to our understanding of molecular events (MARCKS), a major substrate of the signaling enzyme in normal brain development and in regeneration of protein kinase C, is a key molecule in the regulation neuronal structure after injury and disease. of spine shape and stability. Alterations in MARCKS MICROTUBULE-ASSOCIATED PROTEINS are implicated in Alzheimer’s disease and in major One project concerns microtubule-associated pro- depression. We have extensively characterized the teins (MAPs). These proteins are important in regulat- effects of either depleting MARCKS or overexpressing ing the assembly and stability of microtubules and the various mutant forms of MARCKS in hippocampal neu- interactions of microtubules with other components of rons. The results revealed several key insights into the cytoskeleton. We found that one microtubule-binding MARCKS function and novel forms of synaptic plastic- protein, MAP2, also directly binds actin filaments and ity in young neurons. induces filament bundling. Using fluorescence-based time-lapse imaging and high-resolution confocal micros- PUBLICATIONS Calabrese, B., Wilson, M.S., Halpain, S. Development and regulation of dendritic copy, we tracked the behaviors of microtubules and actin spine synapses. Physiology (Bethesda) 21:38 2006. filaments in living neuronal cells with normal and mutant Halpain, S., Dehmelt, L. MAP1 family proteins. Genome Biol., in press. forms of MAP2. Recently, we found that the microtubule-based molecular motor dynein plays a key role in transporting microtubules toward the cell periphery. This dynein-dependent Function of Nuclear Receptors activity provides a key force that pushes the cell mem- in Stress and Mitochondrial brane outward during neurite initiation. Currently, we are using proteomic approaches and high-content, micros- Homeostasis copy-based screening technology to identify other cytoskeletal proteins and signal transduction pathways A. Kralli, J. Cardenas, B. Hazen, M.B. Hock, F. Jaramillo, crucial to the initiation of neurites. C. Tiraby-Nguyen, J. Villena DENDRITIC SPINES e are interested in the molecular mechanisms A second project concerns the regulation of den- that relay metabolic stress signals to a net- dritic spines, specialized cellular protrusions that form W work of transcriptional regulators and the CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 37 ensuing transcriptional outputs that mediate adaptive the mice had reduced oxidative capacity, suggesting that metabolic responses to the stress signals. In particular, the effect of ERRα on mitochondrial function is cell we focus on the coactivators peroxisome proliferator– autonomous. This research establishes for the first time activated receptor γ coactivator-1α (PGC-1α) and that ERRα is an important component of the regulatory PGC-1β and the orphan nuclear receptors of the estro- network that supports high levels of mitochondrial biogen-related receptor (ERR) subfamily, which control genesis and oxidative metabolism in vivo. mitochondrial biogenesis and energy homeostasis. Our Interestingly, whereas ERRα is an important down- goals are to elucidate the biology of this specific tran- stream effector of PGC-1α in the stimulation of mitochon- scriptional network, understand how deregulation of the drial biogenesis and oxidative capacity, it counteracts network leads to disease, and ultimately identify the the ability of PGC-1α to induce gluconeogenic genes components of the network that are most suitable for in hepatocytes. Notably, it is required for proper sup- drug intervention to counteract metabolic disease. pression of gluconeogenic enzymes in the liver of fed REGULATION OF THE PGC-1/ERR NETWORK mice. Mitochondrial dysfunction has been implicated Levels of PGC-1α and PGC-1β change in response as an underlying cause of insulin resistance and type to signals that relay metabolic needs. The coactivators 2 diabetes. Moreover, derepression of hepatic gluconeo- then relay such signals, via interactions with ERRs and genesis contributes to high glucose levels in diabetes. other factors, to regulate the expression of specific tar- The opposing effects of ERRα on genes important for get genes. We are interested in the mechanisms that mitochondrial oxidative capacity vs genes important in regulate PGC-1s at the postranslational level via cova- gluconeogenesis suggest that enhancing ERRα activity lent modifications of or interaction with other proteins, could have beneficial effects on glucose metabolism and thereby control the properties of the PGC-1/ERR in patients with diabetes via 2 distinct mechanisms: network. This past year, in collaboration with M. Stall- increasing mitochondrial oxidative capacity in peripheral cup, University of Southern California, we showed that tissues and suppressing inappropriate glucose produc- PGC-1α is methylated by the protein arginine methyl- tion in the liver. transferase 1 and that this methylation increases the activity of PGC-1α. As a result, protein arginine methyl- PUBLICATIONS Cartoni, R., Léger, B., Hock, M.B., Praz, M., Crettenand, A., Pich, S., Ziltener, J., transferase 1 and PGC-1α act cooperatively to activate Luthi, F., Dériaz, O., Zorzano, A., Gobelet, C., Kralli, A., Russell A.P. Mitofusins ERR and to induce ERR target genes with roles in 1/2 and ERRα expression are increased in human skeletal muscle after physical α α exercise. J. Physiol. 567(Pt. 1):349, 2005. mitochondrial biogenesis. Currently, we are investigat- Herzog, B., Cardenas, J., Hall, R.K., Villena, J.A., Budge, P.J., Giguère, V., ing the molecular mechanisms by which methylation Granner, D.K., Kralli, A. Estrogen-related receptor α is a repressor of phospho- regulates PGC-1α activity. enolpyruvate carboxykinase gene transcription. J. Biol. Chem. 281:99, 2006.

ROLE OF ERRα IN MITOCHONDRIAL FUNCTION Teyssier, C., Ma, H., Emter, R., Kralli, A., Stallcup, M.R. Activation of nuclear Our previous studies suggested that the effects of receptor coactivator PGC-1α by arginine methylation. Genes Dev. 19:1466, 2005. PGC-1α and PGC-1β on mitochondrial biogenesis are mediated primarily by ERRα. To determine the physiologic relevance of ERRα for mitochondrial function, we Structural and Functional are studying brown adipose tissue. This tissue is rich in mitochondria, it expresses high levels of ERRα, and its Proteomics function is readily assayed in the context of the whole P. Kuhn, J. Nieva,* E. Abola, A. Brooun, R. Bruce, C. Chen, organism. Compared with wild-type mice, mice lack- J. Chrencik, P. Clark, S. Coon, J. Dupuy, S. Foster, J. Joseph, ing ERRα have a decrease in the expression of genes L. Kim, A. Kolatkar, M. Kraus, N. Lazarus, M. Leach, associated with oxidative phosphorylation, lipid oxida- D. Marrinucci, E. Rayon, K. Saikatendu, V. Subramanian, tion, and the tricarboxylic acid cycle. Morphologic analy- A. Tang, M. Yadav sis by electron microscopy revealed that brown adipose * Department of Molecular and Experimental Medicine, Scripps Research tissue from mice lacking ERRα has reduced mitochondrial density and increased lipid accumulation. When uring the past 3 years, we have focused on 4 exposed to cold, mice lacking ERRα had impaired adap- major research programs: detection of cancer tive thermogenesis, despite normal induction of the D cells in circulation, structural proteomics of uncoupling protein UCP-1. Adipocytes isolated from cancer drug targets, structural and functional proteomics 38 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE of the coronavirus that causes severe acute respiratory syndrome (SARS-CoV), and development of novel approaches in miniaturization, integration, and automation to lower the overall cost of moving from the synthesis of genes to examination of the structures encoded by the genes. These programs involve collaborations with R.C. Stevens, Department of Molecular Biology; other researchers at Scripps Research; and scientists at the Palo Alto Research Center and Lyncean Technolo- gies in Palo Alto, California, the Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, and deCODE biostructures, Bainbridge Island, Washington. DETECTING RARE CELLS IN THE CIRCULATION Many clinically important cells in blood occur at frequencies of less than 1 cell per 1 million cells. Detecting and characterizing these rare cells requires the development of new technology that operates with exceptional specificity. Scientists at the Scripps-PARC Institute for Advanced Biomedical Sciences have developed an instrument, based on fiber-optic array scanning technol- Fig. 1. An image of a single circulating tumor cell from a ogy (FAST), that provides rapid and accurate identifica- patient with breast cancer identified from a background of 50 million peripheral blood mononuclear cells by using a FAST cytometer. tion of rare cells in the circulation in humans. Potential clinical applications include finding circulating tumor rapidly identify ideal constructs for expression, crystalli- cells, circulating endothelial cells, and circulating fetal zation, and biophysical studies. The results are provid- cells in the maternal circulation. ing general insights into the range of different physical Malignant cells from solid tumors begin to circulate interactions for a given protein-protein interaction. at the earliest stages in cancer formation. Because the The Eph-ephrin interaction is particularly interest- circulating cells are quite rare, technology to detect and ing, because it has been implicated in cancer progres- characterize them can be valuable in screening for can- sion and in pathologic forms of angiogenesis. We have cer and in guiding individualized cancer therapy. During solved the crystal structure of the ligand-binding domain the past year, we used FAST to accurately enumerate of EphB4 in complex with an antagonistic peptide that circulating cancer cells in blood samples obtained from inhibits ephrin binding and has antitumorigenic prop- patients with metastatic breast or lung cancer (Fig. 1). erties in vivo. We have also solved the cocrystal struc- In collaboration with J. Kroener, Scripps Clinic, we found ture of an EphB4–ephrin-B2 complex. Structural and that accurate detection of circulating tumor cells by biophysical analysis are providing the first insights into FAST can be used to predict prognosis in patients with how we can modulate pathways involved in tumorigene- metastatic breast cancer. We are developing molecular sis and angiogenesis that rely on EphB4–ephrin-B2 tools to characterize these rare tumor cells after they signaling.These results will deepen our understanding have been detected. Our goal is to determine their tis- of the basic biology behind protein-protein interactions sue of origin, their potential anatomic destination, and and aid in the development of novel therapeutic their metastatic potential. STRUCTURAL PROTEOMICS AND DRUG DISCOVERY approaches for modulating protein-protein interactions. We are collaborating with the Novartis Institutes for STRUCTURAL AND FUNCTIONAL PROTEOMICS Biomedical Research, Cambridge, Massachusetts, in ANALYSIS OF SARS-CoV a project to understand and modulate therapeutically We are generating a structure-function-interaction relevant protein-protein interactions. Analysis of an map of the SARS-CoV proteome and its interactions initial subset of 5 therapeutically relevant protein-pro- with the host cell to provide a comprehensive set of tein interaction pairs will provide the guiding principles targets for rational, structure-based drug and vaccine for the selection of a feasible set of drug targets to be design. We use bioinformatics, structural biology, genetic examined. We used a high-throughput approach to methods, and functional assays. CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 39

So far, we have determined the structures of 7 logs thereof) that belong to superfamilies of integral SARS-CoV proteins. We used crystallography for 3 membrane proteins and transcription factors. nonstructural proteins, nuclear magnetic resonance for PUBLICATIONS 3 other nonstructural proteins, and electron cryomi- Chrencik, J.E., Brooun, A., Kraus, M.L., Recht, M.I., Kolatkar, A.R., Han, G.W., croscopy for a large surface glycoprotein spike. These Seifert, J.M., Widmer, H., Auer, M., Kuhn, P. Structural and biophysical characterization of the EphB4–ephrinB2 protein-protein interaction and receptor specificity. studies are providing important information on the for- J. Biol. Chem. 281:28185, 2006. mation of the replicase complex, transcription, and Chrencik, J.E., Brooun, A., Recht, M.I., Kraus, M.L., Koolpe, M., Kolatkar, A.R., RNA-processing events unique to the SARS-CoV life Bruce, R.H., Martiny-Baron, G., Widmer, H., Pasquale, E.B., Kuhn, P. Structure and thermodynamic characterization of the EphB4/ephrin-B2 antagonist peptide cycle. A total of 7 other proteins have been success- complex reveals the determinants for receptor specificity. Structure 14:321, 2006. fully expressed in soluble, folded forms, and their Hsieh, H.B., Marrinucci, D., Bethel, K., Curry, D.N., Humphrey, M., Krivacic, structures are being determined. Together, the results R.T., Kroener, J., Kroener, L., Ladanyi, A., Lazarus, N., Kuhn, P., Bruce, R.H., from these studies should enable identification of Nieva, J. High speed detection of circulating tumor cells. Biosens. Bioelectron. 21:1893, 2006. compounds that may be effective agents for treatment Joseph, J.S., Saikatendu, K.S., Subramanian, V., Neuman, B.W., Brooun, A., of infections caused by SARS-CoV. Griffith, M., Moy, K., Yadav, M.K., Velasquez, J., Buchmeier, M.J., Stevens, R.C., ACCELERATED TECHNOLOGIES CENTER FOR GENE Kuhn, P. Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J. TO 3D STRUCTURE Virol. 80:7894, 2006. Scientists at the Accelerated Technologies Center Marrinucci, D.C., Bethel, K., Bruce, R.H., Curry, D.N., Hsieh, B., Humphrey, M., for Gene to 3D Structure are simultaneously develop- Krivacic, R.T., Kroener, J., Kroener, L., Ladanyi, A., Lazarus, N.H., Nieva, J., Kuhn, P. Case study of the morphologic variation of circulating tumor cells. Hum. ing, operating, and deploying 3 key technologies to Pathol., in press. improve the costs of using x-ray crystallography to Neuman, B.W., Stein, D.A., Kroeker, A.D., Chruchill, M.J., Kim, A.M., Kuhn, P., determine the structure of experimental proteins. The Dawson, P., Moulton, H.M., Bestwick, R.K., Iverson, P.L., Buchmeier, M.J. Inhibi- first technology, computer-aided design of expression- tion, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers. J. Virol. 79:9665, 2005. optimized synthetic genes and protein constructs for Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson, crystallography, improves the success rate for gene M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural geno- isolation and allows researchers to engineer the gene mics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsP7. J. Virol. 79:12905, 2005. sequence of interest to be optimized for protein production in a desired heterologous expression system. The Ratia, K., Saikatendu, K.S., Santarsiero, B.D., Barretto, N., Baker, S.C., Stevens, R.C., Mesecar, A.D. Severe acute respiratory syndrome coronavirus papain-like second technology, microfluidic plug-based nanovolume protease: structure of a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S. protein crystallization in microcapillaries for in situ x- A. 103:5717, 2006. ray screening and data collection, is economical and Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K., Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structural greatly broadens the range of useful quantities of pro- basis of severe acute respiratory syndrome coronavirus ADP-ribose-1′′-phosphate teins required for crystal growth. This technology also dephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005. allows for fine control over chemical gradients in crystal Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W.W., Roach L.S., Roach, R.F., Ismagilov, F., Kuhn, P., Stevens, R.C. In situ data collection and structure refinement growth, thereby expanding the coverage of crystalliza- from microcapillary protein crystallization. J. Appl. Crystallogr. 38:900, 2005. tion space without consuming large quantities of protein. The third technology, the compact light source, is a tunable laboratory x-ray source with peak intensity Vascular Imaging and Tumor at x-ray wavelengths that span selenium anomalous absorbance. Having a tunable laboratory x-ray source Targeting With Virus-Based in the same facility where a crystal inventory is held will greatly enhance the ability to efficiently solve new Nanoparticles protein crystal structures. M. Manchester, G. Destito, M. Estrada, M.J. Gonzalez, The future integration of such technologies in a sin- K. Koudelka, E. Powell, C. Rae, P. Singh, D. Thomas gle facility at Scripps Research will enable efficient gene design for improved protein production, small-volume urrent treatment of cancer typically involves crystallization with in situ x-ray diffraction screening, chemotherapies that have severe adverse effects. and tunable x-ray data collection in a single laboratory. C The requirement that patients must withstand the Our target focus is the structural elucidation of human toxic effects of treatment often limits the effectiveness proteins of biomedical relevance (or eukaryotic homo- of the therapy. Further, many promising anticancer 40 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE compounds that are highly effective in vitro are too healthy tissues as well as in disease states such as toxic to be used in vivo. atherosclerosis. The ability to specifically target therapies to the site TUMOR TARGETING WITH VIRUS-BASED of a developing tumor while avoiding healthy tissue is NANOPARTICLES an important goal for cancer research. Similarly, a We have designed virus-based nanoparticles that tremendous need exists to identify, image, and monitor can specifically target tumors in vivo. In collaboration tumors, particularly at early stages and during treatment. with M.G. Finn, Department of Chemistry, we bioconju- Recently, “smart” nanoparticles, which combine these gated CPMV to tumor ligands such as transferrin and multiple targeting, imaging, and drug delivery functions, folic acid, whose receptors are upregulated on meta- have been developed. Therapies based on nanoparticles bolically active tumor cells. The targeted particles had have tremendous potential to increase the sensitivity a high degree of specificity for the tumor ligand and for and specificity of diagnostic imaging and treatment. uptake by tumor cells. In a separate study, we showed Many different classes of nanoparticles are currently in that canine parvovirus, which has a natural affinity for development, including dendrimers, liposomes, para- the transferrin receptor and thus for tumor cells, could magnetic nanoparticles, and quantum dots. specifically deliver small molecules to tumor cells. We focus on virus-based nanoparticles as platforms These studies will allow the further design of anti- for the development of tissue-specific targeting and tumor agents that can provide localized, highly specific imaging agents in vivo. Two of the viruses we study are imaging and therapy in vivo. Use of virus-based nanopar- cowpea mosaic virus (CPMV) and canine parvovirus. ticles may help us visualize and eliminate small tumors CPMV AS A NOVEL BIOMOLECULAR SENSOR FOR before the tumors have a chance to metastasize. In VASCULAR IMAGING addition, the ability of the particles to focus toxic effects CPMV is an icosahedral, 31-nm particle that is to the site of the malignant cells, thereby expanding produced easily and inexpensively in black-eyed pea the range of effective therapies that can be used in plants. In contrast to the structure of most other nano- vivo, holds great promise for reducing cancer-related materials, the structure of the CPMV capsid is defined morbidity and mortality. and can be engineered to display peptides or proteins PUBLICATIONS in controlled orientations on particle surfaces via either Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A., genetic manipulation of the viral genome or by chemical Reddy, V.S. Characterization of polymorphism displayed by the coat protein mutants of tomato bushy stunt virus. Virology 349:222, 2006. attachment to the particle surface. CPMV is bioavail- able and nontoxic, and the capsids are highly stable Lewis, J.D., Destito, G., Zijlstra, A., Gonzalez, M.J., Quigley, J.P., Manchester, M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat. to temperature, pH, and the conditions required for Med. 12:354, 2006. chemical reactions. Manchester, M., Singh, P. Virus-based nanoparticles (VNPs): platform technologies By conjugation to surface lysine residues, CPMV for diagnostic imaging. Adv. Drug Dev. Rev., in press. can be labeled with fluorophores at high densities, Rae, C.S., Khor, I.W., Wang, Q., Destito, G., Gonzalez, M.J., Singh, P., Thomas, resulting in an extremely bright, nontoxic material that D.M., Estrada, M.N., Powell, E., Finn, M.G., Manchester, M. Systemic trafficking is an outstanding tool for imaging vasculature in live of plant virus nanoparticles in mice via the oral route. Virology 343:224, 2005. animals. Working with H. Stuhlmann, Department of Scobie, H.M., Thomas, D., Marlett, J.M., Destito, G., Wigelsworth, D.J., Collier Cell Biology, we showed that CPMV can be used to R.J., Young J.A.T., Manchester, M. A soluble receptor decoy protects rats against anthrax lethal toxin challenge. J. Infect. Dis. 192:1047, 2005. effectively image the complete vasculature in the embryos of several species and that it is superior to Sen Gupta, S., Kuzelka, J., Singh, P., Lewis, W.G., Manchester, M., Finn, M.G. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse other imaging particles such as lectins, fluorescent functional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16:1572, dextrans, or polystyrene microspheres. 2005.

CPMV particles have also been highly useful in high- Singh, P., Destito, G., Schneemann, A., Manchester, M. Canine parvovirus-like lighting angiogenesis in developing tumors. Uptake of particles, a novel nanomaterial for tumor targeting. J. Nanobiotechnol. 4:2, 2006. particles into endothelial cells occurs, yielding a bright Singh, P., Gonzalez, M.J., Manchester, M. Viruses and their uses in nanotechnol- imaging signal that can be used to differentiate between ogy. Drug Dev. Res., in press. arterial and venous vessels. Such endothelial uptake is mediated by a cellular membrane protein, and uptake can be observed in the endothelium of a variety of CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 41 Translational Regulation in Compared with bacterial 70S ribosomes, the chloroplast ribosome has unique structural domains located Chloroplasts and Expression of primarily on the small ribosomal subunit (Fig. 1). This Human Monoclonal Antibodies in Eukaryotic Algae

S.P. Mayfield, D. Barnes, A. Manuell, M. Beligni, J. Marin-Navarro, M. Muto, M. Tran, D. Siefker, R. Henry

ene expression in chloroplasts is primarily controlled during the translation of plastid mRNAs G into proteins, and understanding how this process is regulated is key to understanding plant development and function. Controlling chloroplast translation is also an essential component in optimizing the pro- Fig. 1. Structure of the chloroplast ribosome from C reinhardtii duction of human therapeutic proteins in algae. calculated to 20-Å resolution. Structures identified as chloroplast Using proteomics and bioinformatics analyses, we unique through comparison with bacterial ribosomes are labeled. identified the set of proteins that function in chloroplast The chloroplast-unique structures are found on the path of mRNA translation. These studies indicated that the core trans- through the ribosome, which is labeled as mRNA entrance and exit lational apparatus of chloroplasts is highly related to tunnels. These structures are positioned for interaction with mRNAs before and during translation; such interactions most likely are that of bacteria but that chloroplasts have incorporated used to select and position mRNA for initiation of translation and additional protein components that allow more com- to increase the rate and fidelity of mRNA translation. plex regulatory mechanisms. Some of these additional components are ribosomal proteins; others are protein finding is supported by our proteomics results that translation factors. Chloroplast mRNAs also contain a indicated that the mass of the small (30S) subunit of number of RNA regulatory elements that are not found the chloroplast ribosome is 25% larger than the bac- in bacteria, as well as conserved RNA elements, such terial 30S subunit. Chloroplast-unique structures are as ribosome-binding sequences, but even these con- found on the solvent side of the small subunit; the served elements appear to function in chloroplast trans- large subunit interacting face is similar to that in bac- lation differently than in bacterial translation. The unique terial ribosomes. components of chloroplast translation provide the oppor- The largest of the chloroplast-unique domains occurs tunity for regulation of chloroplast translation, for examin the vicinity of the mRNA exit tunnel but also extends ple in response to exposure to light, that cannot be up alongside the head and down across the platform. achieved in simpler bacterial systems. This structure has multiple lobes and possibly spans the To better understand translation in plants, we are entire solvent-exposed face of the chloroplast small ribo- examining the structure of both the chloroplast and somal subunit, connecting with a small region of chlo- cytoplasmic ribosomes from Chlamydomonas reinhardtii, roplast-unique density below the shoulder. Another a unicellular photosynthetic eukaryote. Using electron distinct region of chloroplast-unique density is located cryomicroscopy and single-particle reconstruction, we in the beak region of the ribosome, near the mRNA determined the structure of the C reinhardtii cytoplas- entrance tunnel. These chloroplast-unique ribosomal mic 80S ribosome and found that it is nearly identical structures are poised to interact with chloroplast to ribosomes from animals, including the human 80S mRNAs early in message recognition, a key point for ribosome. Parallel proteomics analysis supported this translational regulation. These studies have revealed finding. We also determined the structure of the chlo- the structural basis from which we can pursue identi- roplast ribosome to 20 Å and found that although it is fication of the molecular and biochemical interactions conserved with bacterial 70S ribosomes, it has large of mRNAs, translation factors, and the chloroplast unique structural domains, as predicted by our pro- ribosome that result in regulated translation of chloro- teomics analysis. plast mRNAs. 42 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

In addition to these basic studies on translation, we childhood forms of mental retardation to psychiatric have developed a system for the expression of recom- disorders such as schizophrenia with onsets in late binant proteins, including human therapeutic proteins, adolescence and early adulthood to diseases of aging in C reinhardtii chloroplasts. We have expressed a num- such as Alzheimer’s disease. We use genetic manipu- ber of mammalian proteins, including human mono- lation in mice to investigate the molecular events clonal antibodies. We recently expressed 83K7C, a involved in learning and memory. human monoclonal antibody that binds to protective CALCIUM SIGNALING AND MEMORY antigen 83 of anthrax toxin. We showed that 83K7C We know relatively little at a molecular level about assembles in the cell to form functional antibodies that how the brain stores new information. One hypothesis, bind the antigen and thus potentially could block the which we tested, is that calcium-regulated changes in toxic effects of Bacillus anthracis during infection. We the strength of synaptic connections between nerve cells also showed that this algal-based system can be used can store information. The enzyme calcium/calmodu- to produce high levels of a number of other proteins lin-dependent protein kinase is abundant at synapses with potential human therapeutic value. and when activated by calcium can strengthen synaptic These studies indicate that eukaryotic algae have connections. We used genetic manipulations in mice tremendous potential for the expression of recombinant to indiscriminately activate this kinase at all synapses human therapeutic proteins, because algae can be grown in the entorhinal cortex, a part of the brain that is economically at large scale. Our continued genetic, bio- important for memory and is affected in the earliest chemical, and structural studies should lead to a greater stages of Alzheimer’s disease in humans. understanding of the mechanism of chloroplast translation We found not only that the formation of new memo- and enable us to design appropriate transgenes to achieve ries is impaired but also that previously established higher levels of expression of therapeutic proteins. memories could be erased. If memories are stored as precise patterns of synaptic weights, then the indiscrim- PUBLICATIONS inate strengthening of synapses might be expected to Barnes, D., Franklin, S., Schultz, J., Henry, R., Brown, E., Coragliotti, A., May- field, S.P. Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the erase memories in a manner similar to the way writing expression of Chlamydomonas reinhardtii chloroplast genes. Mol. Genet. Genomics 274:625, 2005. all 1’s in computer memory will erase previously stored information. We also examined where calcium/calmodu- Fletcher, S.P., Muto, M., Mayfield, S.P. Optimization of recombinant protein expression in the chloroplast of green algae. In: Transgenic Microalgae as Green lin-dependent protein kinase functions within cells. We Cell Factories. León, R., Gaván, A., Fernández, E. (Eds.). Landes Bioscience, found that the synthesis of this kinase from RNA located Austin, TX, in press. specifically at synapses is necessary for the stabilization Manuell, A.L., Mayfield, S.P. A bright future for Chlamydomonas. Genome Biol. of memories that last several months. 7:327, 2006. GENETIC MODELS OF DISEASE Manuell, A.L., Yamaguchi, K., Haynes, P.A., Milligan, R.A., Mayfield, S.P. Compo- The determination of the complete sequences of sition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals. J. Mol. Biol. the mouse and human genomes indicates that humans 351:266, 2005. are highly similar to mice at the genetic level. One approach for investigating a genetic disease in humans is to introduce the same mutations into mice to produce Molecular Basis of Cognitive a model of the disease for better understanding of the molecular pathology and for testing possible treatments. Function and Dysfunction Rubenstein-Taybi syndrome is a developmental and cognitive disorder that results from mutation in the M. Mayford, E. Korzus, G.J. Reijmers, M. Yasuda, R. Yasuda, gene CBP. We produced a strain of mice with a defect S. Miller, N. Matsuo in CBP and found that the mice were impaired in several he ability to remember is perhaps the most sig- learning and memory tasks. More important, we showed nificant and distinctive feature of our cognitive that these impairments were not due to problems in T life. We are who we are in large part because of development of the brain because they could be reversed what we have learned and what we remember. Impair- by providing a normally functioning CBP gene to adult ments in learning and memory are a component of dis- mice. The protein encoded by CBP chemically modifies orders that affect human beings throughout life, from histones to allow the expression of a large variety of other CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 43 genes. We found that the memory deficits in the mice ulated when monocytoid cells undergo apoptosis. There- could be reversed by treatment with a drug that targets fore, we are investigating the ability of plasminogen to this histone-modifying function, suggesting a possible modulate monocyte apoptosis. treatment for Rubenstein-Taybi syndrome and possibly We cultured monocytoid cells (freshly isolated human other cognitive disorders. monocytes and U937 cells) in plasminogen-deficient

MOLECULAR ANATOMY OF MEMORY serum and induced the cells to undergo apoptosis by When humans learn new information, they use only using either TNF-α or cycloheximide. When induced in a tiny fraction of the neurons in the brain. One of the the presence of increasing concentrations of plasminogen, difficulties in studying memory is an inability to identify apoptosis was inhibited in a dose-dependent manner; full and specifically manipulate those neurons that partici- inhibition occurred at a concentration of plasminogen pate in a particular memory trace. We developed a equal to its normal physiologic concentration. Treatment genetic technique for use in mice that enables us to with plasminogen also markedly reduced intranucleo- specifically introduce genetic changes into neurons somal DNA fragmentation and the active caspase-3, that are activated by behavioral stimuli. We are using caspase-8, and caspase-9 induced by TNF-α or by this approach to introduce marker proteins that enable cycloheximide. us to see the connections between neurons that have Because monocytoid cells synthesize plasminogen been activated during learning. activators, we examined the role of plasmin proteolytic We have used this approach to study extinction, a activity in the antiapoptotic effects of plasminogen. A process used in the treatment of phobias by which mem- plasminogen active-site mutant did not recapitulate the ories are weakened by repeated exposure to a relevant cytoprotective effect of wild-type plasminogen. In addi- stimulus. We found that the neurons originally activated tion, the antiapoptotic activity of plasminogen was by a fearful stimulus were no longer activated after blocked by increasing concentrations of α2-antiplasmin, extinction. This finding suggests that extinction train- with full reversal at a 2-µM concentration of α2-antiplas- ing actually erases or interferes with some component min, suggesting that the cytoprotective effect of plasmin- of the original memory trace. ogen requires activation of plasminogen to plasmin. Furthermore, antibodies against protease-activated recep- PUBLICATIONS tor 1 blocked the antiapoptotic effects of plasminogen. Colvis, C.M., Pollock, J.D., Goodman, R.H., Impey, S., Dunn, J., Mandel, G., Champagne, F.A., Mayford, M., Korzus, E., Kumar, A., Renthal, W., Theobald, Our results suggest that plasminogen protects mono- D.E., Nestler, E.J. Epigenetic mechanisms and gene networks in the nervous system. J. Neurosci. 25:10379, 2005. cytic cells from apoptosis via a mechanism that requires the proteolytic activity of plasmin and that the protection Reijmers, L.G., Coats, J.K., Pletcher, M.T., Wiltshire, T., Tarantino, L.M., Mayford, M. A mutant mouse with a highly specific contextual fear-conditioning deficit found in is mediated by protease-activated receptor 1. Because an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Learn. Mem. 13:143, 2006. monocyte apoptosis regulates inflammation and ather-

Yasuda, M., Mayford, M.R. CaMKII activation in the entorhinal cortex disrupts pre- osclerosis, these results provide insight into a novel role viously encoded spatial memory. Neuron 50:309, 2006. for plasminogen in these processes. An emerging area of research has indicated a novel role for the plasminogen activation system in regulating Regulation of the Plasminogen the release of neurotransmitters. Prohormones, secreted by cells within the sympathoadrenal system, are proc- Activation System essed by plasmin to bioactive peptides that mediate feedback inhibition of secretagogue-stimulated release L.A. Miles, A. Baik, J.W. Mitchell, H. Bai, F.J. Castellino,* of neurotransmitters. Catecholaminergic cells of the R.J. Parmer** sympathoadrenal system are prototypic prohormone- * University of Notre Dame, Notre Dame, Indiana secreting cells. Processing of prohormones by plasmin ** University of California, San Diego, California is enhanced in the presence of catecholaminergic cells, ssembly of plasminogen and plasminogen acti- and the enhancement requires binding of plasmin(ogen) vators on cell surfaces is a key control point for to cellular receptors. Consequently, modulation of the A positive regulation of cell-surface proteolytic local cellular fibrinolytic system of catecholaminergic activity necessary in physiologic and pathologic pro- cells results in substantial changes in catecholamine cesses. Plasminogen-binding sites are markedly upreg- release. However, mechanisms for enhancing prohor- 44 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE mone processing and cell-surface molecules that medi- vide insight into the operation of the machines. During ate the enhancement on catecholaminergic cells have the past year, we continued our work on members of the not been investigated. myosin and kinesin superfamilies, microtubule-stabi- We found that plasminogen activation was enhanced lizing proteins, and membrane proteins. more than 6.5-fold on catecholaminergic cells. Treat- Although the mechanism of plus end–directed, pro- ment with carboxypeptidase B decreased cell-dependent cessive motion by conventional kinesins is now well plasminogen activation by almost 90%, suggesting that understood, the mechanism by which members of the the binding of plasminogen to proteins exposing C-ter- kinesin 14 class move toward the minus ends of micro- minal lysines on the cell surface is required to promote tubules is not. Likewise, in the myosin superfamily, plasminogen activation. Using a novel strategy of targeted how nucleotide-mediated conformational changes in specific proteolysis with carboxypeptidase B combined the motor domain of class VI myosins result in “back- with a proteomics approach involving 2-dimensional gel ward” motility is not known. We are elucidating the electrophoresis, radioligand blotting, and tandem mass molecular mechanisms of these more unusual members spectrometry, we identified catecholaminergic plasmino- of the myosin and kinesin superfamilies. (Movies show- gen receptors required for enhancing plasminogen acti- ing the motions of conventional myosin and kinesin can vation. Two major plasminogen-binding proteins that be viewed at www.scripps.edu/milligan/projects.html.) exposed C-terminal lysines on the cell surface contained The kinesin Ncd belongs to the kinesin 14 class of amino acid sequences corresponding to β- and γ-actin. motor proteins. Compared with the situation with plus A monoclonal antibody to actin inhibited cell-dependent end–directed kinesins, the nature and timing of the plasminogen activation and also enhanced nicotine- structural changes that underlie the motility of kinesin dependent catecholamine release. Our results suggest 14 motors are poorly understood. We used electron that forms of actin expressed on the cell surface bind cryomicroscopy and image analysis to calculate 3-dimen- plasminogen, thereby promoting plasminogen activa- sional maps of Ncd bound to microtubules in various tion and increased prohormone processing leading to stages in its mechanochemical cycle. The maps revealed inhibition of neurotransmitter release. a minus end–directed rotation of approximately 70° of a coiled coil mechanical element of microtubule-bound PUBLICATIONS Ncd upon ATP binding. In parallel with these struc- Mitchell, J.W., Baik, N., Castellino, F.J., Miles, L.A. Plasminogen inhibits TNFα apoptosis in monocytes. Blood 107:4383, 2006. tural studies, our collaborators, N. Endres and R. Vale at the University of California, San Francisco, showed that extending or shortening this mechanical element Structure and Action of respectively increases or decreases movement velocity, without affecting ATPase activity. These results indicate Molecular Machines that as with other kinesins, the force-producing conformational change of Ncd occurs upon ATP binding R.A. Milligan, J. Chappie, P. Chowdhury, R. Coleman, T. Dang, but, unlike the situation with other kinesins, involves S. Falke,* E. Gogol,** M.B. Lee, S. Mulligan, M. Reedy,*** the swing of a rigid, lever arm–like mechanical ele- M.K. Reedy,*** A.B. Ward, E.M. Wilson-Kubalek, C. Yoshioka ment similar to that described for myosins. * William Jewel College, Liberty, Missouri Whereas most kinesins move along intact micro- ** University of Missouri, Kansas City, Missouri tubules, members of the kinesin 13 class, such as *** Duke University Medical Center, Durham, North Carolina KinI, destabilize and depolymerize microtubules and acromolecular assemblies may be composed of do not appear to have motile properties. We found from 2 to perhaps scores of proteins and are the that a KinI fragment consisting of only the conserved M functional units—the molecular machines—of motor core is necessary and sufficient for ATP-depen- the cell. We use electron cryomicroscopy and image dent depolymerization. The motor core binds along analysis to study the structure and mechanism of action microtubules in all nucleotide states, but in the pres- of several of these machines. We combine the 3-dimen- ence of a nonhydrolyzable ATP analog, depolymerization sional maps calculated from electron images of the also occurs. Structural characterization of the analog- machines with biochemical data and high-resolution induced depolymerization products provided a snapshot x-ray structures of the individual components to proof the disassembly machine at the microtubule ends. CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 45

Our data indicate that whereas conventional kinesins that we are using to study the virulence factor per- use the energy of ATP binding to execute a power stroke fringolysin O from Clostridium perfringens. Perfringo- that results in unidirectional motion along the microtu- lysin O is a cytolysin, an important class of proteins bule surface, KinIs at the ends of microtubules use the that oligomerize and embed within membranes as part energy to bend the underlying protofilament, thereby of the proteins’ lytic functions. We obtained helical destabilizing the microtubule lattice and leading to crystals of wild-type and several mutant forms of the microtubule depolymerization. Furthermore, when the cytolysin on nickel-lipid tubules. Three-dimensional motor core is associated with the microtubule wall, the maps of these proteins derived from images of the core is stalled in a weakly bound, nucleotide-free state. helical crystals will be used to complement our stud- Progression to the strongly bound, ATP-containing state ies of pore formation by perfringolysin O on lipid lay- is possible only when the KinI encounters a microtubule ers. These studies will provide a better understanding end, where it can catalyze deformation of protofilaments of the pathogenic function of cytolysins. Additional and disassembly of microtubules. The unusual mechano- studies involving tubular crystallization of membrane chemical coupling of this kinesin provides an elegant proteins and other bacterial toxins are opening up prom- mechanistic basis for its microtubule-depolymerizing ising new areas for future research. activity. Our current research focuses on understanding the role of the second head in these dimeric molecules. PUBLICATIONS The protein doublecortin is expressed in migrating Dang, T.X., Milligan, R.A., Tweten, R.K., Wilson-Kubalek, E.M. Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein. J. Struct. Biol. and differentiating neurons. In humans, mutations in 152:129, 2005. this protein disrupt brain development, causing lissen- Endres, N.F., Yoshioka, C., Milligan, R.A., Vale, R.D. A lever-arm rotation drives cephaly. Although doublecortin is associated with and motility of the minus-end-directed kinesin Ncd. Nature 439:875, 2006. stabilizes the microtubule cytoskeleton, it has no homol- Manuell, A.L., Yamaguchi, K., Haynes, P.A., Milligan, R.A., Mayfield, S.P. Compo- ogy with other microtubule-binding proteins such as sition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals. J. Mol. Biol. MAP2 or tau. We found that doublecortin preferentially 351:266, 2005. nucleates and binds to 13-protofilament microtubules. This specificity was explained when we discovered that the protein binds in the valleys between the protofilaments of the microtubule wall. This binding site is CNS Development and unique and appears to be ideally located for microtu- Mechanosensory Perception bule stabilization. In this location, doublecortin most likely contributes to both the longitudinal and the lat- U. Müller, C. Barros, R. Belvindrah, F. Conti, S. Franco, eral interactions that stabilize the microtubule wall. N. Grillet, S. Hankel, P. Kazmierczak, R. Radakovits, We are now investigating the binding of proteins that C. Ramos, A. Reynolds, A. Sczaniecka, M. Schwander, track with the tips of dynamic microtubules. S. Webb In collaboration with G. Chang, Department of Molecular Biology, we have grown well-ordered arrays disproportionately large number of genes in the of several membrane proteins that are involved in mul- genomes of vertebrates encode cell recognition tidrug resistance. These arrays, helical tubes and 2- A molecules that mediate cell-cell interactions and dimensional crystals of membrane-embedded proteins, interactions between cells and the extracellular matrix. are suitable for structural studies via electron micros- This finding most likely reflects an evolutionary trend copy. In one instance, we trapped a drug transporter toward increasingly more complex cellular interactions in in various stages of its mechanistic cycle and with higher metazoans. The highest diversity of such interac- substrates bound. We anticipate that 3-dimensional tions occurs in the CNS, where thousands of different electron microscopy maps of membrane-embedded neuronal subtypes are connected into defined neuronal transporters in various states, together with the high- circuits. We use mouse genetics, genomics, cell biol- resolution x-ray structures of the detergent-solubilized ogy, biochemistry, and imaging technology to analyze the protein, will provide insights into the mechanisms used function of cell recognition molecules during the develop- to transport metabolites and drugs across membranes. ment of neuronal circuits in the CNS. In another project, In other studies, we developed a general method we are elucidating the mechanisms by which cell recogni- for helical crystallization of proteins on lipid tubules tion molecules contribute to mechanosensory perception. 46 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

FORMATION OF CORTICAL STRUCTURES IN THE CNS component of the so called tip-link, which has been The establishment of the 3-dimensional cytoarchitec- predicted to transmit force onto mechanically gated ture of the nervous system depends on interactions of ion channels in the stereocilia of hair cells. We are receptors on neuronal cells with molecules presented analyzing the function of cadherin 23, proteins that within the extracellular matrix and by neighboring cells. interact with this cell adhesion molecule, and proteins Integrins are a class of neuronal receptors that mediate encoded by additional “deafness” genes for mechano- interactions with glycoproteins secreted by the extracellu- transduction. We are also doing genetic screens in mice lar matrix and with membrane-anchored counterreceptors. to identify novel recessive deafness traits. Using this Recently, we found that integrins cooperate with strategy, we have already identified several novel genes secreted signaling molecules such as sonic hedgehog that may be associated with deafness. and Reelin to regulate important steps during CNS PUBLICATIONS development, such as cell proliferation and formation Barros, C., Müller, U. Cell adhesion in nervous system development: integrin func- of neuronal layers during the development of the cerebral tions in glial cells. In: Integrins and Development. Danen, E.H.J. (Ed.). Landes Bio- science, Austin, TX, 2006, p. 185. and cerebellar cortex. We are identifying the downstream signaling pathways activated by integrins during corti- Belvindrah, R., Müller, U. Integrin signaling and central nervous system development. In: Extracellular Matrix in Development and Disease. Miner, J.H. (Ed.). Else- cal development. We are also studying signaling inter- vier, St. Louis, 2005, p. 153. Advances in Developmental Biology and Biochemistry; Vol. 15. actions between integrins and other receptors such as receptor tyrosine kinases. Finally, we have extended Belvindrah, R., Nalbant, P., Chuanyue, W., Bokoch, G.M., Müller, U. Integrin- linked kinase regulates Bergmann glial differentiation during cerebellar develop- our studies to the analysis of integrin functions in the ment. Mol. Cell. Neurosci., in press.

CNS in adults. Escher, P., Lacazette, E., Courtet, M., Blindenbacher, A., Landmann, L., Beza- CELL RECOGNITION MOLECULES, MECHANOSENSORY kova, G., Lloyd, K., Müller, U., Brenner H.R. Synapses form in skeletal muscles lacking neuregulin receptors. Science 308:1920, 2005. PERCEPTION, AND DEAFNESS Mechanosensation, the transduction of mechanical Naylor, M.J., Li, N., Cheung, J., Lowe, E.T., Lambert, E., Marlow, R., Wang, P., Schatzmann, F., Wintermantel, T., Schuetz, G., Clarke, A.R., Müller, U., Hynes, force into an electrochemical signal, allows living organ- N.E., Streuli, C.H. Ablation of β1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation. J. Cell Biol. isms to detect touch, hear, register movement and 171:717, 2005. gravity, and sense changes in cell volume and shape. Senften, M., Schwander, M., Kazmierczak, P., Lillo, C., Shin, J.B., Hasson, T., In mammals, the hair cells of the inner ear are the Geleoc, G.S.G., Gillespie, P.G., Williams, D., Holt, J.R., Müller, U. Physical and principle mechanosensors for the detection of sound functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells. J. Neurosci. 26:2060, 2006. and movement. Hair cells elaborate stereocilia that contain mechanosensitive ion channels. The stereocilia of a hair cell are interconnected by extracellular Molecular Mechanisms of bridges into a bundle and are situated next to specialized extracellular matrix assemblies. Sound waves or Thermosensation head movements lead to deflection of the stereocilia bundle, changes in the ion permeability of the mech- A. Patapoutian, M. Bandell, A. Dhaka, A. Dubin, T. Earley, anosensitive channels, and depolarization of the hair J. Grandl, H. Hu, L. Macpherson, T. Miyamoto, V. Uzzell cells. The molecules that regulate development and e are interested in the molecular description function of hair cells are poorly defined. of the function of sensory neurons. Of the 5 Because defects in hair cells cause inherited forms W popularly characterized senses—sight, hearing, of deafness, we use human and mouse genetics as a taste, smell, and touch—touch is among the most var- guideline to identify and study molecules that regulate ied and least understood. Within this sense is the ability the development and function of mechanosensory hair to sense mechanical forces, chemical stimuli, and tem- cells. Currently, about 70 genes have been identified perature, and the molecules that mediate this ability in which mutations lead to deafness. Many of these have been a long-standing mystery. Temperature sensa- genes encode molecules secreted into the extracellular tion in particular has received relatively little attention matrix and membrane-anchored cell adhesion molecules. from biologists and yet is critical for interactions with Mutations in the genes for the cell adhesion molecule the environment. cadherin 23 in mice and humans cause deafness. Our We recently discovered proteins that may enable findings provide strong evidence that cadherin 23 is a sensory neurons to convey information about tempera- CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 47 ture. These proteins are ion channels activated by spe- analysis of the thermoTRPs, will provide important clues cific changes in temperature; thus they act as the molec- about how cold or heat activates these ion channels. ular thermometers of the body. Specifically, our results Our long-term goal is to synthesize an integrated have led to the identification and characterization of 1 picture of sensory neuron function. By identifying the novel warm-activated transient-receptor potential (TRP) proteins that initiate the molecular cascade leading to channel, TRPV3 (33°C threshold) and 2 novel cold-acti- temperature perception, we have provided the basis for vated TRP channels, TRPM8 (25°C threshold) and probing the foundation of the sense of temperature. We TRPA1 (ANKTM1, 17°C threshold). We found that now have the opportunity to extend these insights into TRPM8 is also the receptor for the compound menthol, important areas of human health, such as pain patho- providing a molecular explanation of why mint flavors physiology. For example, TRPA1 is a potential target are typically perceived as cooling. Furthermore, we for treating pain, and we are identifying small-molecule discovered that TRPA1 is activated by cinnamaldehyde, inhibitors of TRPA1 in collaboration with scientists at allicin (garlic), and other compounds with a burning sen- the Genomics Institute of the Novartis Research Founda- sory quality, consistent with a role of TRPA1 in the tion, San Diego, California. Therefore, the approaches detection of noxious cold sensations. Together these tem- we are using will yield insights into the basic biology perature-activated channels represent a new subfamily of of the peripheral nervous system and may also have TRP channels that we have dubbed thermoTRPs. an effect on novel treatments for pain. In agreement with a role in initiating temperature sensation, most of the thermoTRPs are normally found in PUBLICATIONS subsets of neurons in dorsal root ganglia. A surprisingly Bandell, M., Dubin, A.E., Petrus, M.J., Orth, A., Mathur, J., Hwang, S.W., Pat- apoutian, A. High-throughput random mutagenesis screen reveals TRPM8 residues distinct expression pattern was observed for TRPV3, the specifically required for activation by menthol. Nat. Neurosci. 9:493, 2006. warm receptor. In mice, high levels of TRPV3 occur solely Dhaka, A., Viswanath, V., Patapoutian, A. TRP ion channels and temperature sen- in skin keratinocytes, suggesting that skin cells might be sation. Annu. Rev. Neurosci. 29:135, 2006. able to “sense” temperature and then communicate this Macpherson, L.J., Geierstanger, B.H., Viswanath, V., Bandell, M., Eid, S.R., information to neurons in dorsal root ganglia. How tem- Hwang, S.W., Patapoutian, A. The pungency of garlic: activation of TRPA1 and TRPV1 in response to allicin. Curr. Biol. 15:929, 2005. perature information is coded from the skin to the spinal cord is not well understood, and we are using a variety of approaches to answer this question. For example, data from mice lacking the gene for TRPV3 suggest that Functional Proteins in Tumor TRPV3 is indeed required for proper heat sensation in Metastasis and Angiogenesis vivo, reinforcing a role of skin in thermosensation. All organisms have a need for thermosensation. J.P. Quigley, E.I. Deryugina, A. Zijlstra, J.P. Partridge, Because some invertebrate species are more amenable T. Kupriyanova, M. Madsen, V. Ardi, M.C. Subauste to genetic studies than mammals are, we asked whether nonvertebrates also use thermoTRPs to sense tempera- e have established a number of in vivo model ture. We showed that the Drosophila ortholog of TRPA1 systems that can recapitulate the major cellu- is an ion channel activated by warm temperatures, sug- W lar and tissue events that occur during tumor gesting an evolutionarily conserved role of TRP chan- metastasis and angiogenesis. The model systems allow nels in temperature sensing. In collaborative efforts quantitative measurements, microscopic analysis in real with P. Garrity, Massachusetts Institute of Technology, time, biochemical and immunologic probing, and direct Cambridge, Massachusetts, and W. Shafer, University molecular and therapeutic interventions. of California, San Diego, we are using genetic studies Recently, use of small interfering RNA molecules to examine the role of TRPA family members in inver- directed against specific expressed genes and applied tebrate species. directly into the models provided insights into the con- Another key question is what makes thermoTRPs tributory role of the gene products in tumor dissemination temperature sensitive whereas other TRPs are not? and neovascularization. In addition, use of subtractive Answering this question requires insight into the funda- immunization, which is used to generate unique func- mental biophysical mechanism of how temperature acti- tion-blocking monoclonal antibodies, in combination with vates ion channels. Our ongoing structure-function immunoproteomics enables us to identify specific anti- experiments, including mutagenesis and chimeric protein genic molecules that are functionally active in metas- 48 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE tasis and angiogenesis. Finally, use of activity-based ANGIOGENESIS protein profiling, in collaboration with B.F. Cravatt, One of the most commonly used in vivo assays for Department of Cell Biology, enables us to detect, iso- angiogenesis is the chick embryo chorioallantoic mem- late, and identify active proteolytic enzymes that are brane assay. We developed a quantitative variation of distinctively and differentially activated during metas- this assay that enables us to detect and measure the tasis and angiogenesis. newly sprouting blood vessels responding to an angio- METASTASIS genic stimulus such as a specific growth factor or a Selected human tumor cells inoculated onto the growing tumor. A highly specific metalloproteinase, chorioallantoic membrane of developing chick embryos MMP-13, has been implicated in the tissue remodel- form primary tumors on the membrane in 4–7 days. A ing that occurs during the formation of the new blood small percentage of the cells in the primary tumor dis- vessels. We characterized this specific proteolytic event seminate through the vasculature and within 3–4 days and found that specific collagen-cleaving metallopro- arrest and proliferate in secondary organs of the embryo. teinases are implicated directly in the outgrowth of Measuring a small number of early-arriving metastatic new vessels. cells (<200) growing and expanding in the secondary We also found that another metalloproteinase, organ has always been technically difficult. We now use MMP-9 (gelatinase B), most likely is involved in angio- an approach in which unique regions of human DNA, genic tissue remodeling. The proteolytic activity of this known as Alu repeat sequences, are amplified by poly- enzyme also appears to be necessary for a full angio- merase chain reaction from the total DNA extracted genic response. Interestingly, these 2 critical enzymes from various organs of the tumor-bearing chick embryo. are actively imported into the vascular/stromal tissue Chicken DNA contains no Alu sequences, so any prod- by distinct inflammatory cells responding to the angio- uct generated by the polymerase chain reaction indi- genic stimulation. Neutrophil-like heterophils rapidly cates that human tumor cells are present in the chick and almost immediately import MMP-9 into the tissue, embryo organ and would have arrived there via the whereas monocyte/macrophages actively deliver MMP- known sequential steps in metastasis. We can now 13 1–2 days later, possibly in response to specific detect as few as 25–50 human tumor cells present in secreted products of the early-arriving heterophils. Thus, the entire chick embryo lung, liver, or brain and can normal angiogenesis and tumor angiogenesis are closely measure the expansion of these metastatic cells by using linked to an accompanying host inflammatory response real-time polymerase chain reaction. that contributes critical functional molecules to the We are using various screening procedures in this angiogenic process. model system to identify molecules that enhance, or We are dissecting out and identifying the specific conversely inhibit, the appearance of metastatic human molecules and cells that link the inflammatory response tumor cells in organs of chick embryos. The screening to the angiogenic process and to the progression of procedures include direct inoculation of primary tumor malignant neoplasms. We are also trying to decipher cells that have been transfected with various small inter- whether the relevant functional molecules are derived fering RNA constructs to silence specific genes that might from host cells or tumor cells. contribute to metastatic dissemination. Inoculating monoclonal antibodies directly into the tumor-bearing embryo INTRAV ASATION We are also investigating intravasation, the entry and monitoring the influence of the antibodies on metastasis are also part of our screening procedures. of primary tumor cells into the host vasculature, often We are also using a more conventional method of the vasculature that is newly formed during tumor angio- monitoring human tumor metastasis in specific immun- genesis. Intravasation appears to be the least-studied odeficient mice. However, compared with our chick process in the metastatic cascade but most likely is a embryo metastasis assay, this method is less quantitative, rate-limiting step in tumor dissemination. We recently requires more time (3–5 weeks), and is more difficult isolated 2 isogenic variants of a human fibrosarcoma to use for inhibitor screening and molecular intervention. cell line that differ 100-fold in their ability to enter the We are using the mouse metastasis assay to take advan- vasculature in vivo and in their ability to metastasize. tage of mouse genetics and to confirm the efficacy of We are using array technology, proteomics approaches, various effector molecules and inhibitors that initially and intravital microscopy in the cellular and molecular are identified in the chick embryo metastasis assay. analysis of these 2 variants. The results should indi- CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 49 cate specific molecules that are functionally important tion system and a new biochemical complementation in interactions between tumor cells and the vascula- assay, we explored the limiting cytosolic requirements ture and contribute to intravasation. Using activity- for endocytosis of the low-density lipoprotein recep- based protein profiling, we found that the proteolytic tor–related protein (LRP) from isolated plasma mem- enzyme urokinase is differentially activated during branes. LRP, also known as a scavenger receptor, intravasation and catalytically contributes to the binds multiple, distinct ligands and participates in enhanced entry of tumor cells into the vasculature. constitutive endocytosis and signal transduction. We found that clathrin, adaptor protein-2, and PUBLICATIONS dynamin do not support efficient LRP uptake; additional Deryugina, E.I., Zijlstra, A., Partridge, J.J., Kupriyanova, T.A., Madsen, M.A., Papagiannakopoulos, T., Quigley, J.P. Unexpected effect of matrix metalloproteinase factors present in a 30% ammonium sulfate supernatant down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination. Cancer Res. 65:10959, 2005. fraction of bovine brain cytosol are required. Fraction- ation of the supernatant revealed that multiple and Lewis, J.D., Destito, G., Zijlstra, A., Gonzalez, M.J., Quigley, J.P., Manchester, M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat. redundant factors are required to support LRP endocy- Med. 12:354, 2006. tosis. Our data suggest that LRP, which has several

Madsen, M.A., Deryugina, E.I., Niessen, S., Cravatt, B.F., Quigley, J.P. Activity- distinct endocytic motifs in its cytoplasmic domain, based protein profiling implicates urokinase activation as a key step in human may use multiple pathways for endocytosis in vitro. fibrosarcoma intravasation. J. Biol. Chem. 281:15997, 2006. The factors we identified, 70-kD heat-shock cognate Zijlstra, A., Seandel, M., Kupriyanova, T.A., Partridge, J.J., Madsen, M.A., Hahn- protein, synaptojanin, and collapsin response mediator Dantona, E.A., Quigley, J.P., Deryugina, E.I. Proangiogenic role of neutrophil-like inflammatory heterophils during neovascularization induced by growth factors and protein-2, are all implicated in clathrin-mediated endo- human tumor cells. Blood 107:317, 2006. cytosis in vivo, thus validating the assay. However, we found that these factors were sufficient but not necessary for formation of CCVs. Thus, functional redun- Regulators of Clathrin-Mediated dancy and complexity make this assay biochemically intractable, and we have chosen, at least for the Endocytosis moment, to abandon it. We continue to analyze the structure and function S.L. Schmid, J. Chappie, S.D. Conner, M. Ishido, M. Leonard, of dynamin. Dynamin is a multidomain protein con- R. Ramachandran, F. Soulet, B.D. Song, M.C. Surka, D. Yarar sisting of an N-terminal GTPase domain, a middle lathrin-mediated endocytosis is essential for the domain of previously unknown function, a PH domain efficient uptake of nutrients and other macro- that binds phosphatidylinositol 4,5-biphosphate, a C molecules into cells and for the regulation of GTPase effector domain (GED) required for dynamin signaling by cell-surface receptors. The process occurs self-assembly that functions as an assembly-dependent at clathrin-coated pits, which concentrate receptor- GTPase-activating protein for dynamin, and a C-terminal ligand complexes, deform the membrane, invaginate, proline-arginine–rich domain. Dynamin self-assembles in and eventually pinch off, forming clathrin-coated vesicles vitro into spirals on liposomes containing phosphatidyli- (CCVs). The major components involved in formation of nositol 4,5-biphosphate to tubulate the liposomes, CCVs are clathrin, adaptor proteins, and dynamin, an resulting in a greater than 100-fold stimulation of atypical GTPase. GTPase activity. Self-assembly and assembly-stimu- Clathrin self-assembles into a polygonal lattice lated GTPase activity at the necks of deeply invagi- and serves as a scaffold for the formation of coated nated coated pits are thought to drive conformational pits. Adaptor protein-2 is a heterotetrameric protein changes that mediate membrane fission. that triggers clathrin assembly at the plasma mem- Our collaborators recently solved the crystal struc- brane and interacts directly with the cytoplasmic tails ture of the isolated, unoccupied GTPase domain of rat of surface receptors to concentrate the receptors into dynamin-1. Unlike the situation in other GTPases, the the assembling coated pit. We view dynamin as the normally unstructured switch 1 and switch 2 regions master regulator of endocytosis. that surround the unoccupied GTP binding pocket were Previously, we developed a cell-free assay in which ordered. On the basis of the structure, 2 arginines near CCVs are reconstituted from sheets of purified plasma the active site were predicted to be required for catal- membranes from rat liver. Using this in vitro reconstitu- ysis, but our enzymatic analysis of lysine and alanine 50 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE substitutions of these residues suggested otherwise. PUBLICATIONS Leonard, M., Song, B.D., Ramachandran, R., Schmid, S.L. Robust colorimetric Thus, the mechanism of GTP hydrolysis remains obscure. assays for dynamin’s basal and stimulated GTPase activities. Methods Enzymol. However, the structure revealed the existence of a hydro- 404:490, 2005. phobic groove created by the N- and C-terminal α-helices Miwako, I., Schmid, S.L. A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP endocytosis. Exp. Cell Res. of the GTPase domain, which was suggested to be a 312:1335, 2006. docking site for the GED. Miwako, I., Schmid, S.L. Clathrin-coated vesicle formation from isolated plasma In a recent study, we identified mutations in the membranes. Methods Enzymol. 404:503, 2005. GED that resulted in reduced GTPase activity without Reubold, T.F., Eschenburg, S., Becker, A., Leonard, M., Schmid, S.L., Vallee, affecting self-assembly. Because these mutations mapped R.B., Kull, F.J., Manstein, D.J. Crystal structure of the GTPase domain of rat to a predicted amphipathic helix at the extreme C termi- dynamin 1. Proc. Natl. Acad. Sci. U. S. A. 102:13093, 2005. nus of GED, we speculated that this GED helix formed a 3-helical bundle with the GTPase domain helices. To test this hypothesis, we have generated a construct con- Molecular Biology of Olfaction sisting of the GTPase of dynamin together with a C-terminal extension composed of a short sequence of L. Stowers, I.S. Bharati, P. Chamero, J. Cruz, K. Flanagan, turn-preferring residues followed by the 20 amino acid J. Lin, D. Logan, T. Marton, C. Ramos C-terminal GED helix. This construct, unlike GTPase very breath samples the environment for olfac- domain or GED constructs is largely soluble when tory chemical information, determining the qual- expressed in Escherichia coli and has basal GTPase E ity of food, warning of danger, and confirming activity equivalent to that of full-length dynamin. safety. The neurons that mediate olfaction are of 2 types: These exciting results suggest that we have fully those that mediate an evocative perception that varies reconstituted the GED-GTPase interactions. We are with each individual’s experience and those that regu- currently expressing the GTPase-GED peptide construct late stereotyped innate social behaviors such as aggres- for high-resolution x-ray crystallography studies, and sion and mating. Neurons that elicit odorant perception mutagenesis is under way to probe the mechanisms of reside in the olfactory epithelium and relay chemical GED-stimulated GTPase activity. information through activation of cAMP-responsive We have also identified a new class of mutants in channels. Recently, we showed that behavior-generat- the middle domain that alter the quaternary structure ing neurons are located in the vomeronasal organ and of dynamin. Native dynamin exists as a tetramer, but respond to pheromones through a cascade that ultimately analytical ultracentrifugation and gel filtration chromatog- activates C-type transient-receptor potential 2 (TRP2) raphy coupled to multiangle light scattering have con- channels. We are using a molecular genetic approach firmed that the middle domain mutants are dimeric. to characterize the function of these pheromone-respon- Kinetic studies established that the basal GTPase activity sive neurons. of dynamin requires a highly cooperative, GTP-depen- Through electrophysiologic recordings, we have dent conformational change in dynamin tetramers. The shown that mutant mice lacking C-type TRP2 channels dimeric dynamin mutants are defective in both activa- do not depolarize in response to natural sources of tion in the basal state and self-assembly into higher pheromones. Behavioral assays with these animals order structures. revealed that this pheromone response is necessary for Finally, in following up our earlier discovery that both intermale aggression and gender recognition. We actin dynamics are required for multiple stages of are identifying other unique molecular subpopulations clathrin-mediated endocytosis, we found that the proof pheromone-responsive neurons, and through genetic tein sorting nexin 9 is a molecular link between dynamin ablation, biochemistry, and electrophysiology, we are and actin assembly. Sorting nexin 9 binds dynamin assigning biological function to each neuron type. through the nexin’s SH3 domain and activates neural A full characterization of the repertoire of chemo- Wiskott-Aldrich syndrome protein to trigger actin-related sensory neurons will be essential in understanding the protein 2/3–dependent actin assembly into branched logic of olfactory information coding. To this end, we filaments. Thus, sorting nexin 9 may trigger the burst are investigating a novel class of olfactory neurons that of actin assembly that accompanies the scission of lack both the cAMP and C-type TRP2 signaling com- coated vesicles. ponents. Analysis of these neurons by transcriptional CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 51 profiling and then molecular genetics and biochemistry in vitro differentiation of embryonic stem cells into is being used to identify their role in olfactory function. embryoid bodies. Our results indicate that Vezf1 affects Elucidation of the function of specific neural circuits vascular differentiation by regulating cell proliferation, that regulate mammalian behavior has been hindered differentiation, and deposition of extracellular matrix. because the pheromone compounds that signal each We are examining the molecular pathways of Vezf1 behavior have not been identified from their complex function. In collaborative studies with L. Benjamin, Beth natural sources. To obtain these important molecules, Israel Deaconess Medical Center, Boston, we found that we are fractionating natural sources of pheromones and VEZF1, the protein encoded by Vezf1, interacts with then using biobehavioral assays to identify the mole- Rho GTPases to modulate the function of Rho in the cules that initiate activity. The results will enable us to endothelium. Microarray cDNA analysis with RNA from both activate specific neural circuits and analyze the wild-type embryos and embryos lacking Vezf1 sug- natural production and regulation of the signaling gested that genes for fibrinogen and claudin and sev- ligands. In total, we expect to define the pheromone eral genes involved in metabolite transport are target response pathway of mice and to reveal general princi- genes for Vezf1. ples of neurons that govern complex social behavior. AN EARLY MARKER FOR ENDOTHELIAL CELLS AND THEIR PROGENITORS Expression of a second endothelial gene identified Molecular Regulation of in our screen, Egfl7, is restricted to the vascular endothelium and endothelial progenitors in the yolk sac Vascular System Development mesoderm. Egfl7 is also expressed in multipotent stem cells in embryos, in primordial germ cells, and during in Mammals spermatogenesis. In the quiescent vasculature in adults, overall Egfl7 expression is downregulated. During physi- H. Stuhlmann, M.J. Fitch, Z. Zou, S. Chitnis, J.D. Lewis, ologic angiogenesis in the uterus during pregnancy and A. Durrans, W. LeVine in the regenerating endothelium after vascular injury, stablishment of a functional circulatory system expression of Egfl7 is upregulated. EGFL7, the protein during development is crucial for the delivery of encoded by Egfl7, is partially secreted and acts as a E nutrients and oxygen to embryos. Defects in the chemoattractant on both endothelial cells and embry- development of blood vessels result in death before birth onic fibroblasts in in vitro migration assays. EGFL7 is or in congenital cardiovascular abnormalities. We exam- a compact 278 amino acid protein with an amino-ter- ine the molecular and genetic pathways that regulate minal signal peptide; an EMI domain; and 2 central the 3 principal processes of vascular development: deter- epidermal growth factor–like domains, one of which mination of vascular lineage, vasculogenesis, and angio- contains a putative Notch interaction domain. Impor- genesis. We focus on the mouse model because of the tantly, recent collaborative studies with J. Kitajewski, ready availability of genetic information on mice and Columbia University, New York City, provide support experimental tools and because of similarities between for our hypothesis that EGFL7 binds to Notch recep- mice and humans. Using an expression-based “gene tors and acts as an agonist for Notch signaling during trap” screen in mouse embryonic stem cells and vascular development. embryos, we identified 2 novel genes involved in these DEVELOPMENT OF MULTIVALENT VIRAL processes: Vezf1 and Egfl7. NANOPARTICLES FOR IN VIVO VASCULAR

A ZINC FINGER GENE ESSENTIAL FOR NORMAL TARGETING AND IMAGING V ASCULAR AND LYMPHATIC DEVELOPMENT In collaboration with M. Manchester, Department Vezf1 is the gene for an early zinc finger transcrip- of Cell Biology, we have developed viral nanoparticles tion factor that controls the development of blood ves- based on the cowpea mosaic virus (CPMV) for nonin- sels and the lymphatic system in mice. Using functional vasive imaging and targeting of the mammalian cardio- genetic studies, we previously showed that Vezf1 plays vascular system. CPMV can be fluorescently labeled to an essential and dosage-dependent role in the prolifer- high densities with no quenching, resulting in bright ation, remodeling, and integrity of the developing vas- particles that allow high-resolution intravital imaging culature. We recently extended these studies by using of the vascular endothelium and blood flow deep inside 52 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE mouse or chick embryos for up to 72 hours. Using a of β-sheets packed in a curled β-sandwich, as in the human fibrosarcoma model for tumor angiogenesis, we related soluble pentameric acetylcholine-binding profound that fluorescent CPMV can be used to distinguish tein. Each of the subunits in the membrane-spanning between arterial and venous vessels and to monitor the domain is made from 4 α-helical segments. The heli- neovascularization of the tumor microenvironment. We cal segments arrange symmetrically, forming an inner are extending these studies by conjugating peptides to ring of helices that shape a water-filled pore and an the CPMV capsid to target the peptides to the vascula- outer shell of helices that coil around each other and ture, both during development and in disease models. shield the inner ring from the lipids. In the closed channel, the helices in the inner ring come together PUBLICATIONS Lewis, J.D., Destito, G., Gonzalez, M.J., Zijlstra, A., Quigley, J.P., Manchester, near the middle of the membrane and make a con- M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat. stricting hydrophobic girdle. This girdle, which is about Med. 12:354, 2006. 50 Å from the acetylcholine-binding sites, constitutes Kuhnert, F., Stuhlmann, H. Role of Vezf1 during vascular and lymphatic develop- an energetic barrier to ion permeation and functions ment. In: Endothelial Biomedicine. Aird, W.C. (Ed.), Cambridge University Press, New York, in press. as the gate of the channel. These details, together with those obtained earlier Zijlstra, A., Lewis, J.D., DeGryse, B., Stuhlmann, H., Quigley, J.P. Inhibition of tumor cell intravasation and subsequent metastasis through the regulation of from studies of the receptor trapped in the open-chan- CD151-mediated in vivo tumor cell motility. Cancer Cell, in press. nel form, have enabled us to understand in outline the structural mechanism by which acetylcholine opens the pore. In the absence of acetylcholine, the pore is Ion Channels and Fast normally closed. When acetylcholine enters the binding sites, localized rearrangements in the α-subunits Synaptic Transmission occur that stabilize an alternative extended conformation of the channel in which the inner sets of β-sheets N. Unwin are rotated by about 10° about axes perpendicular to on channels play a central role in the rapid trans- the membrane plane, relative to the orientations of the mission of electrical signals throughout the nervous sheets in the closed channel. These rotations are com- I system. To determine how these membrane proteins municated through the inner membrane-spanning work, my colleagues and I are using electron micros- helices and open the pore by breaking the hydropho- copy to analyze the structures of the proteins trapped bic girdle apart. in different physiologic states. Current studies center Improvements in resolution of the 3-dimensional on the nicotinic acetylcholine receptor at the nerve- structure, in both the closed- and open-channel forms, muscle synapse. We wish to find out how this ion chan- are now being attempted so that the structural mecha- nel achieves its ion selectivity and high transport rate nism of gating of the channel can be described in and how it opens and desensitizes in response to greater detail. The knowledge gained from the refined acetylcholine released into the synaptic cleft. For our structure of the locations of amino acid residues, in studies, we use postsynaptic membranes isolated from relation to the ion pathway, is also being used to the (muscle-derived) electric organ of the Torpedo ray, develop quantitative explanations of how the high which form tubular crystals of acetylcholine receptors. cation selectivity and high conduction rates of this The acetylcholine receptor is a member of a channel are achieved. These studies are yielding cru- superfamily of transmitter-gated ion channels, which cial insight into the nature of a number of neuromus- includes the receptors for serotonin 5-HT3, γ-aminobu- cular disorders, including several well-characterized tyric-acids A and C, and glycine. It has a cation-selec- congenital myasthenic syndromes. They are also pro- tive pore, delineated by a ring of 5 similar subunits, viding important 3-dimensional information about the that opens upon binding of acetylcholine to the 2 binding sites for drugs that affect the brain by modu- ligand-binding (α) subunits at the subunit interfaces. lating the function the related γ-aminobutyric acid, Recently, we obtained a refined atomic model of serotonin, glycine, and neuronal acetylcholine receptors. the acetylcholine receptor in the closed-channel form. PUBLICATIONS We found that the individual subunits in the N-termi- O’Brien, J., Unwin, N. Organization of spines on the dendrites of Purkinje cells. nal ligand-binding domain are organized around 2 sets Proc. Natl. Acad. Sci. U. S. A. 103:1575, 2006. CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 53 Microscopes and Motility: have a biphasic response to increasing strength of cell adhesion, with slow migration occurring at low and high Systems Integration in strengths and fast migration at intermediate strength. The assumption of the model was that migrating cells Cell Migration have an asymmetry in adhesion strength from the front part of the mass of cells to the rear part, with the cells C.M. Waterman-Storer, O. Rodriguez, S.L. Gupton, K. Kita, connected by symmetric contractile elements, but R. Littlefield, K. Hu, A. Wheeler, W. Shin, M.L. Gardel, dynamic organizational states of F-actin and focal I.C. Schnieder, A. Parapera, J. Lim adhesions were not considered. This biphasic depen- ell migration is critical to development, the dence of migration velocity on increasing adhesion immune response, and wound healing. In can- strength has since been supported experimentally, and C cer cells, loss of regulation of cell motility results a front-to-rear gradient in cell adhesion strength has in deadly metastasis. The locomotion of vertebrate tis- also been shown. We sought to determine if distinct organizational sue cells is thought to require complex and dynamic states of F-actin, myosin II, and focal adhesions accom- interactions between the microtubule and actin cyto- pany adhesion-dependent changes in velocity. We skeletal polymers, the endomembrane trafficking system, characterized a unique phenotype for optimal migra- and focal adhesions to the extracellular environment. tion at intermediate adhesion strength, entailing rapid We develop quantitative light microscopy methods to flow convergence and local depolymerization of F-actin, analyze the dynamic interactions between these complex local activation of myosin II, rapid renewal of the com- macromolecular systems in living cells to understand how ponents of focal adhesions, and intermediate lifetime the systems are spatiotemporally coordinated to drive and turnover rates of focal adhesions. We recapitulated directed cell movement. We then use these microscopic this phenotype and fast migration at a nonoptimal adhe- assays to analyze cells with specific perturbations of sion strength by manipulating the activity of myosin II. cytoskeletal, membrane, or adhesive proteins to dis- In contrast to the results with simple models, we found sect the molecular mechanisms of the regulation of that a complex spatiotemporal integration and feed- the proteins and their contribution to cell morphogene- back between F-actin, myosin II, and focal adhesions sis and migration. mediates the classically observed biphasic migration We pioneered fluorescent speckle microscopy, a velocity response to increasing adhesion strength, so powerful method that allows quantitative analysis of the that a specific balance between adhesion and contrac- dynamics of macromolecular assemblies in living cells. tion induces maximal migration velocity. Recently, we extended the technology to multispectral The microtubule and actin cytoskeletons interact total internal fluorescence reflection fluorescence micros- in cells to promote a coordinated effort to drive protru- copy, allowing analysis of the integration of proteins sion of the leading edge of cells during cell migration. within focal adhesions with the actin cytoskeleton dur- The tumor suppressor protein adenomatous polyposis ing cell migration. In collaboration with G. Danuser, coli and its binding partner EB1 accumulate on the Department of Cell Biology, we developed correlational ends of microtubules. The tumor suppressor protein fluorescent speckle microscopy to measure the coupling specifically collects on microtubules in cell protrusions, of focal adhesion proteins to the actin cytoskeleton. suggesting that it may promote cell protrusion. We We found that different classes of focal adhesion simultaneously visualized dynamics of the suppressor structural and regulatory molecules have different degrees protein and microtubules in living cells. We found that of correlated motions with actin filaments, indicating dif- the association of the protein with the ends of micro- ferential transmission of actomyosin motion through focal tubules correlates with the increased growth stability adhesions. Our results suggest that transient interactions of the microtubules and that this stabilization can occur between focal adhesion proteins and actin filaments con- independent of the association of the protein with EB1. stitute a friction clutch between the cytoskeleton and the We also found that the protein and EB1 associate with extracellular environment that is regulated during the mor- the ends of microtubules by distinct mechanisms. Thus, phodynamic transitions of cell migration. cancer-causing mutations in this tumor suppressor On the basis of a mathematical model, researchers protein may arise from defects in microtubule stability predicted 15 years ago that migration speed would and cell migration. 54 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

PUBLICATIONS are used to optimize normalization methods and cluster Danuser, G., Waterman-Storer, C.M. Quantitative fluorescent speckle microscopy of cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35:361, 2006. boundaries so that the largest number of any given gene type is found in the smallest cluster size. deRooij, J., Kerstens, A., Danuser, G., Schwartz, M.A., Waterman-Storer, C.M. Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J. This analysis resulted in the identification of a sexual Cell Biol. 171:153, 2005. development cluster containing 246 genes, of which Gupton, S.L., Waterman-Storer, C.L. Live-cell fluorescent speckle microscopy approximately 75% were unclassified and which con- (FSM) of actin cytoskeletal dynamics and their perturbation by drug perfusion. In: Cell Biology: A Laboratory Handbook, 3rd ed. Celis, J., et al. (Eds.). Academic tained most known sexual stage genes. These genes had Press, San Diego, 2005, Vol. 3, p. 137. highly correlated, gametocyte-specific expression pat-

Gupton, S.L., Waterman-Storer, C.L. Spatiotemporal feedback between actomyosin terns. Statistical analysis of the upstream promoter and focal-adhesion systems optimizes rapid cell migration. Cell 125:1361, 2006. regions of these 246 genes revealed putative cis regula-

Kita, K., Wittmann, T., Nathke, I.S., Waterman-Storer, C.L. Adenomatous polypo- tory elements. In addition, we extended the ontology- sis coli on microtubule plus ends in cell extensions can promote microtubule net based pattern identification by using current annotations growth with or without EB1. Mol. Biol. Cell 17:2331, 2006. provided by the Gene Ontology Consortium to identify Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman-Storer, C.M., Danuser, G. 380 statistically significant clusters containing genes with Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J. expression patterns characteristic of various biological 89:3456, 2005. processes, cellular components, and molecular functions. We are also studying genetic diversity by using hybridization-based approaches to further characterize Systems Biology and Malaria parasite genes. By performing a full genome scan of allelic variability of 14 field and laboratory strains of E.A. Winzeler, C. Kidgell, V. Ramachandran, N. Kato, P falciparum, we showed that 10% of the genome K. Henson, J. Young, J. Johnson has higher than neutral rates of diversity at tens of espite the widespread impact of malaria on the thousands of loci. We found that whereas many genes world’s health and economies, relatively little are exceptionally well conserved across parasite iso- D is known about the function of the majority of lates, paralog genes (i.e., genes related by duplication the 5300 genes in the genome of Plasmodium falci- within a genome that have different functions), genes parum, the causative agent of the most severe form of near the ends of chromosomes, genes that encode pro- malaria in humans. This lack of knowledge retards the teins that are trafficked to the surface of the infected development of drugs and vaccines against the parasite. red cell, and genes that encode known and potential We use systematic discovery-based approaches to pre- drug targets are exceptionally diverse. These data sug- dict the function of uncharacterized Plasmodium genes; gest that rates of mitotic recombination are elevated our goal is to facilitate the discovery of new treatments. among genes with paralogs and that selection pres- We are using mRNA and protein expression to reveal sure on those without paralogs is strong. genetic regulatory networks and to suggest protein-pro- We also revealed gene amplification events, includ- tein interactions. We are also developing new methods ing one associated with pfmdr1, the gene for multidrug that can be used in systems biology research. resistance in P falciparum, and a previously uncharac- In addition to our past work on blood-stage parasites, terized amplification centered on the gene for GTP we have characterized the expression program of the cyclohydrolase, the first enzyme in the folate biosynthe- sexual stages of malarial parasites. These stages, which sis pathway. Although GTP cyclohydrolase is not the are essential for the mosquito transmission of the dis- known target of any current drugs, downstream mem- ease, are the focus of the development of drugs and bers of the pathway are targeted by several widely used vaccines that block transmission. To better understand antimalarial agents. We propose that amplification of genes important to sexual development, we used a full- the GTP cyclohydrolase enzyme in the folate biosynthe- genome high-density oligonucleotide microarray to profile sis pathway may facilitate increased flux through this the transcriptomes of P falciparum gametocytes. To pathway and increase resistance to antifolate drugs. interpret this transcriptional data, we developed and These data and recent publications indicating that used a novel knowledge-based data-mining algorithm 90% of a small eukaryote’s genetic variation can be termed ontology-based pattern identification. With this captured in a single microarray hybridization, suggest algorithm, published or custom gene classifications that population genomics will be a fruitful approach CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 55 for discovering new determinants of drug resistance in hypothesized that a subset of insulin-signaling targets, a variety of infectious agents. specifically, daf-2 (the gene for an insulin receptor) and daf-16 (the gene for a transcription factor nega- PUBLICATIONS tively regulated by daf-2) mutants, might be differen- Carret, C.K., Horrocks, P., Konfortov, B., Winzeler, E., Qureshi, M., Newbold, C., Ivens, A. Microarray-based comparative genomic analyses of the human malaria tially expressed in wild-type C elegans and thus could parasite Plasmodium falciparum using Affymetrix arrays. Mol. Biochem. Parasitol. 144:177, 2005. be identified by using our quantitative proteomics approach. We identified 104 proteins that were pre- Kidgell, C., Winzeler, E.A. Using the genome to dissect the molecular basis of drug resistance. Future Microbiol. 1:185, 2006. sent in higher or lower levels in daf-2 mutants than in wild-type and daf-16 mutant organisms, including the Winzeler, E.A. Applied systems biology and malaria. Nat. Rev. Microbiol. 4:145, 2006. known targets superoxide dismutase 3 and catalases. Gene ontology analysis revealed that the upregulated Young, J.A., Fivelman, Q.L., Blair, P.L., de la Vega, P., Le Roch, K.G., Zhou, Y., Carucci, D.J., Baker, D.A., Winzeler, E.A. The Plasmodium falciparum sexual proteins in daf-2 mutants were overrepresented in the development transcriptome: a microarray analysis using ontology-based pattern identification. Mol. Biochem. Parasitol. 143:67, 2005. metabolism of reactive oxygen species, metabolism of carbohydrates, and biosynthesis of amino acids; the downregulated proteins were enriched in proteins Advancing Applications in Mass involved in translation and lipid transport. We confirmed by genetics analysis that some of the Spectrometry–Based Proteomics possible insulin-signaling components identified in the study play a role in regulating life span and/or formation J.R. Yates III, A.O. Bailey, G.T. Cantin, E. Chen, D. Cociorva, of dauer larvae, both of which are regulated by C ele- J. Coppinger, C. Delahunty, M.Q. Dong, J. Hewel, gans insulins. Among the confirmed targets is a protein J.R. Johnson, L. Liao, B.W. Lu, I. McLeod, D. McClatchy, phosphatase. Using a green fluorescent protein as a R. Park, E. Romijn, H. Prieto, C.I. Ruse, J. Venable, label, we found that the phosphatase was upregulated in J. Wohlschlegel, C. Wong, T. Xu worms in which daf-2 was inactivated via RNA interference, consistent with the results of mass spectrometry. ass spectrometry has emerged as a powerful Further genetic analysis indicated that this phosphatase technique for cellular proteomics, comple- acted upstream of and/or in parallel to the protein DAF- menting traditional gene-by-gene approaches M 16 in regulation of aging and dauer formation. with a comprehensive description of the molecular fac- Taken together, our data suggest that this protein tors that contribute to a biologically relevant system. phosphatase is both a downstream target and a regu- We remain at the forefront of this field, developing new lator of insulin signaling. Therefore it may be part of a strategies to address more sophisticated scientific ques- feedback loop. This study illustrates the effectiveness tions through proteomics, such as how to measure of combining quantitative mass spectrometry and C global changes in protein abundance and how to char- elegans genetics. Such an approach can be extended acterize complex posttranslational modifications. to other studies beyond insulin signaling. QUANTITATIVE PROTEOMIC ANALYSIS OF INSULIN PROTEIN SUMOYLATION SIGNALING The characterization of posttranslational modifica- Quantitative mass spectrometry–based proteomics tions is also an emerging application of mass spectrom- relies on internal isotopic standards. Metabolic label- etry–based proteomics. We are developing proteomic ing with nitrogen 15 is a preferred method of intro- tools to study the family of small ubiquitin-like modi- ducing internal standards. It produces a standard for fiers (SUMOs). Recently, we focused on Smt3p, the every peptide or protein to be characterized. In addi- budding yeast homolog of a human SUMO. Although tion, it is stable, nonradioactive, less error-prone than genetic studies have indicated that Smt3p is required chemical labeling, and relatively inexpensive. We have for proper regulation of a variety of different cellular used metabolic labeling with nitrogen 15 and quanti- processes, including transcription, intracellular trans- tative mass spectrometry to study insulin signaling in port, progression of the cell cycle, and the mainte- the worm Caenorhabditis elegans. nance of genome integrity, the mechanisms by which Insulin regulates a wide range of processes, includ- it does so remain largely unknown. ing metabolism, development, and aging, but only a Using proteomic approaches, we addressed 2 major handful of its downstream targets are known. We areas in protein sumoylation: the large-scale identifi- 56 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE cation of sumoylation targets and the development of Macromolecular Assemblies strategies for mapping SUMO modification sites. By combining different affinity chromatography strategies Visualized by Electron with our multidimensional protein identification platform, we identified 271 new SUMO targets. These Cryomicroscopy and Image substrates play roles in a diverse set of biological processes and greatly expand the known scope of SUMO Analysis: Membrane Proteins regulation in eukaryotic cells. This research also and Viruses revealed coordinated SUMO modification of multiple proteins in well-defined macromolecular complexes. M. Yeager, R. Abagyan,**** B.D. Adair, K. Baker, K. Altieri, This intriguing result suggests that sumoylation may A. Cheng, M.J. Daniels, K.A. Dryden, B. Ganser, J. Harless, target protein complexes rather than individual pro- Y. Hua, M. Matho, F.A. Palida, M.A. Arnaout,* teins. We are also characterizing the mechanism that A.R. Bellamy,** N. Ben-Tal,*** M.J. Buchmeier,**** underlies this observation. F.V. Chisari,**** K. Coombs,***** H.B. Greenberg,† Characterization of many of the new substrates iden- J.E. Johnson,**** S. Matsui,† L.H. Philipson,†† tified in our study was limited by difficulties in identifying T.D. Pollard,††† A. Rein,†††† A. Schneemann,**** SUMO attachment sites in the target of interest. To solve J.A. Tainer,**** J.A. Taylor,** V.M. Unger††† this problem, we recently developed a method for the * Harvard Medical School, Boston, Massachusetts rapid and efficient identification of SUMO attachment ** University of Auckland, Auckland, New Zealand sites in cellular proteins. In this method, different *** Tel-Aviv University, Tel-Aviv, Israel SUMO mutants are used in combination with various **** Scripps Research protease digestion strategies, and then mass spec- ***** University of Manitoba, Winnipeg, Manitoba trometry is used to directly and specifically map the † locations of the modified lysine residues. We showed the Stanford University, Stanford, California †† usefulness of this method by identifying SUMO modifi- University of Chicago, Chicago, Illinois ††† cation sites in an assortment of model SUMO sub- Yale University, New Haven, Connecticut †††† strates and in complex mixtures. The development of National Cancer Institute, Frederick, Maryland a method for identifying SUMO attachment sites will he ultimate goal of our studies is to gain a deeper be a powerful tool for the characterization of the new understanding of the molecular basis of impor- substrates identified in our initial global analysis. T tant human diseases, such as sudden death, heart attacks, and HIV infection, that cause substantial mor- PUBLICATIONS Cantin, G.T., Venable, J.D., Cociorva, D., Yates, J.R. III. Quantitative phosphopro- tality and suffering. The structural details revealed by teomic analysis of the tumor necrosis factor pathway. J. Proteome Res. 5:127, 2006. our research may provide clues for the design of more

Chen, E.I., Hewel, J., Felding-Habermann, B., Yates, J.R. III. Large scale protein effective and safer medicines. profiling by combination of protein fractionation and multidimensional protein iden- At the basic science level, we are intrigued by ques- tification technology (MudPIT). Mol. Cell. Proteomics 5:53, 2006. tions at the interface between cell biology and struc- Sadygov, R., Wohlschlegel, J., Park, S.K., Xu, T., Yates, J.R. III. Central limit theorem tural biology: How do membrane proteins fold? How as an approximation for intensity-based scoring function. Anal. Chem. 78:89, 2006.. do membrane channels open and close? How are sig- Venable, J.D., Xu, T., Cociorva, D., Yates, J.R. III. Cross-correlation algorithm for calculation of peptide molecular weight from tandem mass spectra. Anal. Chem. nals transmitted across a cellular membrane when an 78:1921, 2006. extracellular ligand binds to a membrane receptor?

Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Improved identifica- How do viruses attach to and enter host cells, repli- tion of SUMO attachment sites using C-terminal SUMO mutants and tailored pro- cate, and assemble infectious particles? To explore tease digestion strategies. J. Proteome Res. 5:761, 2006. such problems, we use high-resolution electron cryomicroscopy and computer image processing. With this approach, we can examine the molecular architecture of supramolecular assemblies such as membrane proteins and viruses. In electron cryomicroscopy, biological specimens are quick frozen in a physiologic state to preserve CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 57 their native structure and functional properties. A special advantage of this method is that we can capture dynamic states of functioning macromolecular assemblies, such as open and closed states of membrane channels and viruses actively transcribing RNA. Three- dimensional density maps are obtained by digital image processing of the high-resolution electron micrographs. The rich detail in the density maps exemplifies the power of this approach to reveal the structural organization of complex biological systems that can be related to the functional properties of such assemblies. Ongoing research projects include the structure analysis of (1) membrane proteins involved in cell-to- cell communication (gap junctions), water transport (aquaporins), ion transport (potassium channels), transmembrane signaling (integrins), and viral recognition (rotavirus NSP4); (2) viruses responsible for sig- Fig. 1. Intercellular gap junction channels have a diameter of nificant human diseases (retroviruses, hepatitis B virus about 65 Å and are formed by the end-to-end docking of 2 hemi- [HBV], rotavirus, astrovirus); and (3) viruses used as channels, each composed of a hexamer of connexin subunits. A Cα model systems to understand mechanisms of patho- model (ribbons) for the membrane-spanning α-helices of the hemi- genesis (arenaviruses, reoviruses, nodaviruses, tetra- channels was derived by combining the information from a compu- viruses and sobemoviruses). The following sections tational analysis of connexin sequences, the results of more than a decade of biochemical studies, and the constraints provided by a highlight selected projects that exemplify the themes 3-dimensional map derived by using electron cryocrystallography. of our research program. Although individually, none of these approaches provided high-reso- GAP JUNCTION MEMBRANE CHANNELS lution information, their sum yielded an atomic model that predicts Gap junction channels connect the cytoplasms of how connexin mutations (spheres), which result in diseases such adjacent cells by means of an intercellular conduit as nonsyndromic deafness and Charcot-Marie-Tooth disease, may formed by the end-to-end docking of 2 hexameric interfere with formation of functional channels by disrupting helix- helix packing. hemichannels called connexons. Gap junctions play an essential functional role by mediating metabolic and nates of Cα atoms in the transmembrane domain, pro- electrical communication within tissues. For instance, vides a structural basis for understanding the different in the heart, gap junction channels organize the pat- physiologic effects of almost 30 mutations and poly- tern of current flow to allow a coordinated contraction morphisms in terms of structural deformations at the of the muscle. interfaces between helices, revealing an intimate con- We expressed a recombinant cardiac gap junction nection between molecular structure and disease. protein, termed connexin 43, and produced 2-dimen- INTEGRINS sional crystals suitable for electron cryocrystallography. Integrins are a large family of heterodimeric trans- Our previous findings indicated that each hexameric membrane receptor proteins that modulate important connexon is formed by 24 closely packed α-helices. biological processes such as development, cell adhe- We have now extended this analysis to 5.7-Å in-plane sion, angiogenesis, wound healing, and neoplastic and 19.8-Å vertical resolution, a step that enables us to transformation. The ectodomain of the integrin αvβ3 identify the positions and tilt angles for the 24 α-helices crystallizes in a bent, genuflexed conformation, which within each hemichannel (Fig. 1). The 4 hydrophobic is considered to be inactive (i.e., unable to bind physi- segments in connexin sequences were assigned to the ologic ligands in solution) unless it is fully extended α-helices in the map on the basis of biochemical and by activating stimuli. To assess whether the bent inte- phylogenetic data. Evolutionary conservation and an grin can bind physiologic ligands, we collaborated analysis of compensatory mutations in connexin evolu- with M.A. Arnaout, Harvard Medical School, Boston, tion were used to identify the packing interfaces between Massachusetts, to generate a stable, soluble complex the helices. The final model, which specifies the coordi- of the manganese-bound αvβ3 ectodomain with a frag- 58 CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE ment of fibronectin containing type III domains 7–10 core protein has been studied intensively. However, lit- and the EDB domain. Electron microscopy and single- tle is known about the structure and assembly of native particle image analysis were used to determine the capsids present in infected cells, and even less is known 3-dimensional structure of this complex (Fig. 2). about the structure of mature virions. We used electron cryomicroscopy and image analysis to examine HBV virions (also called Dane particles) isolated from the serum of a patient with hepatitis B and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice (Fig. 3).

Fig. 2. The 3-dimensional density map (grayscale transparency) of the integrin αvβ3 in a complex with fibronectin was determined by using electron microscopy and image analysis. The x-ray structures of the αv and β3 proteins have been docked into the electron microscopy density envelope. Additional density (lower right) can accommodate fibronectin domain 10 adjacent to the ligand-binding site as well as domain 9 at the synergy site. The complex is shown adjacent to the white box, which represents the 30-Å-thick hydrophobic part of the cellular membrane across which signals are transmitted.

Most αvβ3 particles, whether unliganded or bound to fibronectin, had compact, triangular shapes. A difference map comparing ligand-free and fibronectin-bound integrin revealed density that could accommodate the fibronectin type III domain 10 containing arginine– glycine–aspartic acid in proximity to the ligand-binding site of β3, with domain 9 just adjacent to the synergy site binding region of α v. Fig. 3. A model of the HBV virion (diameter ~450 Å) based on This study suggests that the ectodomain of αvβ3 has electron cryomicroscopy and image analysis. A, The double-stranded a bent conformation that can stably bind a physiologic DNA genome is encapsidated by an icosahedral capsid shell com- ligand in solution. These results are relevant for under- posed of 120 spikes. The surface is studded with glycoproteins standing how binding of ligands to the extracellular spaced about 60 Å apart that bind to membrane receptors on liver domain leads to conformational changes that transmit cells. B, In the close-up view, the x-ray crystal structure of a recombinant capsid has been docked into the electron cryomi- signals across the plasma membranes of cells, culminat- croscopy density map of the virion capsid. The core spikes are in ing in changes in gene transcription in the nucleus. close apposition but do not penetrate the envelope. HEPATITIS B VIRUS HBV currently infects more than 350 million peo- Both types of capsids assembled as icosahedral ple, of which 1 million will die every year. The infec- particles indistinguishable from previous image recon- tious virion is an enveloped capsid containing the viral structions of capsids. Likewise, the virions contained polymerase and the double-stranded DNA genome. The capsids with either T = 3 or T = 4 icosahedral sym- structure of the capsid assembled in vitro from expressed metry. Projections extending from the lipid envelope CELL BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 59

were attributed to surface glycoproteins. The packing of the projections was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.

PUBLICATIONS Daniels, M.J., Wood, M.R., Yeager, M. In vivo functional assay of a recombinant aquaporin in Pichia pastoris. Appl. Environ. Microbiol. 72:1507, 2006.

Daniels, M.J., Yeager, M. Phosphorylation of aquaporin PvTIP3;1 defined by mass spectrometry and molecular modeling. Biochemistry 44:14443, 2005.

Dryden, K.A., Wieland, S.F., Whitten-Bauer, C., Chisari, F.V., Yeager, M. Native hepatitis B virions and capsids visualized by electron cryomicroscopy. Mol. Cell 23:843, 2006.

Greig, S.L., Berriman, J.A., O’Brien, J.A., Taylor, J.A., Bellamy, A.R., Yeager, M., Mitra, A.K. Structural determinants of rotavirus subgroup specificity mapped by cryo-electron microscopy. J. Mol. Biol. 356:209, 2006.

Wiedenheft, B., Mosolf, J., Willits, D., Yeager, M., Dryden, K.A., Young, M., Douglas, T. An archaeal antioxidant: characterization of a Dps-like protein from Sulfolobus solfataricus. Proc. Natl. Acad. Sci. U. S. A. 102:10551, 2005.

Chemistry

Coiled and extended sodium dodecyl sulfate conformations in the water-soluble cavitand. Nuclear magnetic resonance evidence supports the "strained" structure in which 8 carbon atoms adopt a coiled conformation to maximize interaction with the host. Artwork was done by Michael P. Schramm, Ph.D., The Skaggs

Institute for Chemical Biology. Julius Rebek, Jr., Ph.D.

Professor, Department of Chemistry

Director, The Skaggs Institute for Chemical Biology CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 63

DEPARTMENT OF Ramanarayanan Andrew Bin Zhou, Ph.D. Stellios Arseniyadis, Ph.D.**** CHEMISTRY Krishnamurthy, Ph.D. Assistant Professor Ecole Supérieure de Associate Professor Physique et de Chimie Industrielle STAFF Richard A. Lerner, M.D.*** STAFF SCIENTISTS Paris, France President, The Scripps K.C. Nicolaou, Ph.D.* Research Institute Professor and Chairman Byeong D. Song, Ph.D. Gonen Ashkenasy, Ph.D.**** Lita Annenberg Hazen Aline W. and L.S. Skaggs Ben-Gurion University of the Professor of Professor of Chemical Biology Lubica Supekova, Ph.D. Negev Immunochemistry Darlene Shiley Chair in Beer-Sheeva, Israel Cecil H. and Ida M. Green Chemistry Chair in Chemistry Wen Xiong, Ph.D. Nurit Ashkenasy, Ph.D.**** Phil Baran, Ph.D. Ben-Gurion University of the Masayuki Matsushita, INSTRUMENTATION/ Associate Professor Negev Ph.D.**** SERVICE FACILITIES Chugai Pharmaceutical Co., Beer-Sheva, Israel Dale L. Boger, Ph.D.* LTD. Raj K. Chadha, Ph.D. Richard and Alice Cramer Masato Atsumi, Ph.D.**** Tokyo, Japan Director, X-Ray Professor of Chemistry Kyocera JMM Crystallography Facility Michael Meijler, Ph.D. Osaka, Japan Tobin J. Dickerson, Ph.D. Assistant Professor Dee H. Huang, Ph.D. Assistant Professor Elizabeth Barrett, Ph.D. Director, Nuclear Magnetic Jorge J. Nieva, M.D. Resonance Facility Albert Eschenmoser, Ph.D.* Assistant Professor Christoph Behrens, Ph.D.**** Professor University of Gottingen Gary E. Siuzdak, Ph.D. Evan Powers, Ph.D. Gottingen, Germany Director, Mass Spectrometry Sheng Ding, Ph.D. Assistant Professor Facility Assistant Professor Clay Bennett, Ph.D. Julius Rebek, Jr., Ph.D.* M.G. Finn, Ph.D.* Professor Jan Bieschke, Ph.D.**** Associate Professor Director, The Skaggs Institute SENIOR RESEARCH Max Delbrück Center for for Chemical Biology ASSOCIATES Molecular Medicine Valery Fokin, Ph.D. Berlin, Germany Associate Professor Ed Roberts, Ph.D. Ashraf Brik, Ph.D. Professor Babu Boga, Ph.D.**** M. Reza Ghadiri, Ph.D.* Yanping Chen, Ph.D. Schering Plough Professor Floyd E. Romesberg, Ph.D. Redminster, New Jersey Associate Professor Gunnar Kaufmann, Ph.D. William A. Greenberg, Ph.D. Anthony Boitano, Ph.D. Assistant Professor Peter G. Schultz, Ph.D.* Professor RESEARCH ASSOCIATES Venkataiah Bollu, Ph.D. Inkyu Hwang, Ph.D. Scripps Family Chair Assistant Professor Ramzey Abujarour, Ph.D.**** K. Barry Sharpless, Ph.D.* Genomics Institute of the Brant Boren, Ph.D. Hayato Ishikawa, Ph.D. Professor Novartis Research Foundation W.M. Keck Professor of Assistant Professor San Diego, California Daryl Bosco, Ph.D.**** Chemistry Serono, Inc. Kim D. Janda, Ph.D.** Dariush Ajami, Ph.D. Boston, Massachusetts Vaughn Smider, Ph.D. Professor Assistant Professor Ely R. Callaway, Jr., Chair in Lital Alfonta, Ph.D. Andrew Brogan, Ph.D. Chemistry Anita Wentworth, Ph.D. Xaiver Alvarez-Mico, Ph.D. Adrian Brunkhorst, Director, The Worm Institute Assistant Professor for Research and Medicine Ph.D.***** Rahesh Ambasudhan, Ph.D. Paul Wentworth, Jr., Ph.D. Jeffery W. Kelly, Ph.D.* Professor Paul Bulger, Ph.D. Lita Annenberg Hazen Narendra B. Ambhaikar, Professor of Chemistry Chi-Huey Wong, Ph.D.* Ph.D.**** Kevin Bunker, Ph.D. Dean, Graduate and Ernest W. Hahn Professor Vertex Pharmaceuticals Postgraduate Studies and Chair in Chemistry San Diego, California Antonio Burtoloso, Ph.D. 64 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Mark Bushey, Ph.D. Jan Elsner, Ph.D. Sayam Sen Gupta, Ph.D.**** Steven Johnson, Ph.D. Technical University Munich Sara Butterfield, Ph.D. Nil Emre, Ph.D. Munich, Germany Jaroslaw Kalisiak, Ph.D.

Alexandre Carella, Ph.D. Lisa Eubanks, Ph.D. Richard Guy, Ph.D. Michael Kelso, Ph.D.**** University of Wollongong Michael Cassidy, Ph.D.**** Cyrine Ezzili, Ph.D. Clemens Haas, Ph.D.***** Wollongong, Australia Bristol-Myers Squibb New Brunswick, New Jersey Raffaella Faraoni, Ph.D.**** Akiyuki Hamasaki, Ph.D. Gyungyoun Kim, Ph.D.**** Ambit Biosciences SK Biopharmaceuticals Sukbok Chang, Ph.D. San Diego, California Wooseok Han, Ph.D.**** Fairfield, New Jersey Chiron Shuo Chen, Ph.D. Simon Ficht, Ph.D. Emeryville, California F. Scott Kimball, Ph.D.

Yanping Chen, Ph.D.***** Laura Flatauer, Ph.D.**** Frank Hauke, Ph.D.**** Ravinder Reddy Kondreddi, Genomics Institute of the University of Erlangen Ph.D. Jodie Chin, Ph.D. Novartis Research Foundation Erlangen, Germany San Diego, California Larisa Krasnova, Ph.D. Charles Cho, Ph.D. Jason Hein, Ph.D. Ted Foss, Ph.D. Jane Kuzelka, Ph.D. So-Hye Cho, Ph.D. Mark Hixon, Ph.D.**** Joseph Rodolph Fotsing, Takeda Andreas Lanver, Ph.D. Scott Cockroft, Ph.D. Ph.D. San Diego, California Brian Lawhorn, Ph.D. Kevin Cole, Ph.D. Rebecca Fraser, Ph.D.**** Jiyong Hong, Ph.D.**** Novartis Pharma AG Duke University Jinq-Chyi Lee, Ph.D. Antonella Converso, Ph.D.**** Horsham, England Durham, North Carolina Merck & Co., Inc. Jongkook Lee, Ph.D. West Point, Pennsylvania Graeme Freestone, Ph.D. Sukwon Hong, Ph.D.**** University of Florida JongSeok Lee, Ph.D. Jeromy Cottell, Ph.D. Jim Fuchs, Ph.D. Gainsville, Florida Ki-Bum Lee, Ph.D. James Crawford, Ph.D. Amelia Fuller, Ph.D. Zhangyong Hong, Ph.D. Kooyeon Lee, Ph.D. Matthew Cremeens, Ph.D. Jianmin Gao, Ph.D. Richard Hooley, Ph.D. Sang Hyup Lee, Ph.D.**** Jesse Dambacher, Ph.D. Muyun Gao, Ph.D. Daniel Horne, Ph.D.**** LG Life Sciences Amgen Sandra De Lamo Marin, Ph.D. Daejeon, South Korea Ola Ghoneim, Ph.D. Thousand Oaks, California Ross Denton, Ph.D. Sejin Lee, Ph.D. Nathan Gianneschi, Ph.D. Tsui-Ling Hsu, Ph.D. Caroline Desponts, Ph.D. Lucas Leman, Ph.D. Romelo Gibe, Ph.D. Zheng-Zheng Huang, Ph.D. David Diaz-Diaz, Ph.D.**** Institute of Chemical and Alexandre Lemire, Ph.D. Universidad Autónoma de Engineering Sciences Amy Hurshman, Ph.D.**** Madrid Jurong Island, Singapore, Joint Science Department of Edward Lemke, Ph.D. Madrid, Spain China the Claremont Colleges Claremont, California Achim Lenzin, Ph.D. Christine Dierks, Ph.D.**** Christina Gil-Lamaignere, Genomics Institute of the Ph.D. Der-Ren Hwang, Ph.D. Hongming Li, Ph.D. Novartis Research Foundation San Diego, California Neill Gingles, Ph.D. Giltae Hwang, Ph.D. Ke Li, Ph.D.

David Edmonds, Ph.D. Naran Gombosuren, Ph.D. Tetsuo Iwasawa, Ph.D. Pi-Hui Liang, Ph.D.

Greg Elliott, Ph.D.**** Rajesh K. Grover, Ph.D. Michael Jahnz, Ph.D. Jiayu Liao, Ph.D.**** Moore Cancer Center University of California La Jolla, California Jan Grunewald, Ph.D. Wei Jin, Ph.D. Riverside, California CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 65

Yeon-Hee Lim, Ph.D. Robert Milburn, Ph.D.**** Charles Papageorgiou, F. Anthony Romero, Ph.D. Amgen Ph.D.**** Troy Lister, Ph.D. Thousand Oaks, California Amgen Youngha Ryu, Ph.D. Cambridge, Massachusetts Chris Liu, Ph.D. Kyung-Hoon Min, Ph.D.***** Riccardo Salvio, Ph.D. Doron Pappo, Ph.D. Haitian Liu, Ph.D.**** Christos A. Mitsos, Ph.D.**** Anu Sawkar, Ph.D.**** Senomyx, Inc. Athens, Greece Junguk Park, Ph.D. Ropes & Gray La Jolla, California New York, New York Gopi Kumar Mittapalli, Ph.D. Laxman Pasunoori, Ph.D. Jun Liu, Ph.D.**** Patrick Schanen, Ph.D.**** Genomics Institute of the Lionel Moisan, Ph.D. Richard Payne, Ph.D. ETH Zürich, Laboratory of Novartis Research Foundation Organic Chemistry San Diego, California Ana Montero, Ph.D. Murali Peram Surakattula, Zurich, Switzerland Ph.D. Lei Liu, Ph.D. Miguel Morales, Ph.D. Stefan Schiller, Ph.D. Roshan Perera, Ph.D. Wenshe Liu, Ph.D. Tingwei Mu, Ph.D. Daniel Schlawe, Ph.D.**** Goran Petrovic, Ph.D. Syncom BV Yi Liu, Ph.D.**** Mridul Mukherji, Ph.D. Groningen, the Netherlands Jared Piper, Ph.D.**** Lawrence Berkeley National Eli Lilly Laboratory Andrew Myles, Ph.D.**** Michael Schramm, Ph.D. Indianapolis, Indiana Berkeley, California University of Alberta Edmonton, Canada Laura Segatori, Ph.D. Suresh Pitram, Ph.D. Ying (Cindy) Liu, Ph.D. Kenichiro Nagai, Ph.D. Mary Sever, Ph.D. Tülay Polat, Ph.D. Dimitrios Lizos, Ph.D.**** Novartis Pharmaceuticals Yuya Nakai, Ph.D. Alex Shaginian, Ph.D. Damien Poliet, Ph.D. Basil, Switzerland Joonwoo Nam, Ph.D. Guido Pontremoli, Ph.D. David Shaw, Ph.D. Jon Loren, Ph.D.**** Sridhar Narayan, Ph.D.**** Genomics Institute of the Junhwa Shin, Ph.D.**** Eisai Research Institute Mariceli Puga, Ph.D. Novartis Research Foundation Korea Atomic Energy Wilmington, Massachusetts San Diego, California Sreenivas Punna, Ph.D.**** Research Institute Seoul, Korea Daniel Nicoletti, Ph.D. ChemoCentryx, Inc. Hongzheng (Eric) Ma, Ph.D. Mountain View, California Sebastian Steiniger, Ph.D. Alain Noncovich, Ph.D.**** Roman Manetsch, Ph.D.**** Senomyx Inc. Daniela Radu, Ph.D. University of South Florida Shula Stokols, Ph.D.**** La Jolla, California Tampa, Florida Nicole Rahe, Ph.D.**** W. L. Gore & Associates Yasuo Norikane, Ph.D.**** Tesa AG Flagstaff, Arizona Enrique Mann, Ph.D. Nanotechnology Research Hamburg, Germany Institute, AIST Ji Young Suk, Ph.D.**** Felix Marr, Ph.D.***** Tsukuba, Japan Shai Rahimipour, Ph.D.**** Merck Research Laboratories Bar-Ilan University Boston, Massachusetts Carol Lamenca Martinez, Mehdi Numa, Ph.D.**** Ramat-Gan, Isreal Ph.D.**** Vertex Pharmaceuticals Daniel Summerer, Ph.D. Berlin, Germany Incorporated Praveen Rao, Ph.D. San Diego, California Takahiro Suzuki, Ph.D. Shigeo Matsuda, Ph.D. Dalit Rechavi-Robinson, Severin Odermatt, Ph.D. Ph.D.**** Leo Takaoka, Ph.D. Klaus-Dieter Michael Maue, University of Geneva Ph.D. Barun Okram, Ph.D. Geneva, Switzerland Eric Tippmann, Ph.D.

Laura McAllister, Ph.D. Peter Orahovats, Ph.D. Stefanie Roeper, Ph.D.***** Jonathan Tripp, Ph.D. Vertex Pharmaceuticals Kathleen McKenzie, Ph.D. Yazmin Osornio, Ph.D. Cambridge, Massachusetts Meng-Lin Tsao, Ph.D. 66 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Craig Turner, Ph.D. Heiko Wurdak, Ph.D. VISITING INVESTIGATORS Masaaki Sawa, Ph.D. **** Dainippon Pharmaceuticals Jim Turner, Ph.D.**** Jian Xie, Ph.D. Mohammad Al-Sayah, Ph.D. Co., Ltd Fish & Richardson P.C. American University of Osaka, Japan San Diego, California Yue Xu, Ph.D. Sharjah Sharjah, United Arab Masakazu Sugiyama, Ph.D. Andrew Udit, Ph.D. Ajinomoto Co., Inc. Ryu Yamasaki, Ph.D. Emirates Kawasaki-Shi, Japan Taiki Umezawa, Ph.D. Luda Bazhenova, Ph.D.**** Shuyan Yao, Ph.D.**** University of California Shin-Ichi Takanashi, Yasuyuki Ura, Ph.D. Sangamo BioScience, Inc. San Diego, California Ph.D.**** Richmond, California Kenji Usui, Ph.D. Sheila Fleming, Ph.D.**** Erich Uffelman, Ph.D.**** Robert Yeh, Ph.D. **** University of California Washington and Lee Juraj Velcicky, Ph.D.**** Siemens Los Angeles, California University Novartis Torrance, California Lexington, Virginia Basel, Switzerland Masakazu Fujio, Ph.D. Yong Sik Yoo, Ph.D.**** Mitsubishi Pharma Makoto Yamashita, Alessandro Volonterio, Ph.D.**** Cheil Industries, Inc. Corporation Ph.D.**** Takeda Chemical Industries, Seoul, Korea Yokohama, Japan Politencino di Milano Ltd. Milano, Italy Osaka, Japan Zhanqian Yu, Ph.D. Luigi Gomez Paloma, Hong Wang, Ph.D. Ph.D.**** Mark Zak, Ph.D.**** University di Salerno Fisciano, Italy SCIENTIFIC ASSOCIATES Jiangyun Wang, Ph.D. Genentech San Francisco, California Jon Ashley Weidong Wang, Ph.D. Andrew S. Grant, Ph.D.**** Mount Allison University Felix Zelder, Ph.D.**** Gina Dendle Xiaolong Wang, Ph.D. Sackville, New Brunswick, Canada Timo Weide, Ph.D. Huaqiang Zeng, Ph.D.**** Suresh Mahajan, Ph.D. National University of Yoshiyuki Hari, Ph.D. Lisa Whalen, Ph.D.**** Singapore Nagoya City University * Joint appointment in The Skaggs University of New Mexico Singapore Nagoya, Japan Albuquerque, New Mexico Institute for Chemical Biology ** Joint appointments in the Qisheng Zhang, Ph.D. Akira Ino, Ph.D. Matthew Whiting, Ph.D. Skaggs Institute for Chemical Shionogi & Co., LTD. Biology and the Department of Yingchao Zhang, Ph.D. Osaka, Japan Immunology Aarron Willingham, Ph.D.**** *** Joint appointments in The Affymetrix Lisa Landino, Ph.D. Skaggs Institute for Chemical Santa Clara, California Yuanxiang Zhao, Ph.D.**** Biology and the Department of College of William and Mary Cellular Dynamics Molecular Biology Williamsburg, Virginia Albert Willis, Ph.D. International Inc. **** Appointment completed; new Madison,Wisconsin location shown Jason Moss, Ph.D.**** Chung-Yi Wu, Ph.D.**** ***** Appointment completed PDL BioPharma, Inc. Academia Sinica Heyue Henry Zhou, Ph.D. The Genomics Research Fremont, California Institute Min Zhou, Ph.D. Taipei, Taiwan Poul Nielsen, Ph.D. University of Southern Douglass Wu, Ph.D. Xiuwen Zhu, Ph.D. Denmark Optimer Pharmaceuticals Odense, Denmark San Diego, California Joerg Zimmermann, Ph.D. Manuela Rodriguez, Margarita Wuchrer, Ph.D.**** Caterina Zoni, Ph.D. Ph.D.**** Merck KgaA University di Salerno Frankfurt, Germany Fisciano, Italy CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 67

Chadha (x-ray crystallography), are second to none and continue to provide crucial support to our research programs. In addition, the Mabel and Arnold Beckman Cen- ter for the Chemical Sciences constantly receives high praise from visitors from around the world for its architectural design and operational aspects, both highly conducive to research. Research in the Department of Chemistry goes on unabated, establishing international visibility and attracting attention as evidenced by numerous lecture invita- tions, visits by outside scholars, and headline news in the media. As of 2005, the Institute for Scientific Infor- mation ranked 3 members of our department as highly cited researchers (in the top 100 worldwide); 2 of the 3 are among the top 51 positions. Richard Lerner and his group continue to make advances in catalytic antibodies, with new antibodies that catalyze important synthetic and biological reactions and novel applications in chemical synthesis. The group’s research has recently expanded to include the funda- K.C. Nicolaou, Ph.D. mental chemistry of the polyoxygen species. Barry Sharpless and his group continue endeavors to Chairman's Overview discover and develop better catalysts for organic synthesis and to construct, through innovative chemistry and biol- s the "central science," chemistry stands between ogy, libraries of novel compounds for biological screening. biology and medicine and between physics and Scientists in Albert Eschenmoser’s La Jolla-based A materials science and provides the crucial bridge group advance their experimental studies on the chemical for drug discovery and development. But chemistry has etiology of nucleic acid structure by investigating nucleic a much more profound and useful role in science and acid alternatives that have novel backbones and recogni- society. It is the discipline that continually creates the tion elements unrelated to the canonical phophodiester- myriad of new materials that we all encounter in our based oligonucleotide systems. everyday lives: pharmaceuticals, high-tech materials, Members of my own group continue explorations of polymers and plastics, insecticides and pesticides, fab- chemical synthesis and chemical biology, focusing on the rics and cosmetics, fertilizers, and vitamins—basically total synthesis of new anticancer agents, antibiotics, everything we can touch, feel, and smell. marine-derived neurotoxins, antimalarial compounds, Chemists at Scripps Research focus on chemical syn- antifeedant agents, and other biologically active natural thesis and chemical biology, the areas most relevant to and designed molecules. biomedical research and materials science. The mem- The members of Julius Rebek’s group devise bio- bers of our faculty are distinguished teacher-scholars mimetic receptors for studies in molecular recognition. who maintain highly visible and independent research These include molecules that bind neurotransmitters and programs in areas as diverse as biological and chemical membrane components. Larger host receptors can sur- catalysis, synthesis of natural products, combinatorial round 3 or more molecular guests and act as chambers chemistry, molecular design, supramolecular chemistry, where the chemical reactions of the guests are acceler- chemical evolution, materials science, and chemical ated. The group synthesizes small molecules that act as biology. The chemistry graduate program attracts some protein helix mimetics for pharmaceutical applications. of the best-qualified candidates from both the United Peter Schultz and his group have continued to expand States and abroad. Our major research facilities, under the number of genetically encoded amino acids to include the direction of Dee H. Huang (nuclear magnetic reso- fluorescent, photocaged, metal binding, thioester, sul- nance), Gary Siuzdak (mass spectrometry), and Raj fated, and long-chain alkane side chains. They have 68 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE also adapted this technology to mammalian cells and ratory also develop and investigate new organic and are applying it to a number of basic and applied prob- organometallic reactions and use these processes to lems in cell biology. In addition, they have used cell-based synthesize biologically active compounds. screens to identify small molecules that selectively dif- Jeffery Kelly and his group are exploring the inter- ferentiate and expand embryonic and adult stem cells face between chemistry, biology, and medicine. Their and reprogram lineage-committed cells, as well as novel projects aim to understand the physical and biological genes that control cell cycle, cell migration, and devel- basis of protein folding, and the misfolding and aggre- opmental pathways. gation processes leading to age-onset neurodegenera- Chi-Huey Wong and his group further advance the tive diseases. Comprehension of the latter processes is fields of chemoenzymatic organic synthesis, chemical gly- used to develop new small-molecule therapeutic strate- cobiology, and the development of enzyme inhibitors. A gies for a variety of neurodegenerative diseases. new strategy for the synthesis of glycoproteins has been Anita Wentworth and her group are investigating developed. The programmable 1-pot synthesis of oligosac- the chemical basis of complex disease states and are charides developed by this group has been further used in synthesizing peptide and small molecule–based thera- the assembly of glycoarrays for study of saccharides that peutics. Their research is focused on disease states bind to proteins. This group also developed new probes that have a prominent inflammatory and reactive oxy- to study glycosyltransferases and their role in cancer. gen-species chemical component, such as atherosclero- Members of Dale Boger’s group continue their work sis, Alzheimer's disease, and other diseases of aging. on chemical synthesis; combinatorial chemistry; hetero- Researchers in Floyd Romesberg’s laboratory are cycle synthesis; anticancer agents such as vinblastine, using diverse techniques ranging from bioorganic and fostriecin, and yatakemycin; and antibiotics such as biophysical chemistry to bacterial and yeast genetics to vancomycin, teicoplanin, and ramoplanin. understand and manipulate the process of evolution. Scientists in Kim Janda’s laboratory are focusing on Major efforts include designing unnatural base pairs and the impact of organic chemistry in specific biological the directed evolution of DNA polymerases to efficiently systems. Their targeted programs span a wide range of synthesize unnatural DNA containing the base pairs; interests from immunopharmacotherapy to biological using spectroscopy to understand biological function and and chemical warfare agents to filarial infections, such how it evolves; and understanding how induced and as "river blindness," to quorum sensing in bacteria. Their adaptive mutations contribute to evolution in eukaryotic recent achievements include the discovery of a secondary and prokaryotic cells. nicotine metabolite that alters retinoid homeostasis, a Dr. Baran and his group have recently developed critical component of vision and growth; small molecules extremely concise chemical solutions to the synthetic that "superactivate" botulinum neurotoxin; and a virus- challenges posed by numerous marine natural product based system that can degrade cocaine in the central families, including sceptrin, ageliferin, chartelline, haou- nervous system. amine, welwitindolinones, and the stephacidins. These Reza Ghadiri and his group are making significant syntheses are characterized by striking brevity, new contributions in the design and study of a new genera- biosynthetic postulates, the invention of a new methodol- tion of antimicrobial agents, based on self-assembling ogy, and a minimum use or complete absence of protect- peptide nanotube architecture, to combat multidrug- ing groups and superfluous oxidation-state manipulations. resistant infections. In addition, they continue to make The Frontiers in Chemistry Lecturers (17th Annual novel contributions in several ongoing basic research Symposium) for the 2005–2006 academic year were endeavors, such as biosensor designs, molecular com- Richard Lerner, Scripps Research; Peter Vollhardt, Uni- putation, design of self-reproducing systems, under- versity of California, Berkeley; Dieter Enders, Institute standing the origins of life, and design of emergent of Organic Chemistry, RWTH Aachen, Germany; and chemical systems. K.C. Nicolaou, Scripps Research. Thomas Scanlan M.G. Finn and his group have pioneered the use of (University of California, San Francisco) also visited virus particles as chemical reagents and building blocks Scripps this year as the Novartis Lecturer in Organic for nanochemical structures. This effort is directed toward Chemistry, 2005. the development of new diagnostics for disease and catalysts for organic reactions. Members of Dr. Finn’s labo- CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 69

INVESTIGATORS’REPORTS Practical Total Synthesis of Natural Products

P.S. Baran, N.Z. Burns, M.P. DeMartino, C.A. Guerrero, B.D. Hafensteiner, P.J. Krawczuk, K. Li, D.W. Lin, T.J. Maimone, M.K.-D. Maue, S. Nguyen, D.P. O’Malley, J.M. Richter, R.A. Shenvi, B. Whitefield

rom penicillin to paclitaxel (Taxol), natural products have an unparalleled track record in the F betterment of human health. In fact, 9 of the top 20 best-selling drugs were either inspired by or derived from natural products. Even the best-selling drug of all time, atorvastatin (Lipitor), was based on a natural product lead. Total synthesis, the art and science of recreating these entities in the laboratory, invariably leads to fundamental discoveries in chemistry, biology, and medicine. We focus on solving interesting challenges in the total synthesis of natural products and on bridging gaps in synthetic capabilities by inventing new reactions. Through judicious target selection and creative retrosyn- thetic analyses, total synthesis becomes an engine for discovery that drives the field of organic chemistry to new levels of sophistication and practicality. Synthetic organic chemistry requires tremendous ingenuity, artis- tic taste, experimental acumen, persistence, and character. Not surprisingly, drug development relies on the expertise of researchers who have these characteristics. Although we focus entirely on educating students in fundamental chemistry, we also collaborate with expert biologists to explore the medicinal potential of newly synthesized natural products and the products’ analogs. Recently completed total syntheses (Fig. 1) include the anticancer agents stephacidins A and B and avrainvillamide; the antibacterial agents sceptrin and ageliferin; members of the bioactive fischerindole, hapalindole, and welwitindolinone indole alkaloid family; and the anticancer agent haouamine A. Current natural prod- Fig. 1. Recently completed total syntheses. uct targets (Fig. 2) include chartelline C, axinellamine, strictamine, and sarcodonin. Baran, P.S., Li, K., O’Malley, D.P., Mitsos, C. Short, enantioselective total synthesis of sceptrin and ageliferin by programmed oxaquadricyclane fragmentation. Angew. Chem. Int. Ed. 45:249, 2006. PUBLICATIONS Baran, P.S., Burns, N.Z. Total synthesis of (±)-haouamine A. J. Am. Chem. Soc. Baran, P.S., Richter, J.M. Enantioselective total syntheses of welwitindolinone A 128:3908, 2006. and fischerindoles I and G. J. Am. Chem. Soc. 127:15394, 2005.

Baran, P.S., Hafensteiner, B.D., Ambhaikar, N.B., Guerrero, C.A., Gallagher, J.D. Northrop, B.H., O’Malley, D.P., Zografos, A.L., Baran, P.S., Houk, K.N. The mech- Enantioselective total synthesis of avrainvillamide and the stephacidins. J. Am. anism of the vinylcyclobutane rearrangement of sceptrin to ageliferin and nage- Chem. Soc. 128:8678, 2006. lamide E. Angew. Chem. Int. Ed. 45:4126, 2006. 70 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

SYNTHETIC METHODS Central to much of our work are investigations to develop and apply the hetero Diels-Alder reaction, including the use of heterocyclic and acyclic azadienes (Fig. 1),

Fig. 1. N-Sulfonyl-1-aza-1,3-butadiene Diels-Alder reaction.

the thermal reactions of cyclopropenone ketals, intermolecular and intramolecular acyl radical–alkene addition reactions, medium- and large-ring cyclization technology, and solution-phase combinatorial chemistry. In each instance, the development of the methods represents the investigation of chemistry projected as a key element in the synthesis of a natural or designed agent. TOTAL SYNTHESIS OF NATURAL PRODUCTS Efforts are under way on the total synthesis of a number of natural products that constitute agents in which we have a specific interest. Representative agents currently under study include (+)-CC-1065 and func- Fig. 2. Ongoing natural product total syntheses. tional analogs; the duocarmycin class of antitumor antibiotics, including yatakemycin; tropoloalkaloids; Synthetic and prodigiosin and roseophilin; the deoxybouvardin and RA-I class of antitumor agents; vancomycin, teicoplanin, Bioorganic Chemistry ristocetin, chloropeptins and related agents; ramoplanin; the luzopeptins, quinoxapeptins, thiocoraline, D.L. Boger, S.B. Boga, K. Bunker, R. Clark, D. Colby, BE-22179 and sandramycin; bleomycin A2 and func- J. Cottell, B. Crowley, J. DeMartino, G. Elliott, J. Elsner, tional analogs; HUN-7293; chlorofusin; CI-920 (fos- C. Ezzili, J. Fuchs, J. Garfunkle, A. Hamasaki, W. Han, triecin) and cytostatin; the combretastatins; storniamide N. Haq, S. Hong, D. Horne, I. Hwang, H. Ishikawa, W. Jin, A; phomazarin; ningalins; lamellarin O; lukianol A; D. Kato, D. Kastrinsky, M. Kelso, G. Kim, F.S. Kimball, piericidins; nothapodytine and mappicine; rubrolone; B. Lawhorn, S. Lee, C. Liu, K. MacMillan, J. Nam, P. Patel, vindoline; and vinblastine (Figs. 2 and 3). A. Romero, M. Schnermann, A. Shaginian, C. Slown, BIOORGANIC CHEMISTRY L. Takaoka, H. Tao, M. Tichenor, J. Trzupek, J. Velcicky, The agents listed in the previous paragraph were L. Whiby,Y. Zhang selected on the basis of their properties; in many he research interests of our group include the total instances, they are agents related by a projected prop- synthesis of natural products, development of new erty. For example, (+)-CC-1065, the duocarmycins, T synthetic methods, heterocyclic chemistry, bioor- and yatakemycin are antitumor antibiotics and related ganic and medicinal chemistry, the study of DNA-agent sequence-selective DNA minor groove alkylating agents. interactions, and the chemistry of antitumor antibiotics. Representative of such efforts, studies to determine We place a special emphasis on investigations to define the structural features of yatakemycin and the duo- the structure-function relationships of natural or designed carmycins that contribute to the sequence-selective organic agents. DNA alkylation properties of these agents have resulted CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 71

Fig. 2. Recent total syntheses. Fig. 3. Additional recent total syntheses. in the identification of a unique source of catalysis for DNA-binding and DNA-effector agents. Techniques for the DNA alkylation reaction. Efforts are under way to the evaluation of the agent-DNA binding and alkylation develop DNA cross-linking agents of a predefined cross- properties, collaborative efforts in securing biological link, to further understand the nature of the noncovalent data, nuclear magnetic resonance structures of DNA- and covalent interactions between agents and DNA, and agent complexes, molecular modeling, and studies of to apply this understanding to the de novo design of DNA-agent interactions are integral parts of the program. 72 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Additional ongoing studies include efforts to define Tse, W.C., Boger, D.L. A fluorescent intercalator displacement (FID) assay for establishing DNA binding selectivity and affinity. In: Current Protocols in Nucleic the fundamental basis of the DNA-binding or cleavage Acid Chemistry. Beaucage, S.L., et al. (Eds.). Wiley & Sons, New York, in press. properties of bleomycin A , sandramycin, and the luzo- 2 Walker, S., Chen, L., Hu, Y., Rew, Y., Shin, D., Boger, D.L. Chemistry and biology peptins; to design inhibitors of the folate-dependent of ramoplanin: a lipoglycodepsipeptide with potent antibiotic activity. Chem. Rev. enzymes glycinamide ribonucleotide transformylase and 105:449, 2005.

aminoimidazole carboxamide ribonucleotide transfor- Yuan, Z., Ishikawa, H., Boger, D.L. Total synthesis of natural (+)- and ent-(–)-4- mylase as potential antineoplastic agents; to establish desacetoxy-6,7-dihydrovindorosine [corrected] and natural and ent-minovine: oxadiazole tandem intramolecular Diels-Alder/1,3-dipolar cycloaddition reaction [published the chemical and biological characteristics responsible correction appears in Org. Lett. 7:2079, 2005]. Org. Lett. 7:741, 2005. for the sleep-inducing properties of the endogenous lipid oleamide; to inhibit tumor growth through inhibition of angiogenesis; to inhibit aberrant gene transcrip- Chemical and Functional tion associated with cancer; and to control intracellular signal transduction through the discovery of antago- Genomic Approaches to nists or agonists that affect protein-protein interactions, including receptor dimerization. Regenerative Medicine

PUBLICATIONS S. Ding, R. Abu-Jarour, R. Ambasudhan, C. Desponts, Boger, D.L., Miyauchi, H., Du, W., Hardouin, C., Fecik, R.A., Cheng, H., Hwang, N. Emre, H.S. Hahm, S. Hilcove, J. Hsu, M. Kim, Y. Shi, I., Hedrick, M.P., Leung, D., Acevedo, O., Guimarães, C.R.W., Jorgensen, W.L., Cravatt, B.F. Discovery of a potent, selective, and efficacious class of reversible S. Takanashi, W. Xiong, Y. Xu, S. Yao, D. Yue, Y. Zhao, X. Zhu α-ketoheterocycle inhibitors of fatty acid amide hydrolase effective as analgesics. J. Med. Chem. 48:1849, 2005. ecent advances in stem cell biology may make

Capps, K.J., Humiston, J., Dominique, R., Hwang, I., Boger, D.L. Discovery of possible new approaches for the treatment of a AICAR Tfase inhibitors that disrupt requisite enzyme dimerization. Bioorg. Med. number of diseases, including cardiovascular Chem. Lett. 15:2840, 2005. R disease, neurodegenerative disease, musculoskeletal Cheng, H., Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. Design, synthesis, and evaluation of 10-methanesulfonyl-DDAC- disease, diabetes, and cancer. These approaches could THF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent involve cell replacement therapy and/or drug treatment inhibitors of GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med. Chem. 13:3577, 2005. to stimulate the body’s own regenerative capabilities by promoting survival, migration/homing, proliferation, Cheng, H., Hwang, I., Chong, Y., Tavassoli, A., Webb, M.E., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, D.L. Synthesis and biological evaluation of N-{4-[5- and differentiation of endogenous stem/progenitor cells. (2,4-diamino-6-oxo-6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]ben- However, such approaches will require identification of zoyl}-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway. Bioorg. Med. Chem. 13:3593, 2005. renewable cell sources of engraftable functional cells, an

Choi, Y., Ishikawa, H., Velcicky, J., Elliott, G.I., Miller, M.M., Boger, D.L. Total improved ability to manipulate proliferation and differ- synthesis of (–)- and ent-(+)-vindoline. Org. Lett. 7:4539, 2005. entiation of the cells, and a better understanding of the

Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J, Boger, signaling pathways that control the fate of the cells. D.L. Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10- Equipped with large arrayed molecular libraries— CF CO-DDACTHF. Bioorg. Med. Chem. 13:3587, 2005. 3 combinatorial chemical libraries (>100,000 discrete Chou, T.-C., Gaun, Y., Soenen, D.R., Danishefsky, S.J., Boger, D.L. Potent reversal of and diverse small molecules), cDNA overexpression multidrug resistance by ningalin and its use in drug combinations against human colon carcinoma xenografts in nude mice. Cancer Chemother. Pharmacol. 56:379, 2005. libraries (>30,000 human and mouse genes) and

Du, W., Hardouin, C., Cheng, H., Hwang, I., Boger, D.L. Heterocyclic sulfoxide small interfering RNA libraries (targeting >20,000 and sulfone inhibitors of fatty acid amide hydrolase. Bioorg. Med. Chem. Lett. human and mouse genes)—and a high-throughput 15:103, 2005. screening platform, we are developing and integrating Guimarães, C.R.W., Boger, D.L., Jorgensen, W.L. Elucidation of fatty acid amide chemical and functional genomic tools to study stem hydrolase inhibition by potent α-ketoheterocycle derivatives from Monte Carlo simulations. J. Am. Chem. Soc. 127:17377, 2005. cell biology and regeneration. We screen these libraries to identify small molecules and genes that can control Hamasaki, A., Zimpleman, J.M., Hwang, I., Boger, D.L. Total synthesis of ningalin D. J. Am. Chem. Soc. 127:10767, 2005. the fate of stem cells in various systems, including (1)

Leung, D., Du, W., Hardouin, C., Cheng, H., Hwang, I., Cravatt, B.F., Boger, D.L. self-renewal, as well as directed neuronal, cardiac, and Discovery of an exceptionally potent and selective class of fatty acid amide hydrolase pancreatic differentiations of pluripotent mouse and inhibitors enlisting proteome-wide selectivity screening: concurrent optimization of enzyme inhibitor potency and selectivity. Bioorg. Med. Chem. Lett. 15:1423, 2005. human embryonic stem cells; (2) directed neuronal differentiation and subtype neuron specification of Schnermann, M.J., Boger, D.L. Total synthesis of piercidin A1 and B1. J. Am. Chem. Soc. 127:15704, 2005. human and rodent neural stem cells; (3) directed dif- CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 73 ferentiation of mesenchymal stem cells to osteogenic, adipogenic, chondrogenic, and myogenic lineages; (4) functional proliferation of cardiomyocytes and islets/beta cells in adults; (5) cellular plasticity and dedifferentiation of lineage-restricted somatic cells; and (6) developmental signaling pathways. In addition, we are doing systemic biochemical and cellular studies, including detailed investigations of structure-activity relationships, affinity chromatography for target identification, genome-wide expression analysis with microarrays, and cDNA and/or RNA interference complementation screens to map signaling pathways to characterize the molecular mechanism of these identified small molecules and genes. Recent examples of small molecules of interest include neuropathiazol, which can direct differentiation Fig. 1. Formulas of triazine-tagged oligomers. IDA indicates of primary rat adult neural stem cells selectively toward iminodiacetic acid. neurons; pluripotin, which can sustain self-renewal of murine embryonic stem cells in a chemically defined cross-pairing with complementary RNA and DNA medium; and a purine analog that functions as a syn- oligonucleotide sequences, the corresponding oligo- ergistic Wnt pathway agonist and can induce Xenopus (AspGlu)-dipeptides tagged with 2,4-dioxotriazine, axis duplication in combination with Wnt8. These stud- much to our surprise, showed only weak pairing with ies may ultimately facilitate the therapeutic application the natural oligonucleotides. The intrasystem self-pair- of stem cells and the development of small-molecule ing of 2,4-diaminotriazine–, 2,4-dioxotriazine–, and 2- drugs to stimulate tissue and organ regeneration in vivo. amino-4-oxo-triazine–tagged oligo-(AspGlu)-dipeptides was equally weak. PUBLICATIONS The base-pairing properties of triazine-tagged oligo- Warashina, M., Min, K.H., Kuwabara, T., Huynh, A., Gage, F.H., Schultz, P.G., Ding, S. A synthetic small molecule that induces neuronal differentiation of adult (AspAsp)-dipeptides paralleled the trends observed in hippocampal neural progenitor cells. Angew. Chem. Int. Ed. 45:591, 2006. the oligo-(AspGlu)-dipeptide series, but, as expected, Zhao, Y., Clark J., Ding, S. Genomic studies in stem cell systems. Curr. Opin. Mol. were consistently weaker in base-pairing strength. Ther. 7:43, 2005. A variation of the oligo-(AspAsp)-dipeptide shown in Figure 1B is the achiral oligodipeptoid derived from iminodiacetic acid units (Fig. 1C). Again, oligodipep- Chemical Etiology of the toids containing 2,4-diaminotriazines cross-paired with RNA and DNA oligonucleotides, but no discernible Structure of Nucleic Acids pairing occurred with the 2,4-dioxotriazine–tagged oligodipeptoids. A. Eschenmoser, R. Krishnamurthy, G. Kumar, F. De Our studies indicate that the family of triazine-based Riccardis, R. Kondreddi, Y. Osornio, M. Guerrero recognition elements lacks the balance in pairing strength uring the past year we worked on the following characteristic of the purine-pyrimidine combination in projects. the natural series, presumably because of the imbal- K D ance in protophilicity of dioxotriazines (p a about 6) OLIGOMERS BASED ON TRIAZINE-TAGGED vs diaminotriazines (pKa about 3.9), in an aqueous OLIGODIPEPTIDE AND OLIGODIPEPTOID BACKBONES environment. Such imbalance in pairing potential leads We continued our studies on the self- and cross- to the conclusion that triazines, irrespective of their pairing properties of triazine-tagged oligodipeptides con- generational simplicity, would have been functionally sisting of alternating aspartic acid (Asp) and glutamic incapable of fulfilling the role of recognition elements acid (Glu) residues (Fig. 1). Although the oligo-(AspGlu)- in a primordial genetic system. This realization has led dipeptides tagged with 2,4-diaminotriazine had strong us pursue the following project. 74 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

OLIGOMERS BASED ON 5-AMINOPYRIMIDINE–TAGGED Mittapalli, G.K., Osornio, Y.M., Guererro, M.A., Reddy, K.R., Krishnamurthy, R., Eschenmoser, A. Mapping the landscape of potentially primordial informational OLIGODIPEPTIDE BACKBONES oligomers: oligodipeptides and oligodipeptoids tagged with 2,4-disubstituted-5- Oligomerization of hydrogen cyanide, a potentially amino-pyrimidines as recognition elements. Angew. Chem. Int. Ed., in press. prebiotic reaction generally assumed to have acted as Wagner, T., Han, B., Koch, G., Krishnamurthy, R., Eschenmoser, A. Tautomerism in the primordial source of the canonical nucleobases ade- 5,8-diaza-7,9-dicarbaguanine (“alloguanine”). Helv. Chim. Acta 88:1960, 2005. nine and guanine, produces in addition pyrimidines, not the canonical ones, but mostly 5-aminopyrimidines, which do not play a role in contemporary biology (Fig. 2). Organic, Materials, and Analytical Chemistry

M.G. Finn, J. Kuzelka, D. Prasuhn, S. Presolski, V. Rodionov, Y.-H. Lim, B. Venkataiah

n addition to synthetic chemistry research on viruses, our program encompasses organic, organometallic, I and materials chemistry. Special emphasis is placed on methods of chemical synthesis, the discovery of functional molecules, and catalysis. MECHANISMS AND APPLICATIONS OF CLICK CHEMISTRY The copper-catalyzed azide-alkyne cycloaddition Fig. 2. Top, Formulas of 5-aminopyrimidine heterocycles. Bottom, reaction, discovered in 2002 by V.V. Fokin and Also shown, as a representative example, is the 5-amino-2,4-dioxo– K.B. Sharpless, Department of Chemistry, has been tagged oligomer containing alternating residues of aspartic and glutamic acid. adopted by chemists all over the world for organic synthesis, drug development, and materials science. Chemical reasoning makes a study of the base-pairing We have continued our mechanistic studies of the properties of the members of this family highly desir- reaction and our efforts to apply the reaction to the able; they not only can potentially act as substitutes synthesis of biologically active compounds, materials, in the 2 canonical Watson-Crick base pairs but also and bioconjugates. offer a unique opportunity to tag polypeptide chains A protocol for using the reaction in the polyvalent bearing recurring carboxyl groups by using simple decoration of scaffolds has been optimized (Fig. 1). In (regioselective) amide formation. We have synthesized 5-aminopyrimidine–tagged oligo-(AspGlu)-dipeptides (up to hexadecamers) by using all 4 members of the family and have explored base-paring properties of the tagged dipeptides. Preliminary results indicated cross- pairing between all of these recognition elements with the corresponding complementary RNA and DNA oligonucleotides, although the 5-aminopyrimidine heterocy- Fig. 1. Bioconjugation of alkynes to polyvalent azides via the cles have stark differences in base-pairing strength. copper complex of bathophenanthroline ligand 1. Also, cross-pairing occurs between the 2,4-diaminotriazine–tagged oligo-(AspGlu)-dipeptides and 5-amino- the most demanding situations, with sensitive proteins 2,4-dioxopyrimidine–tagged oligo-(AspGlu)-dipeptides. at micromolar concentrations, the use of sulfonated bathophenanthroline (compound 1 in Fig. 1) is vital. PUBLICATIONS We continue to develop new catalysts to remove the Eschenmoser, A. Searching for nucleic acid alternatives. Chimia 59:836, 2005. last barrier to convenient application of the method, Mittapalli, G.K., Reddy, K.R., Xiong, H., Munoz, O., Han, B., De Riccardis, F., the need to perform the reaction in an inert atmosphere Krishnamurthy,R., Eschenmoser, A. Mapping the landscape of potentially primor- when protein instability prevents the simultaneous use dial informational oligomers: oligodipeptides and oligodipeptoids tagged with triazines as recognition elements. Angew. Chem. Int. Ed., in press. of a reducing agent. In addition, a variation of the CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 75

standard copper-catalyzed process has been uncovered Díaz, D.D., Rajagopal, K., Strable, E., Schneider, J., Finn, M.G. “Click” chemistry in a supramolecular environment: stabilization of organogels by copper(I)-catalyzed in which aromatic azides react with alkynes to give the azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 128:6056, 2006. 1,5-triazole isomer rather than the customary 1,4-tria- Díaz, D.D., Ripka, A.S., Finn, M.G. 1-(tert-Butyl-imino-methyl)-1,3-dimethyl-urea zole. Last, mechanistic studies have revealed changes hydrochloride. Org. Synth. 82:59, 2005. in the rate-limiting steps when certain copper-binding Johnson, J.A., Lewis, D.R., Díaz, D.D., Finn, M.G., Koberstein, J.T., Turro, N.J. ligands and substrates are used. Synthesis of degradable model networks via ATRP and click chemistry. J. Am. Chem. Soc. 128:6564, 2006. SYNTHESIS AND USE OF FORMAMIDINE COMPOUNDS We synthesized amidines, including formamidines Meng, J., Fokin, V.V., Finn, M.G. Kinetic resolution by copper-catalyzed azide- and formamidine ureas, and tested them for binding alkyne cycloaddition. Tetrahedron Lett. 46:4543, 2005. to the acetylcholine-binding proteins of Lymnaea stag- Punna, S., Kaltgrad, E., Finn, M.G. “Clickable” agarose for affinity chromatography. Bioconjug. Chem. 16:1536, 2005. nalis and Aplysia californica, soluble homologs of the nicotinic acetylcholine receptor. Compounds 2, 3, and 4 Punna, S., Meunier, S., Sen Gupta, S., Venkataiah, B., Truong, P., McGavern, D., Finn, M.G. Polyvalent inhibition of the LFA-ICAM interaction. J. Am. Chem. Soc., (Fig. 2) have moderate to high affinities for the target in press.

Rae, C.S., Khor, I.W., Wang, Q., Destito, G., Gonzalez, M.J., Singh, P.R., Thomas, D.M., Estrada, M.N., Powell, E., Finn, M.G., Manchester, M. Systemic trafficking of plant virus nanoparticles in mice via the oral route. Virology 343:224, 2005.

Sen Gupta, S., Kuzelka, J., Singh, P., Lewis, W.G., Manchester, M., Finn, M.G. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16:1572, 2005.

Fig. 2. Amidine derivatives that bind to nicotinic receptor proteins. Sen Gupta, S., Raja, K.S., Kaltgrad, E., Strable, E., Finn, M.G. Virus-glycopoly- mer conjugates by copper(I) catalysis of atom transfer radical polymerization and azide-alkyne cycloaddition. Chem. Commun. (Camb.) 4315, 2005, Issue 34. proteins, representing a new class of receptor ligands. Whereas amidines such as compound 4 are relatively Whiting, M., Muldoon, J., Lin, Y.-C., Silverman, S.M., Lindstrom, W., Olson, A.J., Kolb, H.C., Finn, M.G., Sharpless, K.B., Elder, J.H., Fokin, V.V. Inhibitors of HIV-1 stable, formamidine ureas such as compounds 2 and 3 protease via in situ click chemistry. Angew. Chem. Int. Ed. 45:1435, 2006.

are deactivated during a period of approximately 1 hour Wu, P., Malkoch, M., Hunt, J.N., Vestberg, R., Kaltgrad, E., Finn, M.G., Fokin, by hydrolysis when not bound. Using fluorescence V.V., Sharpless, K.B., Hawker, C.J. Multivalent, bifunctional dendrimers prepared by click chemistry. Chem. Commun. (Camb.) 5775, 2005, Issue 46. spectroscopy and x-ray crystallography, we showed that the bound molecules reside in the canonical hydrophobic pocket. Electrophysiologic measurements indicated that compound 4 is a nicotinic receptor agonist, Design of Functional consistent with its observed binding behavior. We are Synthetic Systems extending this research to molecules specific for subtypes of the nicotinic receptor family. These studies M.R. Ghadiri, G. Ashkenasy, N. Ashkenasy, J. Beierle, are conducted in collaboration with P. Taylor, Univer- A. Chavochi, N. Gianneschi, W.S. Horne, Z.-Z. Huang, sity of California, San Diego, and A. Markou, Molecular P. Imming, L. Leman, A. Loutchnikov, A. Montero, L. Motiei, and Integrative Neurosciences Department. D. Nicoletti, Y. Norikane, J. Picuri, N. Rahe, D. Radu, S. Rahimipour, J. Shin, R. Yamasaki, Y.S. Yoo PUBLICATIONS Díaz, D.D., Converso, A., Sharpless, K.B., Finn, M.G. 2,6-Dichloro-9-thiabicy- e are engaged in a multidisciplinary research clo[3.3.1]nonane: multigram display of azides and cyanides components on a versatile scaffold. Molecules 11:212, 2006. effort to uncover new chemical and biochemical approaches for the design of functional Díaz, D.D., Finn, M.G. Facile synthesis of N,N′-bis[formamidine]ureas and sym- W metrical N,N′-dsubstituted formamidines. Lett. Org. Chem. 2:621, 2005. molecular, supramolecular, and complex self-organized systems. Our endeavors span disciplines ranging from Díaz, D.D., Finn, M.G., Mishima, M. Substituent effects on the gas-phase basicity of formamidine ureas. Eur. J. Org. Chem. 235, 2006, Issue 1. synthetic organic, bioorganic, and physical organic chemistry to nanotechnology, biophysics, enzymology, and Díaz, D.D., Lewis, W.G., Finn, M.G. Acid-mediated amine exchange of N,N- dimethylformamidines: preparation of electron-rich formamidines. Synlett 2214, molecular biology. Current research includes the design of 2005, Issue 14. synthetic peptide catalysts, antimicrobial self-assembling

Díaz, D.D., Lewis, W.G., Finn, M.G. Activation of urea as a leaving group in sub- peptide nanotubes, semisynthetic allosteric enzymes, stitution reactions of formamidine ureas. Chem. Lett. 34:78, 2005. self-replicating molecular systems and emergent net- 76 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE works, single-molecule stochastic DNA sensing, molecular computation, and prebiotic chemistry. ANTIMICROBIAL PEPTIDE NANOTUBES We showed that appropriately designed cyclic peptide subunits can self-assemble through hydrogen bond–directed ring stacking into open-ended hollow tubular structures that have marked antibacterial and antiviral activities in vitro. The effectiveness of this novel supramolecular class of bioactive species as selective Fig. 2. Schematic representation of an intrasterically inactivated antibacterial agents was highlighted by the high effi- inhibitor-DNA-enzyme construct (left) and the DNA hybridization–triggered enzyme activation (right). The construct can be used to sense cacy of one of these antimicrobials against lethal methi- low concentrations of cDNA because of its built-in capacity for sig- cillin-resistant Staphylococcus aureus infections in mice. nal amplification via rapid turnover of substrate. Currently, we are exploring rational design of cyclic glycopeptides and selections from combinatorial librar- membrane protein α-hemolysin as a rapid and highly ies to discover novel antiviral and anticancer supramol- sensitive sensor element for stochastic analysis of the ecular compounds (Fig. 1). molecules lodged or trapped inside the protein pore; the analysis relies on detecting the perturbations in the conductance levels produced in the ion channel in the native protein. Using this technique, we developed an approach by which a single-stranded DNA molecule can be trapped in a specific configuration inside an α- hemolysin channel (Fig. 3), manipulated, and studied

Fig. 1. Antiviral agents based on self-assembling cyclic peptide nanotubes. Cyclic D,L-α-peptides act on endosomal membranes to prevent the development of low pH in endocytic vesicles, arrest the escape of virions from the endosome, and abrogate adenovirus infection.

DESIGN OF SIGNAL SELF-AMPLIFYING DNA SENSORS We constructed a novel sequence-specific DNA Fig. 3. Functional supramolecular chemistry at the single-mole- detection system based on rationally designed semisyn- cule level. Single strands of DNA can be captured inside an α- thetic enzymes. The system is composed of covalently hemolysin transmembrane pore protein to form single-species pseudorotaxanes composed of α-hemolysin and DNA. This process associated inhibitor-DNA-enzyme modules that function can be used to identify a single adenine nucleotide at a specific via DNA hybridization–triggered allosteric enzyme activa- location on a strand of DNA on the basis of the characteristic tion and signal amplification through substrate turnover reductions in the α-hemolysin ion conductance. (Fig. 2). The functional capacity of the system is highlighted by the sequence-specific detection of approxi- with high sensitivity at the single-molecule level. More- mately 10 fmol of DNA in less than 3 minutes under over, a single adenine nucleotide at a specific location physiologic conditions. Our studies suggest that ratio- on a strand of polydeoxycytidine can be detected by nally designed intrasterically regulated enzymes may be its characteristic effect in reducing the ion conduc- a promising new class of reagents for highly sensitive, tance in α-hemolysin. We are extending this approach rapid, 1-step detection of label-free DNA sequences that to the design of rapid single-molecule DNA sensing does not depend on polymerase chain reactions. and sequencing.

STOCHASTIC ANALYSIS OF SINGLE-MOLECULE DNA SYNTHETIC NETWORKS ROTAXANES Living cells use complex networks of evolutionarily We are interested in the study of matter at the level selected biomolecular interactions and chemical trans- of single molecules. For these studies we use the transformations to process multiple extracellular input signals CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 77 rapidly and simultaneously. We are interested in under- marginally prebiotic. We showed that carbonyl sulfide, standing and experimentally modeling the organiza- a simple gas present in the emissions from present-day tional and functional properties of biological networks. volcanoes, is a condensing agent that brings about the We have developed a general strategy for the design formation of peptides from amino acids under mild con- and construction of self-organized synthetic peptide ditions in aqueous solution (Fig. 5). We have studied the networks based on the sequence-selective autocatalytic and cross-catalytic template-directed coiled coil peptide fragment condensation reactions in aqueous solutions. The synthetic networks have some of the basic architectural and dynamic features of the living networks, reorganize in response to changes in environmental conditions and inputs (Fig. 4), and perform

Fig. 5. Peptide formation under plausibly prebiotic reaction conditions. Carbonyl sulfide, a volcanic gas, is the most simple and effective amino acid–condensing agent for the formation of peptides in aqueous solutions.

Fig. 4. Adaptive reorganization in a synthetic peptide network. carbonyl sulfide–mediated condensations of α-amino The graph structure or wiring of a synthetic peptide network acids under aerobic and anaerobic conditions in the responds dramatically to changes in the environmental stimuli (pH or salt content). absence of any added reagents and in the presence of metal ions, oxidizing agents, or alkylating agents. basic Boolean logic functions such as OR, NOR, and Depending on the reaction conditions and additives NOTIF logic. We suggest that the ability to rationally used, exposure of α-amino acids to carbonyl sulfide construct predictable chemical circuitry might be useful generates peptides in yields of up to 80% in minutes in advancing the modeling and better understanding to hours at room temperature. of some of the basic dynamic information-processing characteristics of the more complex cellular networks. PUBLICATIONS Askkenasy, N., Sánchez-Quesada, J., Bayley, H., Ghadiri, M.R. Recognizing a sin- PREBIOTIC CHEMISTRY gle base in an individual DNA strand: a step toward DNA sequencing in nanopores. In almost all discussions of prebiotic chemistry, it Angew. Chem. Int. Ed. 44:1401, 2005. is assumed that amino acids, nucleotides, and possibly Horne, S.W., Ashkenasy, N., Ghadiri, M.R. Modulating charge transfer through cyclic D,L-α-peptide self-assembly. Chemistry 11:1137, 2005. other monomers were first formed on Earth or brought to it in comets and meteorites and that the monomers Horne, S.W., Wiethoff, C.M., Cui, C., Wilcoxen, K.M., Amorin, M., Ghadiri, M.R., Nemerow, G.R. Antiviral cyclic D,L-α-peptides: targeting a general biochemical subsequently condensed nonenzymatically to form pathway in viral infections. Bioorg. Med. Chem. 13:5145, 2005. oligomeric products. Unfortunately, attempts to create Yadav, M.K., Redman, J.E., Leman, L.J., Alvarez-Gutiérrez, J.M., Zhang, Y., plausibly prebiotic polymerization reactions have met Stout, C.D., Ghadiri, M.R. Structure-based engineering of internal cavities in with limited success. Direct heating of solid mixtures coiled-coil peptides. Biochemistry 44:9723, 2005. leads to nonspecific products, and the condensing agents that have been studied, with the possible exception of inorganic polyphosphates, are relatively inefficient and/or 78 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE A Merging of Chemistry and Biology

K.D. Janda, J. Ashley, C. Berndt, G. Boldt, A. Brogan, C. Chung, S. De Lamo Marin, T. Dickerson, L. Eubanks, M. Hixon, A. Ino, G. Kaufmann, J. Kennedy, Y. Kim, J. Liu, Y. Liu, C. Lowery, H. Ma, S. Mahajan, L. McAllister, G. McElhaney, K. McKenzie, J. Mee, M. Meijler, J. Park, S. Steiniger, J. Treweek, A. Willis, Y. Xu, B. Zhou, H. Zhou

uring the past year, we explored various applications of organic chemistry at the interface of D chemistry and biology. Representative examples of our results were obtained in 3 research programs: Fig. 1. Mechanism of nornicotine-catalyzed Z-to-E isomerization. catalysis of retinal isomerization by the nicotine metabolite nornicotine, “superactivation” of botulinum neu- mechanism for the pathologic changes in key smok- rotoxin by small molecules, and the development of a ing-related diseases, because the accumulation of cocaine esterase–bacteriophage construct with suitable compounds such as all-E-retinal feeds the N-retinyli- kinetics for the degradation of cocaine in humans. dene-N-retinylethanolamine biosynthetic pathway,

ALTERED RETINOID HOMEOSTASIS CATALYZED BY forming an undigestible byproduct of the visual cycle

A NICOTINE METABOLITE and a fluorescent chromophore characteristic of the In recent years, we have been studying the role of pathologic changes in age-related macular degenera- long-lived drugs of abuse and their metabolites in drug- tion, a leading cause of blindness. Smoking is accepted related diseases. Much of this effort has centered on as the primary environmental factor contributing to age- the Maillard reaction, a process by which amine-con- related macular degeneration, and thus elucidating the taining molecules irreversibly react with proteins through molecular mechanism of this contribution is clinidand the intermediacy of glucose, the dominant serum mono- retinal compounds, we conclusively showed that nor- saccharide. The mechanism of the Maillard reaction nicotine can indeed catalyze the Z-to-E isomerization parallels that of amine organocatalysts; iminium and/or of unsaturated compounds at rates that could have enamine intermediates are necessary for rate enhance- biological significance in the context of disease. ment. In the past year, we expanded our studies beyond SUPERACTIVATION OF BOTULINUM NEUROTOXIN the Maillard reaction to other biological processes in SEROTYPE A LIGHT-CHAIN METALLOPROTEASE which iminium ion intermediates are critical. The 7 neurotoxins (A–G) of the bacterium Clostrid- Retinoids (vitamin A) play 2 major roles in higher ium botulinum are the most lethal poisons known. animals: light absorption in vision and gene regulation Exposure to these toxins leads to progressive flaccid in growth and development. Specifically, these processes paralysis resulting from cleavage of proteins critical are regulated by the conformation of the double bonds for proper release of neurotransmitters from peripheral in the polyunsaturated hydrocarbon chain. For example, nerve cells. Despite their potent toxicity, botulinum in the visual cycle, 11-Z-retinal is converted to all-E- neurotoxins are widely used in medicine, as well as retinal by a photon of light, ultimately leading to the cosmetically for treating facial wrinkles. Conditions perception of vision. Much of the biosynthetic path- including multiple sclerosis, stroke, cerebral palsy, ways leading up to this reaction are controlled by the migraine, and backache can all be treated with the formation of iminium ions between the retinal terminal neurotoxins. Yet, repeated exposure to the toxins can aldehyde group and a lysine side chain from an appro- result in the development of a marked immune response priate enzyme. to them, thereby compromising their efficacy. Tolerance We hypothesized that nornicotine, a metabolite of develops most rapidly when patients are treated fre- nicotine, could also perform this type of chemistry quently with high doses of the toxins. We speculated (Fig. 1) and thus alter the concentrations of retinal inter- that the coadministration of a botulinum neurotoxin mediates. This reaction would provide an intriguing with a molecule that can “activate” the catalytic activ- CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 79 ity of the toxin would lead to lower doses, thus reduc- the importance of botulinum neurotoxins continues to ing the unintended immune response. expand, methods such as this may ultimately provide In recent investigations of light-chain metallopro- a method for minimizing dosage of the toxins and tease inhibitors of botulinum neurotoxin A, we discov- thereby increase the clinical efficacy of the molecules. ered that the molecule arginine hydroxamic acid is a DEGRADING COCAINE WITH VIRUSES modest inhibitor. Using this compound as a guide, we Cocaine is a powerful stimulant and among the most prepared a small collection of compounds containing a reinforcing of all drugs. Consequently, abuse of cocaine zinc-binding motif (2-acylthiophene) combined with an continues to be a major problem. Cocaine acts as an arginine-side-chain mimetic (acylguanidine). To our sur- indirect dopamine agonist by blocking the dopamine prise, although no inhibition occurred, one compound transporter in the pleasure-reward center of the brain. (compound 1 in Fig. 2) consistently produced a 2-fold This obstruction leads to an excess of dopamine in the synapses, amplifying the sensation of pleasure. Despite intensive efforts, no effective pharmacotherapy for cocaine abuse exists. The inherent difficulties in antago- nizing a blocker have led to the development of protein- based therapeutics designed to treat cocaine abuse. In an approach termed immunopharmacotherapy, we have devoted extensive efforts to the use of antibodies to cocaine that can sequester cocaine, retarding its ability to enter the CNS. We have also developed a parallel strategy that involves use of catalytic antibodies specific for the hydrolysis of the benzoyl ester of cocaine to give the nonpsychoactive products benzoate and methyl ecgonine (Fig. 3).

Fig. 2. Chemical structures of molecules that can superactivate botulinum neurotoxin serotype A. The specific motifs used in the design of these compounds are highlighted. enhancement of activity. Further structure-activity relationship studies revealed that specific features of this compound were critical for activation, such as the thio- phene sulfur atom and the acylguanidine group. When these initial screening efforts were completed, compound 2 (Fig. 2) was the most potent activator. Because of the clinical promise of an activator of Fig. 3. Hydrolysis products resulting from cleavage of cocaine botulinum neurotoxin, we further examined the mecha- esters. Both the uncatalyzed reaction (path a) and the cocaine nism of this phenomenon. Extensive kinetic characteriza- esterase–catalyzed hydrolysis (path b) pathways are shown. tion indicated that these compounds operate primarily by reducing the Michaelis constant (Km), not by alter- Although the potential of this method has been ing the turnover number (kcat). In this context, com- demonstrated in rodent models of cocaine overdose pound 2 is the most potent small-molecule activator and reinforcement, the kinetic constants of these anti- of a protease reported to date, with up to 14-fold rate bodies must be improved before the method will be enhancement at limiting concentrations of substrate. practical as a clinical treatment. Furthermore, these Indeed, as little as 2-fold enzyme activation has previ- approaches are only effective in the periphery, whereas ously been reported as a state of superactivation. a pharmacotherapy that could act in both the CNS and In total, the activation profile and structure-activity the periphery is desirable. relationship for activation suggests the presence of a Bacteriophages are viruses that infect bacteria yet specific “activation domain” on the enzyme. Because lack intrinsic tropism for eukaryotic cells. Because of 80 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

the genetic flexibility of bacteriophages, a wide range Brogan, A.P., Dickerson, T.J., Boldt, G.E., Janda, K.D. Altered retinoid homeostasis catalyzed by a nicotine metabolite: implications in macular degeneration and of proteins and peptides can be expressed on the phage normal development. Proc. Natl. Acad. Sci. U. S. A. 102:10433, 2005. coat in an approach termed phage display. Furthermore, Carrera, M.R.A., Trigo, J.M., Wirsching, P., Roberts, A.J., Janda, K.D. Evaluation phage molecules can penetrate virtually all tissues, of the anticocaine monoclonal antibody GNC92H2 as an immunotherapy for including the CNS. cocaine overdose. Pharmacol. Biochem. Behav. 81:709, 2005.

We recently reported that cocaine-binding antibod- Dickerson, T.J., Beuscher, A.E. IV, Rogers, C.J., Hixon, M.S., Yamamoto, N., Xu, Y., ies displayed on the surface of bacteriophages can be Olson, A.J., Janda, K.D. Discovery of acetylcholinesterase peripheral anionic site ligands through computational refinement of a directed library. Biochemistry administered intranasally and that the treated animals 44:14845, 2005. are protected from the locomotor stimulation associated Dickerson, T.J., Janda, K.D. Recent advances for the treatment of cocaine abuse: with exposure to cocaine. However, because of the req- central nervous system immunopharmacotherapy. AAPS J. 7:E579, 2005. uisite 1:1 stoichiometry of any traditional antibody phar- Eubanks, L.M., Dickerson, T.J., Janda, K.D. Vitamin B2-mediated cellular photoin- macotherapy, obtaining a meaningful concentration of hibition of botulinum neurotoxin A. FEBS Lett. 579:5361, 2005. the therapeutic agent in vivo is difficult. We envisioned Kaufmann, G.F., Sartorio, R., Lee, S.H., Mee, J.M., Altobell, L.J. III, Kujawa, that this limitation could be overcome by displaying a D.P., Jeffries, E., Clapham, B., Meijler, M.M., Janda, K.D. Antibody interference catalyst on the phage surface that can degrade cocaine, with N-acyl homoserine lactone-mediated bacterial quorum sensing. J. Am. Chem. Soc. 128:2802, 2006. yielding a therapeutically practical approach for treating cocaine abuse. Kim, Y., Lillo, A., Moss, J.A., Janda, K.D. A contiguous stretch of methionine residues mediates the energy-dependent internalization mechanism of a cell-penetrat- For these studies, we used cocaine esterase, a ing peptide. Mol. Pharm. 2:528, 2005. globular bacterial enzyme that is the most efficient Lee, B.S., Mahajan, S., Janda, K.D. Asymmetric dihydroxylation catalyzed by ionic protein catalyst for cocaine hydrolysis reported to polymer-supported osmium tetroxide. Tetrahedron Lett. 46:4491, 2005. date. We displayed this enzyme on the phage coat Lee, B.S., Mahajan, S., Janda, K.D. Molecular iodine-catalyzed imine activation and then used high-performance liquid chromatogra- for three-component nucleophilic addition reactions. Synlett 1325, 2005, Issue 8. phy to determine the kinetic parameters. We found Lillo, A.M., McKenzie, K.M., Janda, K.D. Phage-displayed antibody libraries. In: that the catalytic efficiency of cocaine esterase–phage Cell Biology: A Laboratory Handbook, 3rd ed. Celis, J., et al. (Eds.). Academic constructs was reduced relative to the efficiency of the Press, San Diego, 2006, p. 491. native enzyme yet exceeded the postulated therapeuti- Ma, H., Zhou, B., Kim, Y., Janda, K.D. A cyclic peptide-polymer probe for the cally relevant threshold. No reported catalytic anti- detection of Clostridium botulinum neurotoxin serotype A. Toxicon 47:401, 2006. body capable of cocaine hydrolysis achieves this value, Matsushita, M., Meijler, M.M., Wirsching, P., Lerner, R.A., Janda, K.D. A blue flu- and indeed, only recently described “designer” mutants orescent antibody-cofactor sensor for mercury. Org. Lett. 7:4943, 2005. of the enzyme butyrylcholinesterase are comparable to McAllister, L.A., Hixon, M.S., Kennedy, J.P., Dickerson, T.J., Janda, K.D. Superac- tivation of the botulinum neurotoxin serotype A light chain metalloprotease: a new our cocaine esterase–phage constructs. wrinkle in botulinum neurotoxin. J. Am. Chem. Soc. 128:4176, 2006. These results indicate that phage display of clini- McKenzie, K.M., Meijler, M.M., Lowery, C.A., Boldt, G.E., Janda, K.D. A fura- cally relevant enzymes can be achieved without com- nosyl-carbonate autoinducer in cell-to-cell communication of V. harveyi. Chem. promising the catalytic efficacy of the desired enzyme. Commun. (Camb.) 4863, 2005, Issue 38.

We envision that this new technology will stimulate Moss, J.A, Stokols, S., Hixon, M.S., Ashley, F.T., Chang, J.Y., Janda, K.D. Solid- further development of other protein-based treatments phase synthesis and kinetic characterization of fluorogenic enzyme-degradable hydrogel cross-linkers. Biomacromolecules 7:1011, 2006. for CNS-related disorders and will lead to powerful tools to combat drug abuse. Qi, L., Yamamoto, N., Meijler, M.M., Altobell, L.J. III, Koob, G.F., Wirsching, P., Janda, K.D. ∆9-Tetrahydrocannabinol immunochemical studies: haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled ∆9-tetrahydrocannabi- PUBLICATIONS nol. J. Med. Chem. 48:7389, 2005. Boldt, G.E., Dickerson, T.J., Janda, K.D. Emerging chemical and biological approaches for the preparation of discovery libraries. Drug Discov. Today 11:143, 2006. Rogers, C.J., Dickerson, T.J., Janda, K.D. Kinetic isotope and thermodynamic analysis of the nornicotine-catalyzed aqueous aldol reaction. Tetrahedron 62:352, 2006. Boldt, G.E., Eubanks, L.M., Janda, K.D. Identification of a botulinum neurotoxin A protease inhibitor displaying efficacy in a cellular model. Chem. Commun. (Camb.) Rogers, C.J., Dickerson, T.J., Wentworth, P., Jr., Janda, K.D. 3063, 2006, Issue 29. A high-swelling reagent scaffold suitable for use in aqueous and organic solvents. Tetrahedron Boldt, G.E., Kennedy, J.P., Hixon, M.S., McAllister, L.A., Barbieri, J.T., Tzipori, 61:12140, 2005. S., Janda, K.D. Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin A inhibitors. J. Comb. Rogers, C.J., Mee, J.M., Kaufmann, G.F., Dickerson, T.J., Janda, K.D. Toward Chem. 8:513, 2006. cocaine esterase therapeutics J. Am. Chem. Soc. 127:10016, 2005.

Boldt, G.E., Kennedy, J.P., Janda, K.D. Identification of a potent botulinum neuro- Shimomura, O., Lee, B.S., Meth, S., Suzuki, H., Mahajan, S., Nomura, R., toxin A protease inhibitor using in situ lead identification chemistry. Org. Lett. Janda, K.D. Synthesis and application of polytetrahydrofuran-grafted polystyrene 8:1729, 2006. (PS-PTHF) resin supports for organic synthesis. Tetrahedron 61:12160, 2005. CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 81

Shute, T.S., Matsushita, M., Dickerson, T.J., La Clair, J.J., Janda, K.D., Burkart, roxine in the blood and cerebrospinal fluid. More than M.D. A site-specific bifunctional protein labeling system for affinity and fluorescent analysis. Bioconjug. Chem. 16:1352, 2005. 99.5% of the transthyretin binding sites for L-thyrox- ine remain unoccupied in blood, and only about 50% Toker, J.D., Tremblay, M.R., Yli-Kauhaluoma, J., Wentworth, A.D., Zhou, B., Wentworth, P., Jr., Janda, K.D. Exploring the scope of the 29G12 antibody cat- of transthyretin tetramers in plasma are bound to a alyzed 1,3-dipolar cycloaddition reaction. J. Org. Chem. 70:7810, 2005. single holo-retinol binding protein. As a consequence Wu, W., Luo, Y., Sun, C., Liu, Y., Kuo, P., Varga, J., Xiang, R., Reisfeld, R., of a mutation or denaturation stress associated with Janda, K.D., Edgington, T.S., Liu, C. Targeting cell-impermeable prodrug activation to tumor microenvironment eradicates multiple drug-resistant neoplasms. Cancer aging and/or oxidative stress, dissociation of the native Res. 66:970, 2006. transthyretin tetramer, the rate-limiting step for amy-

Xu, Y., Shi, J., Yamamoto, N., Moss, J.A., Vogt, P.K., Janda, K.D. A credit-card loidogenesis, followed by changes in the tertiary struc- library approach for disrupting protein-protein interactions. Bioorg. Med. Chem. ture of the monomer make the monomeric subunits 14:2660, 2006. competent to misassemble into aggregates, including Xu, Y., Yamamoto, N., Ruiz, D.I., Kubitz, D.S., Janda, K.D. Squaric monoamide amyloid fibrils. The deposition of transthyretin amyloid monoester as a new class of reactive immunization hapten for catalytic antibodies Bioorg. Med. Chem. Lett. 15:4304, 2005. is linked with a number of human diseases, including senile systemic amyloidosis, familial amyloid polyneu- Yamashita, M., Lee, S.-H., Koch, G., Zimmermann, J., Clapham, B., Janda, K.D. Solid-phase synthesis of oxazolones and other heterocycles via Wang resin-bound ropathy (FAP), familial amyloid cardiomyopathy, and diazocarbonyls. Tetrahedron Lett. 46:5495, 2005. CNS-selective amyloidosis. Yao, Y., Martinez-Yamout, M., Dickerson, T.J., Brogan, A.P., Wright, P.E., Dyson, A suitable strategy to slow or prevent the formation H.J. Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones. J. Mol. Biol. 355:262, 2006. of aggregates is to inhibit the rate-limiting dissociation of the transthyretin tetramer by making the native state Zhang, L., Long, H., Boldt, G.E., Janda, K.D., Schatz, G.C., Lewis, F.D. α- and β-Stilbenosides as base-pair surrogates in DNA hairpins. Org. Biomol. Chem. more stable than the dissociative transition state. We 4:314, 2006. have designed, synthesized, and characterized several

Zhu, X., Dickerson, T.J., Rogers, C.J., Kaufmann, G.F., Mee, J.M., McKenzie, classes of structurally distinct small molecules that bind K.M., Janda, K.D., Wilson, I.A. Complete reaction cycle of a cocaine catalytic anti- to and stabilize the transthyretin tetramer. One of these body at atomic resolution. Structure 14:205, 2006. molecules is being tested in phase 2/3 clinical trials for treatment of FAP. Compounds discovered in high- Strategies to Ameliorate Protein throughput screening tests, such as genistein, a natural compound present in soy products, also inhibit Misfolding Diseases formation of fibrils of wild-type amyloid, as well as amyloidogenesis by the transthyretin variants V30M and J.W. Kelly, J. Bieschke, D.A. Bosco, E. Culyba, M.T.A. Dendle, V122I, 2 of the most common disease-associated vari- W. D’Haeze, T.R. Foss, D.M. Fowler, Y. Fu, J. Gao, M.-Y. Gao, ants. Furthermore, a clinical study in healthy human S.M. Johnson, T. Mu, E.T. Powers, A.R. Sawkar, L. Segatori, subjects indicated that kinetic stabilization of trans- S. Siegel, J.Y. Suk, R.L. Wiseman, I. Yonemoto, Z. Yu thyretin mediated by orally administered diflunisal, a ur goal is to better understand the molecular nonsteroidal anti-inflammatory agent also discovered mechanisms that lead to protein misfolding by screening, should ameliorate transthyretin amyloido- O diseases, including Alzheimer’s, Parkinson’s, sis. The effect of diflunisal on FAP is currently being and Gaucher diseases, so that we can design new strate- evaluated in a phase 3 clinical study. gies to ameliorate such maladies. To accomplish this Most likely the age-associated nature of neurodegen- goal, we use organismal and cell biological disease erative diseases such as the transthyretin amyloidoses models and spectroscopic and biophysical approaches can be explained by a shift from efficient to inefficient in combination with chemical synthesis and molecular aggregate clearance, leading to increasing concentrations biology techniques. Successful collaborations with of the aggregates and proteotoxic effects. We showed W.E. Balch, Department of Cell Biology, P. Wentworth, that the reassembly of transthyretin homotetramers Jr., Department of Chemistry, and A. Dillin, the Salk occurs via a monomer-dimer-trimer-tetramer pathway Institute for Biological Studies, La Jolla, California, are in which each step depends on the concentration of critical to achieve our goals. folded transthyretin monomers. This finding suggests TRANSTHYRETIN AMYLOIDOGENESIS that partitioning of transthyretin monomers between Transthyretin is a 55-kD homotetrameric protein transthyretin tetramer reassembly and the aggregation that transports holo-retinol binding protein and L-thy- pathway is correlated with the relative concentrations 82 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE of reassembly intermediates and aggregates. The con- tion occurs predominantly via a noncovalent mecha- centration of the aggregates presumably increases with nism. Overexpression of α-synuclein may lead to the aging because of inefficient aggregate clearance. production of reactive oxygen species, which stimulates In another study, we found that subunits of the the production of oxidative cholesterol metabolites that transthyretin variant R104H most likely act as an in then accelerate α-synuclein aggregation. This acceler- vivo trans-suppressor of amyloidogenesis associated ation may enhance local oxidative stress, resulting in with the V30M variant. Compound heterozygotes with a vicious cycle that eventually leads or contributes to genes for the V30M and R104H variants did not show α-synucleinopathies. signs of the pathologic changes associated with FAP Although the exact role of α-synuclein in Lewy body typical of heterozygotes with genes for the V30M vari- disease and Parkinson’s disease remains to be eluci- ant and wild-type transthyretin; however, compound dated, our findings add to an understanding of how heterozygotes with genes for R104H and the aggres- aldehyde-based organic compounds formed as a result sive mutation T59K on their second allele did have of aging and inflammation may contribute to neurode- pathologic FAP effects similar to those of compound generative diseases. heterozygotes with genes for wild-type transthyretin GELSOLIN AMYLOIDOSIS and the T59K mutation. Gelsolin amyloidogenesis occurs in persons who pro- We investigated the energetics of R104H homote- duce D187N/Y plasma gelsolin variants. This disease tramers and mixed tetramers in a fashion analogous to is characterized by amyloid deposits composed of 5- that used to elucidate the mechanism of T119M trans- and 8-kD fragments of plasma gelsolin. The D187N/Y thyretin interallelic trans-suppression. We found that mutation abrogates calcium binding in domain 2, allow- in contrast to T119M, R104H does not suppress aggre- ing aberrant furin cleavage in the Golgi apparatus during gation by a kinetic stabilization mechanism. We showed trafficking and yielding a 68-kD fragment. The fragment that R104H may trans-suppress transthyretin aggrega- is then cleaved by the membrane type matrix metallo- tion by subtle thermodynamic stabilization of the trans- proteinase 1 (MT1-MMP), resulting in 5- and 8-kD thyretin quaternary structure. This discovery suggests fragments that are deposited as amyloid fibrils in the that R104H could protect compound heterozygotes extracellular matrix. Fibroblasts from animals lacking from transthyretin aggregation in situations in which the gene for MT1-MMP are incapable of generating 8- the mutation is mildly destabilizing. This finding sup- and 5-kD fragments from the 68-kD gelsolin fragment. ports the current clinical data associated with R104H Biophysical studies indicated that gelsolin amyloid in compound heterozygotes. formation is substantially accelerated in the presence ABERRANT OXIDATIVE METABOLITES AFFECT of the extracellular matrix component heparin. The α -SYNUCLEINOPATHIES extent of sulfation and the location and relative orien- The α-synucleinopathies are characterized by cyto- tation of sulfate residues and the molecular weight are plasmic α-synuclein-rich aggregates within degenerating important factors in the heparin-mediated acceleration dopaminergic neurons in the substantia nigra. Clinical of gelsolin amyloidogenesis, possibly explaining the heavy observations suggest a correlation between oxidative deposition of gelsolin amyloid in the extracellular matrix. stress/inflammation and protein misfolding diseases. Most likely tissue-selective deposition of gelsolin amy- We wished to determine whether oxidized metabolites loid is correlated with the localization of extracellular accelerate the aggregation of α-synuclein, research that sulfated glycosaminoglycans, a notion supported by the would shed light on the correlation between oxidative colocalization of glycosaminoglycans with gelsolin amy- stress and sporadic α-synucleinopathies. loid in the extracellular space of gelsolin amyloidosis We found that overexpression of α-synuclein in a transgenic mice. These transgenic mice will be used neuronal cell line is sufficient to increase the produc- to evaluate the effect of MT1-MMP inhibitors and gly- tion of oxidative metabolites derived from the oxidation cosaminoglycan amyloid antagonists on gelsolin amyloi- of cholesterol, presumably due to the production of dosis in vivo. reactive oxygen species. We showed that these oxida- CHEMICAL CHAPERONES AND GAUCHER DISEASE tive metabolites are cytotoxic and that they significantly Mutations in glucocerebrosidase, a lysosomal hydro- accelerate aggregation of α-synuclein in vitro. The exper- lase, lead to an accumulation of glucosylceramide in the imental data suggest that the acceleration in aggrega- lysosome, causing Gaucher disease, the most common CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 83

lysosomal storage disorder. Previously, we showed that Powers, E.T., Deechongkit, S., Kelly, J.W. Backbone-backbone H-bonds make context-dependent contributions to protein folding kinetics and thermodynamics: N-(n-nonyl)deoxynojirimycin increases the activity of the lessons from amide-to-ester mutations. Adv. Protein Chem. 72:39, 2005. glucocerebrosidase variant N370S in a cell line derived Premkumar, L., Sawkar, A.R., Boldin-Adamsky, S., Toker, L., Silman, I., Kelly, from tissue from a patient with Gaucher disease. This J.W., Futerman, A.H., Sussman, J.L. X-ray structure of human acid-β-glucosidase “chemical chaperoning” effect most likely is due to the covalently bound to conduritol-B-epoxide: implications for Gaucher disease. J. Biol. Chem. 280:23815, 2005. binding of N-(n-nonyl)deoxynojirimycin to the native state Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.-C., Wong, C.-H., Beutler, E., of N370S, allowing the glucocerebrosidase to be traf- Zimmer, K.-P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show ficked from the endoplasmic reticulum to the lysosomes. mutation-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005.

Interestingly, the activity of G202R glucocerebrosidase, Sawkar, A.R., D’Haeze, W., Kelly, J.W. Therapeutic strategies to ameliorate lysosomal a variant retained in the endoplasmic reticulum, is also storage disorders: a focus on Gaucher disease. Cell. Mol. Life Sci. 63:1179, 2006.

increased in the presence of chemical chaperones, sug- Sekijima, Y., Dendle, M.T., Wiseman, R.L., White, J.T., D’Haeze, W., Kelly, J.W. gesting that those chemical chaperones stimulated R104H may suppress transthyretin amyloidogenesis by thermodynamic stabilization, but not by the kinetic mechanism characterizing T119 trans-suppression. Amyloid transport to the lysosomes. 13:57, 2006. Our results indicate that some chemical chaper- Sörgjerd, K., Ghafouri, B., Jonsson, B.-H., Kelly, J.W., Blond, S.Y., Hammarström, P. ones enhance the activity of distinct glucocerebrosi- Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates dase variants to an extent thought to be sufficient to amyloidogensis. J. Mol. Biol. 356:469, 2006. ameliorate Gaucher disease. Preliminary data suggest Suk, J.Y., Zhang, F., Balch, W.E., Linhardt, R.J., Kelly, J.W. Heparin accelerates gelsolin amyloidogenesis. Biochemistry 45:2234, 2006. that certain glucocerebrosidase mutants most likely will need specifically designed chemical chaperones Wiseman, R.L., Powers, E.T., Kelly, J.W. Partitioning conformational intermediates between competing refolding and aggregation pathways: insights into transthyretin that target the compromised domain in order to facili- amyloid disease. Biochemistry 44:16612, 2005. tate proper trafficking and partial restoration of the function of the glucocerebrosidase. Total Synthesis, New PUBLICATIONS Bosco, D.A., Fowler, D.M., Zhang, Q., Nieva, J., Powers, E.T., Wentworth, P., Jr., Lerner, R.A., Kelly, J.W. Elevated levels of oxidized cholesterol metabolites in Lewy Synthetic Technologies, body disease brains accelerate α-synuclein fibrilization [published correction appears in Nat. Chem. Biol. 2:346, 2006]. Nat. Chem. Biol. 2:249, 2006. and Chemical Biology Deechongkit, S., Nguyen, H., Jäger, M., Powers, E.T., Gruebele, M., Kelly, J.W. β-Sheet folding mechanisms from perturbation energetics. Curr. Opin. Struct. Biol. K.C. Nicolaou, S. Arseniyadis, W. Brenzovich, P. Bulger, 16:94, 2006. A. Burtoloso, J. Chen, K. Cole, A. Converso, J. Crawford, Foss, T.R., Wiseman, R.L., Kelly, J.W. The pathway by which the tetrameric pro- P. Dagneau, R. Denton, A. Estrada, D. Edmonds, C. Fang, tein transthyretin dissociates. Biochemistry 44:15525, 2005. R. Faraoni, M. Frederick, M. Freestone, R. Gibe, S. Harrison, Fowler, D.M., Koulov, A.V., Alory-Jost, C., Marks, M.S., Balch, W.E., Kelly, J.W. V. Jeso, A. Johnson, D. Kim, A. Kislukhin, A. Lanver, K. Lee, Functional amyloid formation within mammalian tissue. PLoS Biol. 4:e6, 2006. S. Lee, A. Lemire, A. Lenzen, A. Li, H. Li, Y. Lim, T. Lister, Fu, Y., Bieschke, J., Kelly, J.W. E-Olefin dipeptide isostere incorporation into a D. Lizos, E. Loizidou, N. Mainolfi, C. Mathison, X. Mico-Alvarez, polypeptide backbone enables hydrogen bond perturbation: probing the requirements for Alzheimer’s amyloidogenesis. J. Am. Chem. Soc. 127:15366, 2005. R. Milburn, A. Nold, A. Noncovich, R. de Noronha, A. Ortiz, C. Papageorgiou, D. Pappo, L. Pasunoori, G. Petrovic, Green, N.S., Foss, T.R., Kelly, J.W. Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis. Proc. Natl. Acad. Sci. U. S. A. J. Piper, D. Polet, G. Pontremoli, B. Pratt, D. Sarlah, 102:14545, 2005. D. Shaw, C. Stathakis, C. Solorio-Alvarado, T. Suzuki,

Johnson, S.M., Wiseman, R.L., Sekijima, Y., Green, N.S., Adamski-Werner, S.L., G. Tria, C. Turner, T. Umezawa, J. Wang, H. Xu, M. Zak Kelly, J.W. Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses. Acc. Chem. Res. e focus on the total synthesis of natural prod- 38:911, 2005. ucts, the discovery and development of new Kelly, J.W., Balch, W.E. The integration of cell and chemical biology in protein W synthetic technologies, and chemical biology. folding. Nat. Chem. Biol. 2:224, 2006. Naturally occurring substances are selected as synthetic Nguyen, H., Jäger, M., Kelly, J.W., Gruebele, M. Engineering a β-sheet protein targets because of their novel molecular architectures, toward the folding speed limit. J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 109:15182, 2005. important biological properties, and interesting mechanisms of action. The projects are designed to optimize Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.-J., Venable, J., Yates, J. III, Kelly, J.W., Balch, W.E. Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. the opportunities for discovery and invention in the areas EMBO J. 24:4124, 2005. of chemistry, biology, and medicine. The drug pacli- 84 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

taxel (Taxol), the antitumor epothilones, the neurotoxins Nicolaou, K.C. Joys of molecules, 1: campaigns in total synthesis. J. Org. Chem. 70:7007, 2005. brevetoxins A and B, the antibiotic vancomycin, the cholesterol-lowering CP-molecules, the antibiotic everni- Nicolaou, K.C. Joys of molecules, 2: endeavors in chemical biology and medicinal chemistry. J. Med. Chem. 48:5613, 2005. nomicin, the TNF α–associated trichodimerol, the Nicolaou, K.C., Brenzovich, W.E., Bulger, P.G., Francis, T.M. Synthesis of iso- tetrahydropyran class of natural products, apoptolidin, epoxy-amphidinolide N and des-epoxy-caribenolide I structures: initial forays. Org. diazonamide A, thiostrepton, and the azaspiracids exem- Biomol. Chem. 4:2119, 2006

plify this philosophy. Current projects include studies Nicolaou, K.C., Bulger, P.G., Brenzovich, W.E. Synthesis of iso-epoxy-amphidino- of the antibiotics nocathiacin and abyssomicin C, the lide N and des-epoxy-caribenolide I structures: revised strategy and final stages. Org. Biomol. Chem. 4:2158, 2006. antifeedant azadirachtin, and the antitumor agents Nicolaou, K.C., Chen, D.Y.-K., Li, Y., Uesaka, N., Petrovic, G., Koftis, T.V., marinomycin and A lomaiviticin B (Fig. 1). Bernal, F., Frederick, M.O., Govindasamy, M., Ling, T., Pihko, P.M., Tang, W., Vyskocil, S. Total synthesis and structural elucidation of azaspiracid-1: synthesis- based analysis of originally proposed structures and indication of their non-identity to the natural product. J. Am. Chem. Soc. 128:2258, 2006.

Nicolaou, K.C., Denton, R.M., Lenzen, A., Li, A., Edmonds, D.J., Milburn, R.R., Harrison, S.T. Stereocontrolled synthesis of model core systems of lomaiviticins A and B. Angew. Chem. Int. Ed. 45:2076, 2006.

Nicolaou, K.C., Frederick, M.O., Loizidou, E.Z., Petrovic, G., Cole, K.P., Koftis, T.V., Yamada, Y.M.A. Second-generation total synthesis of azaspiracids-1, -2, and - 3. Chem. Asian J. 1:245, 2006.

Nicolaou, K.C., Frederick, M.O., Petrovic, G., Cole, K.P., Loizidou, E. Total synthesis and confirmation of the revised structures of azaspiracid-2 and azaspiracid- 3. Angew. Chem. Int. Ed. 45:2609, 2006.

Nicolaou, K.C., Harrison, S.T. Total synthesis of abyssomicin C and atrop-abyssomicin C. Angew. Chem. Int. Ed. 45:3256, 2006.

Nicolaou, K.C., Kim, D.W., Schlawe, D., Lizos, D.E., de Noronha, R.G., Longbot- tom, D.A. Total synthesis of halipeptins A and D and analogues. Angew. Chem. Int. Ed. 44:4925, 2005.

Nicolaou, K.C., Koftis, T.V., Vyskocil, S., Petrovic, G., Tang, W., Frederick, M.O., Chen, D.Y.-K., Li, Y., Ling, T., Yamada, Y.M.A. Total synthesis and structural elucidation of azaspiracid-1: final assignment and total synthesis of the correct structure of azaspiracid-1. J. Am. Chem. Soc. 128:2859, 2006.

Nicolaou, K.C., Lim, Y.H., Papageorgiou, C.D., Piper, J.L. Total synthesis of (+)- rugulosin and (+)-2,2′-epi-cytoskyrin A through cascade reactions. Angew. Chem. Int. Ed. 44:7917, 2005.

Nicolaou, K.C., Lizos, D.E., Kim, D.W., Schlawe, D., de Noronha, R.G., Longbot- tom, D.A., Rodriquez, M., Bucci, M., Cirino, G. Total synthesis and biological evaluation of halipeptins A and D and analogues. J. Am. Chem. Soc. 128:4460, 2006.

Nicolaou, K.C., Mathison, C.J.N. Synthesis of imides, N-acyl vinylogous carbamates and ureas, and nitriles with Dess-Martin periodinane. Angew. Chem. Int. Ed. 44:5992, 2005.

Nicolaou, K.C., Papageorgiou, C.D., Piper, J.L., Chadha, R.K. The cytoskyrin cascade: a facile entry into cytoskyrin A, deoxyrubroskyrin, rugulin, skyrin and flavoskyrin model systems. Angew. Chem. Int. Ed. 44:5846, 2005.

Fig. 1. Selected target molecules. Nicolaou, K.C., Pihko, P.M., Bernal, F., Frederick, M.O., Qian, W., Uesaka, N., Diedrichs, N., Hinrichs, J., Koftis, T.V., Loizidou, E., Petrovic, G., Rodriquez, M., In addition, we are developing synthetic technolo- Sarlah, D., Zou, N. Total synthesis and structural elucidation of azaspiracid-1: construction of key building blocks for originally proposed structure. J. Am. Chem. gies and strategies for chemical synthesis and chemi- Soc. 128:2244, 2006.

cal biology studies. Our overall aims are to advance Nicolaou, K.C., Pratt, B.A., Arseniyadis, S., Wartmann, M., O’Brate, A., Gian- the art and science of chemical synthesis and to develop nakakou, P. Molecular design and chemical synthesis of a highly potent epothilone. enabling technologies for biology and medicine while Chemmedchem 1:41, 2006. maximizing educational opportunities and training of Nicolaou, K.C., Safina, B.S., Zak, M., Lee, S.H., Nevalainen, M., Bella, M., Estrada, A.A., Funke, C., Zécri, F., Bulat, S. Total synthesis of thiostrepton: ret- young men and women in chemistry. rosynthetic analysis and construction of key building blocks. J. Am. Chem. Soc. 127:11159, 2005. PUBLICATIONS Ito, E., Frederick, M.O., Koftis, T.V., Tang, W., Petrovic, G., Ling, T., Nicolaou, Nicolaou, K.C., Schlawe, D., Kim, D.W., Longbottom, D.A., de Noronha, R.G., Lizos, K.C. Structure toxicity relationships of synthetic azaspiracid-1 and analogs in mice. D.E., Manan, R.R., Faulkner, D.J. Total synthesis of halipeptins: isolation of halipeptin Harmful Algae. 5:586, 2006. D and synthesis of oxazoline halipeptin analogues. Chemistry 11:6197, 2005. CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 85

Nicolaou, K.C., Xu, H. Total synthesis of floresolide B and ∆6,7-Z-floresolide B. DUAL OPIOID AGONISTS–CHOLECYSTOKININ Chem. Commun. (Camb.) 600, 2006, Issue 6. ANTAGONISTS FOR TREATMENT OF CHRONIC AND

Nicolaou, K.C., Zak, M., Rahimipour, S., Estrada, A.A., Lee, S.H., O’Brate, A., NEUROPATHIC PAIN Giannankakou, P., Ghadiri, M.R. Discovery of a biologically active thiostrepton Nociception, or the perception of pain, and its mod- fragment. J. Am. Chem. Soc. 127:15042, 2005. ulation depend on the interaction of many endogenous Nicolaou, K.C., Zak, M., Safina, B.S., Estrada, A.A., Lee, S.H., Nevalainen, M. Total synthesis of thiostrepton: assembly of key building blocks and completion of neurotransmitters in the spinal cord. The interaction the synthesis. J. Am. Chem. Soc. 127:11176, 2005. of endogenous peptides such as cholecystokinin with exogenously administered opioids markedly alters activity in acute and chronic pain states. Cholecystokinin Translational Chemistry antagonizes the analgesic effects of morphine, whereas cholecystokinin antagonists enhance them. The inter- and Medicine play between cholecystokinin antagonists and opiates may lead to the development of novel medications E. Roberts, Z.Y. Chen, O. Ghoneim, C. Martinez, S. Sinha, that are more effective and safer than currently used C. Zoni opioids alone. iscovery and development of new medicines Molecules with both opioid agonist and cholecys- require the integration of several scientific dis- tokinin antagonist properties would be useful in condi- D ciplines. For example, medicinal chemistry relies tions in which the effectiveness of opioids is reduced, on iterative in vitro and in vivo biological testing of mol- as in the development of tolerance to opiate pain reliev- ecules. These biological investigations, in turn, rely on ers in chronic pain associated with cancer. The mole- the design and synthesis of new molecules with thera- cules might also be useful in neuropathic pain conditions peutic potential to delineate and validate pathways in which opioids are ineffective. Thus, because of the for therapeutic intervention. The primary focus of our prevention (or reversal) of tolerance, the possibility of research is to identify potential new medicines for the physical dependence on opioids might be diminished or treatment of diseases that currently have inadequate inhibited. The advantages of developing a single com- or no current therapy. pound with dual opioid agonist–cholecystokinin antag- GALANIN LIGANDS FOR THE TREATMENT OF onist activity rather than a combination of an opioid NEUROLOGIC DISEASES agonist taken with a separate cholecystokinin antago- Epilepsy is a disease in which a hyperexcited state nist are clear. Development of a single compound of the CNS is caused by an imbalance between inhibi- involves only a single set of parameters, such as toxi- tory and excitatory neurotransmission. Current epilepsy cology, pharmacokinetics, and formulation, rather than 2 therapy focuses on modulating the classical neurotrans- independent and often unrelated sets of data. mitters glutamate and γ-aminobutyric acid. The neuro- In collaboration with F. Porreca and J. Lai, Univer- peptide galanin antagonizes excitatory glutaminergic sity of Arizona, Tucson, we are using a limited set of neurotransmission in the hippocampus, suggesting that galanin may have a role in seizure activity. Galanin molecular templates that have affinity across a wide and its receptors may be useful in developing novel range of type 1 G protein–coupled receptors. The 3 antidepressant pharmacotherapies. In a recent study cloned opiate receptors (µ, δ, and κ) and the cholecys- in humans with depression, intravenously administered tokinin 1/(A) and 2/(B) receptors are all members of galanin had a rapid and strong antidepressant effect. this subclass of G protein–coupled receptors. Ligands To identify new nonpeptidic ligands for galanin are known for both sets of receptors that contain the receptors, we are using a small set of known molecules diphenylmethyl moiety as a privileged or biased template. that can potently displace the peptide galanin from its Other critical elements for activity may be appended to binding site on the R3 receptor subtype. Selectivity and chemically “silent” sites (Fig. 1). potency can then be established by appending appro- NEUROPHARMACOLOGIC APPROACHES FOR priate accessory binding motifs selective for the desired PREVENTION AND TREATMENT OF AUTISM protein. This research is being done in collaboration Autism is a bioneurologic developmental disability with T. Bartfai, Molecular and Integrative Neurosciences that generally appears before the age of 3 years. This Department, and A.M. Mazarati, University of Califor- disability affects the normal development of the brain nia, Los Angeles. in the areas of social interaction, communication skills, 86 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Fig. 3. A, Known mixed vasopressin V1a/V2 receptor antago- Fig. 1. Proposed opioid agonist–cholecystokinin antagonist hybrids. nists. B, Known vasopressin V1a receptor antagonists. and cognitive function. The prevalence of autistic diseases has reached pandemic proportions. In the United States, autism occurs in an estimated 1 in 166 births, and roughly 1.5 million persons have some form of autism. This rate is increasing significantly and now surpasses that of all types of cancer combined. No drug is consistently effective in treating the signs and symptoms of autism. Vasopressin and oxytocin are Fig. 4. Known oxytocin receptor agonists. 2 nonapeptides (Fig. 2) secreted by the hypothalamus. and A. Roberts, Molecular and Integrative Neuro- sciences Department.

PUBLICATIONS Roberts, E., Sancon, J.P., Sweeney, J.B. A new class of ammonium ylid for [2,3]- sigmatropic rearrangement reactions: end-endo-spiro ylids. Org. Lett. 7:2075, 2005. Fig. 2. Structures of vasopressin and oxytocin. Workman, J.A., Garrido, N.P., Sancon, J., Roberts, E., Wessel, H.P., Sweeney, They differ in only 2 amino acids, and both exert their J.B. Asymmetiric [2,3]-rearrangement of glycine-derived allyl ammonium ylids. J. Am. Chem. Soc. 127:1066, 2005. effects at protein receptors that belong to the G protein–coupled receptor superfamily, namely vasopressin V1a, V1b, and V2 receptors and oxytocin receptors. Chemical, Biological, and When oxytocin and vasopressin exert their agonist effects at the oxytocin and vasopressin V1a receptors, Biophysical Approaches to respectively, in rodents, marked effects occur in the CNS. These effects include behaviors associated with autism, Understanding Evolution such as social behaviors (e.g., bonding, aggression); cognition (e.g., memory and active-passive avoidance); F.E. Romesberg, D.A. Bachovchin, J.K. Chin, R.T. Cirz, and repetitive, patterned movements (e.g., grooming M.E. Cremeens, C. Gil-Lamaignere, N. Gingles, D.A. Harris, or social interaction). Antagonists reverse or have no A.A. Henry, G.T. Hwang, A.M. Leconte, E.T. Lis, S. Matsuda, effect on the observed behaviors. E.L. Oakman, B.A. O’Neill, T.C. Roberts, L.B. Sagle, We are designing and developing small-molecule P.A. Smith, M.C. Thielges, P. Weinkam, W. Yu, oxytocin agonists that can penetrate the CNS and pre- J. Zimmermann viously unknown vasopressin V1a receptor agonists to he molecules of biology are unique because they establish the potential roles for these molecules in the have been evolved for function. We take a unique treatment of autism (Figs. 3 and 4). This research is T and multidisciplinary approach to understanding being done in collaboration with T. Bartfai, G.F. Koob, and manipulating these processes. CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 87

INCREASING THE CHEMICAL AND GENETIC malian cells will revolutionize our understanding of POTENTIAL OF DNA cancer and aging and identify drug targets whose inhi- Biological information storage is based on the nat- bition might actually inhibit these processes. ural genetic alphabet, composed of the 2 base pairs In bacteria, induced mutation can lead to antibi- guanine-cytosine and adenine-thymine. We are inter- otic resistance. We have fully characterized the mech- ested in increasing the information potential of DNA anism of induced mutation and antibiotic resistance in by expanding the genetic alphabet with a third base Escherichia coli (Fig. 2), and we are characterizing pair composed of unnatural nucleobases. Using hydrophobicity, polarity, shape complementarity, and hydrogen bonding, we are developing novel unnatural base pairs, including several that are replicable in vitro. Nature developed the natural genetic code, not only by optimizing DNA and RNA but also by evolving the polymerases that synthesize these nucleic acids. We developed an activity-based selection system (Fig. 1) to

Fig. 2. Induced mutation in E coli is controlled by the transcriptional regulator LexA. Duplex DNA is shown in black; each gray box indicates a double-strand break. The breaks can be repaired through pathway A, B, or C. Derepression of the error-prone polymerases Pol IV and Pol V leads to mutations (pathway D).

these pathways in other bacterial pathogens. We are also designing a drug that inhibits bacterial mutation and thus evolution.

Fig. 1. Activity-based phage display selection system for evolv- EVOLUTION OF PROTEIN DYNAMICS ing polymerases with novel activity. Infection of phage (B) with the The products of evolution are molecules with unique polymerase library (A) leads to production of phage particles that vibrational dynamics. The study of vibrational dynam- display 0–1 copies of the polymerase and 3–5 copies of the acidic ics in proteins and nucleic acids has been limited by peptide. Phage particles are combined with DNA primer–template spectral complexity, but selective deuteration of a pro- (C) and incubated with the desired nucleoside triphosphates. Active mutants are isolated (D) and characterized. tein or a nucleic acid results in a carbon-deuterium oscillator that absorbs light in an otherwise transpar- evolve polymerases for any desired function. Using this ent region of the infrared spectrum. The synthesis of system, we have already evolved polymerases with a selectively deuterated proteins has provided us with a variety of novel functions, including the synthesis of residue-specific probe of flexibility, function, and fold- DNA containing one of the unnatural base pairs. We are ing. We are also using multidimensional femtosecond optimizing these polymerases and evolving new ones. spectroscopy to characterize how protein motion is DNA DAMAGE RESPONSE evolved during the somatic evolution of antibodies. Evolution requires mutation, but mutations also make We discovered that the immune system can manipu- cells susceptible to aging and cancer. It is now under- lating protein dynamics, a finding that suggests a role stood that at times of sufficient stress, cells induce for these dynamics in molecular recognition. error-prone replication to facilitate their own evolution. We used genome-wide high-throughput methods to iden- PUBLICATIONS Chin, J.K., Bashkirov, V.I., Heyer, W.D., Romesberg, F.E. Esc4/Rtt107 and the tify genes involved in both error-free and error-prone control of recombination during replication. DNA Repair (Amst.) 5:618, 2006. responses to DNA stress in budding yeast. Characteri- Cirz, R.T., Gingles, N., Romesberg, F.E. Side effects may include evolution. Nat. zation of the proteins required for mutation in mam- Med. 8:890, 2006. 88 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Cirz, R.T., O’Neill, B.M., Hammond, J.A., Head, S.R., Romesberg, F.E. Defining molecules with defined biological or chemical functions. the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin. J. Bacteriol. 188:7101, 2006. Nature, on the other hand, has produced an array of molecules with remarkably complex functions, ranging Cirz, R.T., Romesberg, F.E. Induction and inhibition of ciprofloxacin resistance-con- ferring mutations in hypermutator bacteria. Antimicrob. Agents Chemother. from photosynthesis and signal transduction to molecu- 50:220, 2006. lar recognition and catalysis. Our aim is to combine the Cremeens, M., Fujisaki, H., Zhang, Y., Zimmermann, J., Sagle, L.B., Matsuda, S., synthetic strategies and biological processes of Nature Dawson, P.E., Straub, J.E., Romesberg, F.E. Efforts toward developing direct probes of protein dynamics. J. Am. Chem. Soc. 128:6028, 2006. with the tools and principles of chemistry to create new molecules with novel chemical and biological functions. Dupradeau, F.-Y., Case, D.A., Yu, C., Jimenez, R., Romesberg, F.E. Differential solvation and tautomer stability of a model base pair within the minor and major By studying the properties of the resulting molecules, grooves of DNA. J. Am. Chem. Soc. 127:15612, 2005. we can gain new insights into the molecular mecha- Henry, A.A., Romesberg, F.E. Evolution of DNA polymerases with novel activities. nisms of complex biological and chemical systems. Curr. Opin. Biotechnol. 16:370, 2005. For example, we have shown that the tremendous Hwang, G.T., Romesberg, F.E. Substituent effects on the pairing and polymerase combinatorial diversity of the immune response can be recognition of simple unnatural base pairs. Nucleic Acids Res. 34:2037, 2006. chemically reprogrammed to generate selective enzyme- Kim, Y., Leconte, A.M., Hari, Y., Romesberg, F.E. Stability and polymerase recognition of pyridine nucleobase analogues: role of minor-groove H-bond acceptors. like catalysts. We have developed antibodies that cat- Angew. Chem. Int. Ed., in press. alyze a wide array of chemical and biological reactions,

Leconte, A.M., Chen, L., Romesberg, F.E. Polymerase evolution: efforts toward from acyl transfer to redox reactions. Characterization expansion of the genetic code. J. Am. Chem. Soc. 127:12470, 2005. of the structure and mechanisms of these catalytic anti- Leconte, A.M., Matsuda, S., Hwang, G., Romesberg, F.E. Efforts towards expan- bodies has led to important new insights into the mech- sion of the genetic alphabet: pyridone and methyl pyridone nucleobases. Angew. Chem. Int. Ed. 45:4326, 2006. anisms of biological catalysis. In addition, the detailed characterization of the properties and structures of Leconte, A.M., Matsuda, S., Romesberg, F.E. An efficiently extended class of unnatural base pairs. J. Am. Chem. Soc. 128:6780, 2006. germ-line and affinity-matured antibodies has revealed fundamental new aspects of the evolution of binding Leconte, A.M., Romesberg, F.E. Amplify this! DNA and RNA get a third base pair. Nat. Methods 3:667, 2006. and catalytic function, in particular, the role of struc-

Lis, E.T., Romesberg, F.E. Role of Doa1 in the Saccharomyces cerevisiae DNA tural plasticity in the immune response. Most recently, damage response. Mol. Cell. Biol. 26:4122, 2006. we have focused on in vitro evolution methods that

Matsuda, S., Henry, A.A., Romesberg, F.E. Optimization of unnatural base pair involve the development of novel chemical screens packing for polymerase recognition. J. Am. Chem. Soc. 128:6369, 2006. and selections for identifying metalloantibodies with Sagle, L.B., Zimmermann, J., Dawson, P.E., Romesberg, F.E. Direct and high-resolu- proteolytic activity. tion characterization of cytochrome c equilibrium folding. J. Am. Chem. Soc., in press. Our work on catalytic antibodies redirects natural Sagle, L.B., Zimmermann, J., Matsuda, S., Dawson, P.E., Romesberg, F.E. Redox- combinatorial diversity to produce new function. We coupled dynamics and folding in cytochrome c. J. Am. Chem. Soc. 128:7909, 2006. are extending this combinatorial approach to many Zimmermann, J., Oakman, E.L., Thorpe, I.F., Shi, X., Abbyad, P., Brooks, C.L. III, other problems, including the generation of genetic Boxer, S.G., Romesberg, F.E. Antibody evolution constrains conformational het- ereogeneity by tailoring protein dynamics. Proc. Natl. Acad. Sci. U. S. A. “microcalorimetes” and yeasts lacking mitochondrial 103:13722, 2006. genomes and the ab initio evolution of novel protein domains. We are also generating structure-based combinatorial libraries of small heterocycles that are being Biological Chemistry used in conjunction with novel cellular and organismal screens to identify important proteins involved in such P.G. Schultz, A. Boitano, E. Brustad, M. Bushey, S. Chen, cellular processes as differentiation, proliferation, and J. Guo, J. Grbic, D. Groff, J. Grünewald, W.Y. Hur, M. Jahnz, signaling. Indeed, we have identified molecules that T. Kuo, J. Lee, J.-S. Lee, K.-B. Lee, E. Lemke, C. Liu, control both adult and embryonic stem cell differentia- W. Liu, C. Lyssiotis, J. Mills, M. Mukherji, T. Nom, B. Okram, tion and self-renewal and that reprogram lineage-com- R. Perera, F. Peters, Y. Ryu, S. Schiller, M. Sever, mitted cells. We are using x-ray crystallographic and D. Summerer, L. Supekova, E. Tippmann, M.-L. Tsao, biochemical studies, together with genomics technolo- J. Wang, T. Young, Q. Zhang, S. Zhu gies, to characterize the mode of action of these com- lthough chemists are remarkably adept at syn- pounds and to study their effects on cellular processes. thesizing molecular structures, they are far less We are also applying genomics tools (cell-based phe- A sophisticated in designing and synthesizing notypic screens of arrayed genomic cDNA and small CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 89 interfering RNA libraries) and proteomics tools (mass Powerful Click Processes for spectrometric phosphoprotein profiling) to a variety of important biomedical problems in cancer biology, neu- Organic Synthesis, Chemical rodegenerative and autoimmune diseases, and virology. In addition, we are investigating the role and regulation Biology, and Materials Research of noncoding RNAs. We have also developed a general biosynthetic K.B. Sharpless, V.V. Fokin, M. Ahlquist, B. Boren, M. Cassidy, method that can be used to site specifically incorporate S. Chang, A. Feldman, J. Fotsing, R. Fraser, A. Grant, J. Hein, unnatural amino acids into proteins in vitro and in vivo. J. Kalisiak, L. Krasnova, S.-W. Kwok, Y. Liu, J. Loren, Using this method, we effectively expanded the genetic R. Manetsch, K. Nagai, S. Narayan, S. Pitram, L.K. Rasmussen, J. Raushel, S. Roeper, W. Sharpless, codes of both prokaryotic and eukaryotic organisms by A. Sugawara, J. Tripp, X. Wang, J. Wassenaar, T. Weide, adding new components to the biosynthetic machinery M. Whiting, P. Wu of living cells. We have genetically encoded amino acids with novel spectroscopic and chemical properties (e.g., he driving forces in our research are the discov- metal-binding, glycosylated, and fluorescent amino acids ery and understanding of chemical reactivity, the and photocross-linking and photoisomerizable amino T harbingers of all new reactions. Our main goal acids) in response to unique 3- and 4-base codons. is to develop practical transformations that facilitate These amino acids are being used to explore protein synthesis of novel molecules with desired functions and structure and function both in vitro and in vivo and to allow manipulation of complex biological systems at the evolve proteins with novel properties. Our results have molecular level. removed a billion-year constraint imposed by the genetic CLICK CHEMISTRY code on the ability to chemically manipulate the struc- Among the many factors that determine the suc- tures of proteins. cess of a search for compounds with desired properties, 2 stand out: the degree of diversity of the building PUBLICATIONS Bose, M., Groff, D., Xie, J., Brustad, E., Schultz, P.G. The incorporation of a photoiso- blocks that can be used and the speed with which merizable amino acid into proteins in E. coli. J. Am. Chem. Soc. 128:388, 2006. synthesis, screening for the desired function, and lead

Chen, S., Do, J., Zhang, Q., Yao, S., Yan, F., Peters, E., Scholer, H., Schultz, optimization can be performed. The greater the variety P.G., Ding, S. Self-renewal of embryonic stem cells by a small molecule. Proc. of scaffolds and functional groups that can be used in Natl. Acad. Sci. U. S. A., in press. the rapid construction of candidate compounds, the more Mukherji, M., Cho, C., Supekova, L., Wang, Y., Batalov, S., Bell, R., Martin, C., likely it is that new and useful function will be discov- Sahasrabuhde, S., Orth, A.P., Chanda, S.K., Schultz, P.G. Functional analysis of human genome for cell-cycle regulators. Proc. Natl. Acad. Sci. U. S. A., in press. ered. Because of the enormous number of compounds to explore (the number of small druglike compounds Summerer, D., Chen, S., Wu, N., Deiters, A., Chin, J.W., Schultz, P.G. A genetically encoded fluorescent amino acid. Proc. Natl. Acad. Sci. U. S. A. 103:9785, 2006. may be as high as 1064), the size of a given collection

Turner, J.M., Graziano, J., Spraggon, G., Schultz, P.G. Structural characterization becomes much less important than the ability to rapidly of a p-acetylphenylalanyl aminoacyl-tRNA synthetase. J. Am. Chem. Soc. probe the collection for a desired activity. However, 127:14976, 2005. many chemical methods often have restrictions such as Warashina, M., Min, K.H., Kuwabara, T., Huynh, A., Gage, F.H., Schultz, P.G., limited scope, inaccessibility of starting materials, Ding, S. A synthetic small molecule that induces neuronal differentiation of adult hippocampal neural progenitor cells. Angew. Chem. Int. Ed. 45:591, 2006. requirements for protecting groups, and difficult purifica- tions. In addition, inert atmospheres and anhydrous sol- Willingham, A.T., Orth, A.P., Batalov, S., Peters, E.C., Wen, B.G., Aza-Blanc, P., Hogenesch, J.B., Schultz, P.G. A strategy for probing the function of noncoding vents are usually required, a situation that makes these RNAs finds a repressor of NFAT. Science 309:1570, 2005. methods impractical for manipulating biological molecules in the molecules’ natural, aqueous environment. In the past several years, we have sought to develop and use only the best reactions for the synthesis of functional molecules. The reactions that fulfill the most stringent criteria of usefulness and convenience have been grouped under the name click chemistry. Most click reactions form carbon-heteroatom bonds, are tol- erant of water, and are often accelerated when water 90 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE is used as the sole medium, even if the reagents are Although a number of copper(I) complexes can be not soluble in water. used to catalyze the reaction, we found that the cata- COPPER-CATALYZED CYCLOADDITIONS lyst is often better prepared in situ by reduction of The copper-catalyzed 1,3-dipolar cycloaddition of copper(II) salts, which are readily available and are azides and alkynes has emerged as a premiere click reac- easier to handle than most copper(I) salts. As the tion that enables reliable assembly of complex molecules reductant, ascorbic acid (vitamin C) or sodium ascor- by means of the 1,2,3-triazole heterocycle. Although bate is excellent. Remarkably, even copper metal can both alkynes and azides are highly reactive, their reac- be used as a source of the catalytic species, making tivity profiles are quite narrow, that is, “orthogonal” to the experimental procedure even simpler: a small piece an unusually broad range of reagents, solvents, and other of copper metal (wire or turning) is all that is added to functional groups. These features allow clean sequen- the reaction mixture, which is then shaken or stirred tial transformations of broad scope without the need for 12–48 hours. This protocol is particularly convenient for protecting groups, even if the reactions are performed in parallel synthesis, because triazole products are under physiologic conditions. generally isolated in high yields and can often be sub- The 1,2,3-triazoles have the advantageous proper- mitted for screening without further purification. ties of high chemical stability (in general, being inert to Our studies of reactivity of sulfonyl azides in the severe hydrolytic, oxidizing, and reducing conditions, copper-catalyzed cycloaddition of azides and alkynes even at high temperatures), strong dipole moment, resulted in the development of an experimentally sim- presence of aromatic groups, and the ability to accept ple catalytic procedure for the highly selective conver- hydrogen bonds. Thus, they can interact productively sion of alkynes to N-sulfonyl azetidin-2-imines under in several ways with biological molecules. For example, mild conditions. This 3-component process is thought 1,2,3-triazoles can replace the amide bond in peptides, to proceed via initial reaction of in situ generated cop- preventing proteolytic degradation of the peptides. per(I) acetylides with sulfonyl azides, resulting in tran- The fundamental thermal reaction, involving termi- sient (1-sulfonyltriazolyl) copper intermediates that upon nal or internal alkynes (Fig. 1, top), has been known extrusion of dinitrogen generate N-sulfonyl keteneimines. The azetidinimine products are remarkably stable in a wide range of reaction conditions, and other functional groups can be easily added (Fig. 2). This newly dis-

Fig. 1. Thermal cycloaddition of azides and alkynes (top) requires prolonged heating and results in mixtures of both 1,4- and Fig 2. Synthesis of azetidinimines from alkynes, sulfonyl azides, 1,5-regioisomers, whereas the copper-catalyzed 1,3-dipolar and imines. cycloaddition of azides and alkynes produces only 1,4-disubstituted-1,2,3-triazoles at room temperature (bottom). covered reaction sequence rapidly produces densely for more than a century and has been thoroughly inves- functionalized azetidine derivatives from readily avail- tigated. Although the process is strongly thermodynami- able terminal alkynes in just 2 or 3 simple steps and cally favored, it has a relatively high kinetic barrier should be useful for exploring the usefulness of these that makes the reaction slow at room temperature for 4-atom heterocycles. unactivated reactants and results in the formation of Although useful because of its rate and functional regioisomers. Copper(I) catalysis dramatically accelerates group tolerance, the copper-catalyzed 1,3-dipolar the reaction, by a factor of up to 107, and regiospecifi- cycloaddition of azides and alkynes cannot produce cally produces only 1,4-disubstituted-1,2,3-triazoles 1,5-disubstituted 1,2,3-triazoles, and it is not effec- (Fig. 1, bottom). Because of its experimental simplicity tive with internal alkynes. Therefore, the recent dis- and unusually broad scope, this process has been used covery of ruthenium(II) catalysts that are active in in a number of applications in synthesis, medicinal azide-alkyne cycloaddition and result in the formation chemistry, molecular biology, and materials science. of the complementary 1,5-regioisomers of 1,2,3-tria- CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 91

zoles was a welcome advance. Pentamethyl cyclopen- binding protein concanvalin A and rabbit red blood tadienyl ruthenium(II) complexes are active and easy to cells. The dendrimer had 240-fold greater potency handle and provide triazole products in good to excel- than monomeric mannose, a difference that translates lent yields (Fig. 3). In addition, these catalysts are active to a 15-fold increase in activity per unit. Additionally, our preliminary experiments indicate that this dendrimer binds to the modified surface of Escherichia coli, producing detectable fluorescent signals. These results are a significant advance in dendrimer chemistry and illustrate an evolving synergy between organic chem- Fig. 3. Ruthenium-catalyzed synthesis of fully substituted istry and functional materials. 1,2,3-triazoles. Studies of other applications, ranging from biology with internal alkynes, allowing easy synthesis of fully to materials science, are currently underway in our labo- substituted triazoles. ratories and in collaboration with M.G. Finn, P.K. Vogt, SYNTHESIS OF POLYFUNCTIONAL DENDRIMERS C.-H. Wong, J.H. Elder, and others at Scripps Research. Dendrimers are highly ordered, regularly branched PUBLICATIONS globular macromolecules of defined structure; den- Bourne, Y., Radic, Z., Kolb, H.C., Sharpless, K.B., Taylor, P., Marchot, P. Struc- drimers are ideal building blocks for creating bioactive tural insights into conformational flexibility at the peripheral site and within the active center gorge of AChE. Chem. Biol. Interact. 157-158:159, 2005. macromolecules and nanomaterials. Previously, we exploited the high fidelity of the copper-catalyzed 1,3 Cassidy, M.P., Raushel, J., Fokin, V.V. Practical synthesis of amides from in situ generated copper(I) acetylides and sulfonyl azides. Angew. Chem. Int. Ed. dipolar cycloaddition of azides and alkynes in the effi- 45:3154, 2006. cient synthesis of dendrimers. We used procedures that Hansen, T.V., Wu, P., Fokin, V.V. One-pot copper(I)-catalyzed synthesis of 3,5-dis- involved little more than mixing stoichiometric quantities ubstituted isoxazoles. J. Org. Chem. 70:7761, 2005. of reactants, stirring, and isolating dendrimer products. Loren, J.C., Krasinski, A., Fokin, V.V., Sharpless, K.B., NH-1,2,3-triazoles from In collaboration with M.G. Finn, Department of azidomethyl pivalate and carbamates: base-labile N-protecting groups. Synlett Chemistry, and C.J. Hawker, University of California, 2847, 2005, Issue 18. Santa Barbara, we have extended this approach to Malkoch, M., Schleicher, K., Drockenmuller, E., Hawker, C.J., Russell, T.P., Wu, P., Fokin, V.V. Structurally diverse dendritic libraries: a highly efficient functional- synthesize chemically heterogeneous dendrimers (Fig. 4) ization approach using click chemistry. Macromolecules 38:3663, 2005.

Meng, J.-C., Fokin, V.V., Finn, M.G. Kinetic resolution by copper-catalyzed azide- alkyne cycloaddition. Tetrahedron Lett. 46:4543, 2005.

Petasis, N.A., Akritopoulou-Zanze, I., Fokin, V.V., Bernasconi, G., Keledjian, R., Yang, R., Uddin, J., Nagulapalli, K.C., Serhan, C.N. Design, synthesis and bioac- tions of novel stable mimetics of lipoxins and aspirin-triggered lipoxins. Prostaglandins Leukot. Essent. Fatty Acids 73:301, 2005.

Radic, Z., Manetsch, R., Krasinski, A., Raushel, J., Yamauchi, J., Garcia, C., Kolb, H.C., Sharpless, K.B., Taylor, P. Molecular basis of interactions of cholinesterases with tight binding inhibitors. Chem. Biol. Interact. 157-158:133, 2005.

Rodionov, V.O., Fokin, V.V., Finn, M.G. Mechanism of the ligand-free Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 44:2210, 2005.

Whiting, M., Fokin, V.V. Copper-catalyzed reaction cascade: direct conversion of alkynes to N-sulfonylazetidin-2-imines. Angew. Chem. Int. Ed. 45:3157, 2006.

Whiting, M., Muldoon, J., Lin, Y.-C., Silverman, S.M., Lindstrom, W., Olson, A.J., Kolb, H.C., Finn, M.G., Sharpless, K.B., Elder, J.H., Fokin, V.V. Inhibitors of HIV- 1 protease by using in situ click chemistry. Angew. Chem. Int. Ed. 45:1435, 2006. Fig. 4. Synthesis of polyfunctional dendrimers via copper-catalyzed 1,3-dipolar cycloaddition of azides and alkynes. Wu, P., Hilgraf, R., Fokin, V.V. Osmium-catalyzed olefin dihydroxylation and aminohy- droxylation in the second catalytic cycle. Adv. Synth. Catal. 348:1079, 2006.

that have multiple recognition functions (such as 16 Wu, P., Malkoch, M., Hunt, J., Vestberg, R., Kaltgrad, E., Finn, M.G., Fokin, V.V., α-d-mannose units) and detection elements (2 coumarin- Sharpless, K.B., Hawker, C.J. Multivalent, bifunctional dendrimers prepared by click chemistry. Chem. Commun. (Camb.) 5775, 2005, Issue 46. derived fluorescent chromophores). The performance of one such bifunctional dendrimer was evaluated in a Zhang, L., Chen, X., Xue, P., Sun, H.H.Y., Williams, I.D., Sharpless, K.B., Fokin, V.V., Jia, G. Ruthenium-catalyzed cycloaddition of alkynes and organic azides. J. standard hemagglutination assay with the mannose- Am. Chem. Soc. 127:15998, 2005. 92 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE Chemistry, Biology, and Inflammatory Disease

P. Wentworth, Jr., J.Y. Chang, Y.P. Chen, J. Dambacher, L. Eltepu, R.K. Grover, J. Nieva, M. Puga, A. Shafton, B.D. Song, M.M.R. Peram, J. Rogel, S. Tripurenani, R. Troseth, H. Wang, A.D. Wentworth

ur research is interdisciplinary and involves bioorganic, biophysical, physical organic, synthetic, O and analytical chemistry coupled with biochemical techniques, cell-based assays, and animal models. These diverse approaches are combined to facilitate a better understanding of and generate new therapeutic approaches to complex disease states. Ongoing projects include studies on atherosclerosis, neurodegeneration, ischemia-reperfusion injury, macular degeneration, can- Fig. 1. Schematic presentation of riboflavin within the binding cer, inflammation, and infectious diseases. site of IgGGAR. Residues that form van der Waals interactions with THE ANTIBODY-CATALYZED WATER OXIDATION riboflavin are indicated; those that participate in the hydrogen bonds with the riboflavin are shown in ball-and-stick representa- PATHWAY tions. Hydrogen bonds are illustrated as dotted lines. Our discovery that all antibody molecules, regardless of source or antigenic specificity, can catalyze the wiched between several aromatic side chains. Binding reaction between singlet oxygen and water to give hydro- of riboflavin occurs in a narrow cleft with the isoallox- gen peroxide is causing a revision of the axiom that azine ring stacked between parallel aromatic groups of antibodies are the classical adapter molecule of the tyrosine at position H33 (with the re face of riboflavin), immune system, linking recognition and killing of for- phenylalanine at position H58, and tyrosine at position eign pathogens. Both the chemical and biological aspects H100A (both associated with the si face of the flavin); of this pathway are being explored extensively, and the distances between the isoalloxazine ring and the intriguing new insights into how this pathway may respective aromatic rings from these 3 residues are play a role in immune defense and inflammatory dam- about 3.2, 3.5. and 3.4 Å, respectively. Such π stack- age are emerging. ing is known to quench the excited state of riboflavin, We recently showed that flavins, biologically rele- a feature that a number of flavin-binding proteins vant photoactive molecules, can act as efficient triggers have also evolved, presumably to protect themselves for the antibody-catalyzed water oxidation pathway at from the intrinsic photochemistry of the flavin moiety. physiologically relevant concentrations and a certain We are searching for the active site for the antibody- amount of visible light radiation. This observation indi- catalyzed water oxidation pathway within the antibody cates that immune defense and damage may be linked structure. We have cloned and expressed soluble indi- to an association of flavins, such as riboflavin or flavin vidual domains of the murine Fab 4C6. Initial attempts mononucleotide, with immunoglobulins. The trigger to efficiently express wild-type domains were unsuc- would be antibody-antigen binding on cells exposed to cessful, but by mutating hydrophobic residues involved visible light, such as the skin or retina. in interdomain interactions into soluble hydrophilic resi- In collaboration with I.A. Wilson, Department of dues, we prepared folded domains. All the domains we Molecular Biology, we recently studied, at atomic reso- cloned and expressed have been successfully purified lution, an antibody, IgGGAR, that has riboflavin bound to homogeneity from the periplasm of Escherichia coli in the complementarity-determining regions (Fig. 1). Of transformed with plasmids encoding individual domains. interest, the bound riboflavin is essentially photochemi- In addition, to assess the critical nature of tryptophan cally inert. The x-ray structure of the IgGGAR-flavin residues in the photosensitization of antibody-bound complex offers an insight into the lack of photochemi- triplet oxygen to singlet oxygen, we mutated 2 trypto- cal activity; the isoalloxazine ring is effectively sand- phan residues within domain CH1 to leucine. Interest- CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 93 ingly, neither the single nor the double mutation affected LDL, atheronal-B, but not atheronal-A, induces cul- the ability of ultraviolet-irradiated CH1 to generate tured human monocytes to differentiate into macro- hydrogen peroxide. phage cell lineage. CHOLESTEROL SECO-STEROLS AND INFLAMMATORY These in vitro data and the effects of cholesterol DISEASE 5,6-seco-sterols on the formation of foam cells and We recently discovered that the 5,6-seco-sterols the cytotoxic effects of macrophages indicate that the atheronal-A and atheronal-B (Fig. 2), compounds of a atheronals have biological effects that could lead in class of cholesterol ozonolysis products, are present in vivo to the recruitment, entrapment, dysfunction, and ultimate destruction of macrophages, the major leukocytes in inflammatory artery disease. The ultimate goals of research for genetic and environmental factors that increase the propensity of a specific protein to misfold are the understanding and treatment of disease states as diverse as atherosclerosis, light-chain deposition disease, systemic amyloidosis, Alzheimer’s disease, and Parkinson’s disease. We showed that the inflammation-derived atheronal-A and atheronal-B trigger a deformation in the secondary structure of the normally folded protein apolipoprotein B-100 into a proamyloidogenic form. In collaboration with Dr. Kelly, we extended this model and showed that these cholesterol seco-sterols also trigger the misfolding of amyloid β-peptide(1–40), leading to formation of fibrils similar to those observed in Fig. 2. The cholesterol seco-sterols atheronal-A (top) and patients with Alzheimer’s disease. Interestingly, analysis atheronal-B (bottom). of the structure-activity relationship revealed that among human atherosclerotic plaques and plasma. In addition, a panel of aldehydes, only atheronal-A, atheronal-B, and we found that the atheronals are present in murine mod- 4-hydroxynonenal trigger misfolding of amyloid β-pep- els of atherosclerosis, in a rabbit model of acute respi- tide, suggesting that structural aspects of the aldehyde ratory distress syndrome (studies done in collaboration and not simple protein adduction were critical to this with C. Cochrane, Department of Molecular and Experi- misfolding. More recently, using mutated synthetic mental Medicine), and in brain tissue from patients with sequences of amyloid β-peptide(1–40), we found that Lewy body dementia (studies done in collaboration with the accelerated aggregation of this protein only occurs J. Kelly, Department of Chemistry). when a particular lysine of the sequence is modified. The atheronals have a range of biological properties We have also shown that atheronals and other lipid that in combination would increase the number of mac- aldehydes accelerate the aggregation of several wild-type rophages at sites of vascular inflammation. When cul- amyloidogenic proteins, including immunoglobulin light tured macrophage cells were incubated with atheronal-A chains, mouse prions, and the tumor suppressor pro- and atheronal-B complexed with low-density lipoprotein p53. The generality and specificity of this process tein (LDL), marked upregulation of scavenger receptor suggest that inflammatory aldehydes and their post- class A, but not CD36, occurred, showing that cultured translational modification of amyloidogenic peptides macrophages respond to complexes of atheronals and may be the chemical link between the known associa- LDL in a manner highly analogous to the response to tions of inflammation, oxidative damage, and various acetylated LDL. Both atheronal-A and atheronal-B induce protein misfolding diseases. chemotaxis of cultured macrophages in a dose-depen- INTERACTION BETWEEN PROTOZOAN J-BINDING dent manner. When complexed with LDL, atheronal-A, PROTEIN 1 AND GLYCOSYLATED DNA but not atheronal-B, induces a dose-dependent upreg- Current treatments of parasitic infections such as ulation of the cell-surface adhesion molecule E-selectin leishmaniasis, African trypanosomiasis, and American on vascular endothelial cells. When complexed with trypanosomiasis are limited in terms of their effective- 94 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE ness, increasing drug resistance and the inherent toxic that some loci around the glycan are essential for binding effects of the drugs. Thus, an elucidation of new para- and some are not. This information is being translated site-specific biological targets for new therapeutic agents into structure-based design of chemical libraries as is needed. In this regard, the discovery that DNA from inhibitors of JBP-1 binding. members of the order Kinetoplastida, but not from other PUBLICATIONS D eukaryotes, contains an unusual modified base, β- - Bielsch, J., Wang, Q.T., Bosco, D., Powers, E.T., Wentworth, P., Jr., Kelly, J. glucosyl(hydroxymethyl)uracil, called base J (compound Inflammatory metabolite-initiated protein misfolding. Acc. Chem. Res., in press.

1a in Fig. 3) was a breakthrough. Extracts of several Bosco, D.A., Fowler, D.M., Zhang, Q., Nieva, J., Powers, E.T., Wentworth, P., Jr., kinetoplastids contain a J-binding protein (JBP) that Lerner, R.A., Kelly, J.W. Elevated levels of oxidized cholesterol metabolites in Lewy body disease accelerate α-synuclein fibrilization [published correction appears in Nat. Chem. Biol. 2:346, 2006]. Nat. Chem. Biol. 2:249, 2006.

Nieva, J., Kerwin, L., Wentworth, A.D., Lerner, R.A., Wentworth, P., Jr. Immunoglobulins can utilize riboflavin (vitamin B2) to activate the antibody-catalyzed water oxidation pathway. Immunol. Lett. 103:33, 2006.

Rogers, C.J., Dickerson, T.J., Wentworth, P., Jr., Janda, K.D. A high-swelling reagent scaffold suitable for use in aqueous and organic solvents. Tetrahedron 61:12140, 2005.

Takeuchi, C., Galve, R., Nieva, J., Witter, D.P., Wentworth, A.D., Troseth, R.P., Lerner, R.A., Wentworth, P., Jr. Proatherogenic effects of the cholesterol ozonolysis products, atheronal-A and atheronal-B. Biochemistry 45:7162, 2006.

Toker, J.D., Tremblay, M., Yli-Kauhaluoma, J., Wentworth, A.D., Zhou, B., Went- worth, P., Jr., Janda, K.D. Exploring the scope of the 29G12 antibody catalyzed 1,3-dipolar cycloaddition reaction. J. Org. Chem. 70:7810, 2005.

Witter, D., Wentworth, P., Jr. The antibody-catalyzed water-oxidation pathway from discovery to an emerging role in health and disease. Antioxid. Redox Signal., in press.

Zhu, X., Wentworth, P., Jr., Kyle, R.A., Lerner, R.A., Wilson, I.A. Cofactor-contain- Fig. 3. Base J (1a) and base J analogs (1b–1g) synthesized and ing antibodies: crystal structure of the original yellow antibody. Proc. Nat. Acad. Sci. U. S. A. 103:3581., 2006. used as probes for studying JBP-1–J-DNA recognition. Inset, Image of base J in doubled-stranded DNA shows how glucose sits in the major groove. Bioorganic and specifically binds to J-containing duplex DNA. JBP-1 is essential in Leishmania. Synthetic Chemistry As a drug target, JBP has merit. The protein shares little homology with other proteins in the Protein Data C.-H. Wong, C. Bennett, A. Brik, Y.-H. Chen, S. Dean, Bank, and it has a unique ligand, J-DNA containing S. Ficht, M. Fujio, W. Greenberg, R. Guy, S. Hanson, Z. Hong, telomeric stretches of double-stranded DNA, that does T.-L. Hsu, D.-R. Hwang, J.-C. Lee, P.-H. Liang, L. Liu, not occur in other eukaryotes. However, a preliminary T. Polat, M. Sawa, M. Sugiyama, D. Thayer, S.-K. Wang, high-throughput screen, focused on disrupting binding L. Whalen, C.-Y. Wu, D. Wu, M. Wuchrer, Y.-Y. Yang between JBP-1 and J-DNA, with a library of compounds ur research programs involve development of consisting of all the major drug pharmacophoric groups new chemical and enzymatic strategies and revealed no compounds of interest. O methods for the synthesis of biologically active In parallel, we have studied the nature of the molec- compounds. We use the synthesized compounds as ular recognition that underpins JBP-1 recognition of molecular probes to explore carbohydrate-mediated glycosylated DNA. We synthesized a panel of modified biological recognition events and enzymatic reactions. J-containing bases (compounds 1b–1g in Fig. 3) and ORGANIC AND BIOORGANIC SYNTHESIS incorporated them into a 16-nucleotide telomeric stretch Our work in organic and bioorganic synthesis includes of double-stranded DNA. In collaboration with D. Mil- the development of new chemical reactions and the lar, Department of Molecular Biology, we determined exploitation of native and engineered enzymes for the dissociation constants of these analogs for JBP-1 organic synthesis. In the past year, we developed new and generated a ∆G of binding assessment of each methods for making glycopeptides and glycoproteins hydroxyl around the glucosyl core. This analysis revealed via native chemical ligation methods. We will use these CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 95 methods to synthesize homogenous glycoproteins that We synthesized a series of bacterial glycolipids and are important therapeutic agents in humans. We con- analogs and found that they are active ligands for CD tinue to develop covalent glycoarrays for high-through- cell markers involved in activation of human natural put analysis of protein-carbohydrate interactions and killer T cells. These compounds may be useful as the use of aldolases in the synthesis of glycosyltrans- immunotherapeutic agents for the treatment of bacter- fer enzyme inhibitors. Using directed evolution, we ial and viral infections, as well as cancer, and we are developed new aldolase variants capable of making elucidating the structural basis for their activity. In both enantiomers of sugars. collaboration with Dr. Wilson, we are determining the DEVELOPMENT OF INHIBITORS OF ENZYMES AND structures of the complexes formed by these glycolipids RECEPTORS and CD1d; the results will assist in designing ligands Our goals in the area of enzyme and receptor inhibi- with improved therapeutic potential. In collaboration tors are to develop new strategies to discover inhibitors with J.C. Paulson, Department of Molecular Biology, and ligands with high selectivity as potential therapeutic we developed new methods for microfabrication of agents. Current strategies involve the design and syn- saccharides on glass slides or microtiter plates for use thesis of structure- and mechanism-based inhibitors of in high-throughput analysis of sugar-protein interactions. enzymes associated with a variety of diseases. Targets for investigation include bacterial transglycosidase, sul- PUBLICATIONS fotransferases, retroviral proteases, lethal factor of Bacil- Brigl, M., van den Elzen, P., Chen, X., Meyers, J.H., Wu, D., Wong, C.-H., Red- lus anthracis dington, F., Illarianov, P.A., Besra, G.S., Brenner, M.B., Gumperz, J.E. Conserved , and enzymes involved in the biosynthesis and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors. of carbohydrates essential for biological functions. J. Immunol. 176:3625, 2006. We have developed new iminocyclitols and derivatives Brik, A., Ficht, S., Wong, C.-H. Strategies for the preparation of homogenous gly- as inhibitors of glycosidases and glycosyltransferases coproteins. Curr. Opin. Chem., in press. for potential treatment of inflammatory diseases. In addi- Brik, A., Wu, C.-Y., Wong, C.-H. Microtiter plate based chemistry and in situ tion, using a new strategy based on a rapid microscale screening: a useful approach for enzymatic inhibitor discovery. Org. Biomol. Chem. synthesis coupled with in situ high-throughput screening, 4:1446, 2006. we developed new tight-binding inhibitors of anthrax Brik, A., Yang, Y.-Y., Ficht, S., Wong, C.-H. Sugar-assisted glycopeptide ligation. J. lethal factor, a sulfotransferase, and drug-resistant HIV Am. Chem. Soc. 128:5626, 2006. proteases. We also developed new reactions based on Calarese, D.A., Lee, H.-K, Huang, C.-Y., Best, M.D., Astronomo, R.D., Stanfield, tetrabutylammonium fluoride–mediated N- and O-alky- R.L., Katinger, H., Burton, D.R., Wong, C.-H., Wilson I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. lation and epoxide opening in aqueous solution and Natl. Acad. Sci. U. S. A. 102:13372, 2005. used the reactions to identify potent enzyme inhibitors. Cheng, Y.-S.E., Lo, K.-H., Hsu, H.-H., Shao, Y.-M., Yang, W.-B., Lin, C.-H., CARBOHYDRATE CHEMISTRY AND MOLECULAR Wong, C.-H. Screening for HIV protease inhibitors by protection against activity- GLYCOBIOLOGY mediated cytotoxicity in Escherichia coli. J. Virol. Methods 137:82, 2006.

We continue to improve the programmable 1-pot Chuang, M.-H., Wu, M.-S., Lo, W.-L., Lin, J.-T., Wong, C.-H., Chiou, S.-H. The oligosaccharide synthesis method for convenient and antioxidant protein alkylhydroperoxide reductase of Helicobacter pylori switches from a peroxide reductase to a molecular chaperone function. Proc. Natl. Acad. rapid preparation of oligosaccharides. So far, we have Sci. U. S. A. 103:2552, 2006. designed approximately 600 building blocks and mea- Fujio, M., Wu, D., Garcia-Navarro, R., Ho, D.D., Tsuji, M., Wong, C.-H. Structure- sured the anomeric reactivity of each building block. based discovery of glycolipids for CD1d-mediated NKT cell activation: tuning the Using the computer program OptiMer, developed in adjuvant versus immunosuppression activity. J. Am. Chem. Soc. 128:9022, 2006. our laboratory, we rapidly assembled a number of Hong, Z.-Y., Liu, L., Hsu, C.-C., Wong, C.-H. Three-step synthesis of sialic acids oligosaccharides. We are using this method to define and derivatives. Angew. Chem. Int. Ed., in press. the specificity of interactions between carbohydrates Hsu, H.-Y., Hua, K.-F., Su, Y.-C., Chu, L.-C., Su, S.-C., Chiu, H.-W., Wong, C.-H., and their receptors, with particular focus on optimiza- Chen, S.-T., Shieh, C.-W., Yang, S.-S., Chen, Y.-M., Chao, L.K. Alkali-soluble poly- tion of the cancer antigen Globo H and HIV gp120 saccharides of Rhizoclonium riparium alga induces IL-1 gene expression via protein kinase signaling pathways. J. Agric. Food Chem. 54:3558, 2006. oligomannose as vaccine candidates. In collaboration with D.R. Burton, Department of Immunology, and I.A. Huang, C.-Y., Thayer, D.A., Chang, A.Y., Best, M.D., Hoffmann, J., Head, S., Wong, C.-H. Carbohydrate microarray for profiling the antibodies interacting with Wilson, Department of Molecular Biology, we are eval- Globo H tumor antigen. Proc. Natl. Acad. Sci. U. S. A. 103:15, 2006. uating a designed oligomannose-protein conjugate as Huang, K.-T., Wu, B.-C., Lin, C.-C., Luo, S.-C., Chen, C., Wong, C.-H., Lin, C.-C. an antigen to elicit antibodies for neutralizing HIV gp120 Multi-enzyme one-pot strategy for the synthesis of sialyl Lewis X-containing PSGL-1 and variants. glycopeptide. Carbohydr. Res. 341:2151, 2006. 96 CHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Kinjo, Y., Tupin, E., Wu, D., Fujio, M., Garcia-Navarro, R., Benhnia, M.R.E.I., Whalen, L.J., Wong, C.-H. Enzymes in organic synthesis: aldolase-mediated syn- Zajonc, D.M., Ben-Menachem, G., Ainge, G.D., Painter, G.F., Khurana, A., thesis of iminocyclitols and novel heterocycles. Aldrichim. Acta 39:63, 2006. Hoebe, K., Behar, S.M., Beutler, B., Wilson, I.A., Tsuji, M., Sellati, T.J., Wong, C-H., Kronenberg, M. Natural killer T cells recognize diacylglycerol antigens from Wu, C.-Y., Brik, A., Wang, S.-K., Chen, Y.-H., Wong, C.-H. Tetrabutylammonium pathogenic bacteria. Nat. Immunol. 7:978, 2006. fluoride-mediated rapid alkylation reaction in microtiter plates for the discovery of enzyme inhibitors in situ. Chembiochem 6:2176, 2005. Lee, J.-C., Wu, C.-Y., Apon, J.V., Siuzdak, G., Wong, C.-H. Reactivity-based one- pot synthesis of the tumor-associated antigen N3 minor octasaccharide for the Wu, C.-Y., King, K.-Y., Kuo, C.-J., Fang, J.-M., Wu, Y.-T., Ho, M.-Y., Liao, C.-L., development of a photocleavable DIOS-MS sugar array. Angew. Chem. Int. Ed. Shie, J.-J., Liang, P.-H., Wong, C.-H. Stable benzotriazole esters as mechanism- 45:2753, 2006. based inactivators of the severe acute respiratory syndrome 3CL protease. Chem. Biol. 13:261, 2006. Liang, F.-S., Brik, A., Lin, Y.-C., Elder, J.H., Wong, C.-H. Epoxide opening in water and screening in situ for rapid discovery of enzyme inhibitors in microtiter Wu, D., Zajonc, D.M., Fujio, M., Sullivan, B.A., Kinjo, Y., Kronenberg, M., Wilson, plates. Bioorg. Med. Chem. 14:1058, 2006. I.A., Wong, C.-H. Design of natural killer T cell activators: structure and function of a microbial glycosphingolipid bound to mouse CD1d. Proc. Natl. Acad. Sci. U. S. Liang, P.-H., Cheng, W.-C., Lee, Y.-L., Yu, H.-P. Wu, Y.-T., Lin, Y.-L., Wong, C.-H. A. 103:3972, 2006. Novel five-membered iminocyclitol derivatives as selective and potent glycosidase inhibitors: new structures for antivirals and osteoarthritis. Chembiochem 7:165, 2006. Zajonc, D.M., Maricic, I., Wu, D., Halder, R., Roy, K., Wong, C.-H., Kumar, V., Wilson, I.A. Structural basis for CD1d presentation of a sulfatide derived from Liang, F.-S., Greenberg, W.A., Hammond, J.A., Hoffmann, J., Head, S.R., Wong, myelin and its implications for autoimmunity. J. Exp. Med. 202:1517, 2005. C.-H. Evaluation of RNA-binding specificity of aminoglycosides with DNA microarrays. Proc. Natl. Acad. Sci. U. S. A. 103:12311, 2006.

Lin, K.-I., Kao, Y.-Y., Kuo, H.-K., Yang, W.-B., Chou, A., Lin, H.-H., Yu, A.L-T., Wong, C.-H. Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1. J. Biol. Chem. 281:24111, 2006.

Lin, Y.-C., Brik, A., de Parseval, A., Tam, K., Torbett, B.E., Wong, C.-H., Elder, J.H. Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell- based expression system. J. Virol. 80:7832, 2006.

Liu, L., Bennett, C.S., Wong, C.-H. Advances in glycoprotein synthesis. Chem. Commun. (Camb.) 21, 2006, Issue 1.

Liu, L., Hong, Z.-Y., Wong, C.-H. Convergent glycopeptide synthesis by traceless Staudinger ligation and enzymatic coupling. Chembiochem 7:429, 2006.

Liu, H., Wong, C.-H. Characterization of a transglycosylase domain of Streptococ- cus pneumoniae PBP1b. Bioorg. Med. Chem. 14:7187, 2006.

Qiul, H., Gabrielsen, A., Agardh, H.E., Wan, M., Wetterholm, A., Wong, C.-H., Hedin, U., Swedenborg, J., Hansson, G.K., Samuelsson, B., Paulsson-Berne, G., Haeggstrom, J.Z. Expression of 5-lipoxygenase and leukotriene A4 hydrolase in human atherosclerotic lesions correlates with symptoms of plaque instability. Proc. Natl. Acad. Sci. U. S. A. 103:8161, 2006.

Sanna, M.G., Wang, S.-K., Gonzalez-Cabrera, P.J., Don, A., Marsolais, D., Matheu, M.P., Wei, S.H., Parker, I., Jo, E., Cheng, W.-C., Cahalan, M.D., Wong, C.-H., Rosen, H. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo. Nat. Chem. Biol. 2:434, 2006.

Sawa, M., Hsu, T.-L., Itoh, T., Sugiyama, M., Hanson, S.R., Vogt, P.K., Wong, C.-H. Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo. Proc. Natl. Acad. Sci. U. S. A. 103:12371, 2006.

Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.-C., Wong, C.-H., Beutler, E., Zimmer, K.-P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show mutation-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005.

Scanlan, C., Calarese, D., Lee, H.K., Blixt, O., Wong, C.-H., Wilson, I., Burton, D., Dwek, R., Rudd P. Antibody recognition of a carbohydrate epitope: a template for HIV vaccine design. Adv. Exp. Med. Biol. 564:7, 2005.

Shie, J.-J., Fang, J.-M., Kuo, T.-H., Kuo, C.-J., Liang, P.-H., Huang, H.-J., Wu, Y.-T., Jan, J.-T., Cheng, Y.-S.E., Wong, C.-H. Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic α,β-unsaturated esters. Bioorg. Med. Chem. 13:5240, 2005.

Thayer, D., Wong, C.-H. Vancomycin analogs with improved biological activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy. Chem. Asian J., in press.

Wei, S.H., Rosen, H., Matheu, M.P., Sanna, M.G., Wang, S.-K., Jo, E., Wong, C.-H., Parker, I., Cahalan, M.D. Sphingosine 1-phosphate type 1 receptor agonism inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat. Immunol. 6:1228, 2005. Immunology

Nascent Drosophila Toll-8 crystals. Toll receptors are critical signaling receptors of development and of innate immunity conserved in evolution from flies to humans. Recombinant forms of these receptors are used to grow crystals and determine structures in order to establish a structure-function relationship. Work done in the laboratory of Luc Teyton, M.D., Ph.D. He Zhou, M.D., Ph.D., Research Associate

Ralph A. Reisfeld, Ph.D., Professor

Yunping Luo, M.D., Ph.D., Research Associate

Department of Immunology IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 99

DEPARTMENT OF Peter Ghazal, Ph.D. David Nemazee, Ph.D. R. Anthony Williamson, Ph.D. IMMUNOLOGY Adjunct Associate Professor Professor Associate Professor

Jiahuai Han, Ph.D. Glen R. Nemerow, Ph.D. Curtis B. Wilson, M.D. STAFF Professor Professor Professor Emeritus Richard J. Ulevitch, Ph.D. Wendy L. Havran, Ph.D. Professor and Chairman Per A. Peterson, M.D., Ph.D. Rong Xiang, M.D., Ph.D. Professor Adjunct Professor Assistant Professor Bruce A. Beutler, M.D. Shuang Huang, Ph.D. Professor Pascal Poignard, M.D. Michael Zwick, Ph.D. Assistant Professor Adjunct Assistant Professor Assistant Professor Roberto Baccala, Ph.D. Assistant Professor Julie Jameson, Ph.D. Ralph A. Reisfeld, Ph.D. Assistant Professor Professor STAFF SCIENTISTS Gary M. Bokoch, Ph.D. * Jonathan G. Kaye, Ph.D. Professor Matthias Riewald, M.D. Steven Brown, Ph.D. Associate Professor Assistant Professor Dennis R. Burton, Ph.D. ** Udayan Chatterji, Ph.D. Professor Richard Klemke, Ph.D.*** Hugh Rosen, M.D., Ph.D. Associate Professor Professor Xin Du, Ph.D. Tsung-Hsien Chuang, Ph.D. Moores Cancer Center Assistant Professor San Diego, California Wolfram Ruf, M.D. Colleen Fearns, Ph.D. Professor Charles G. Cochrane, M.D. Norman R. Klinman, M.D., Vladimir Kravchenko, Ph.D. Ph.D. Professor Emeritus Daniel R. Salomon, M.D. Professor Adjunct Associate Professor John Mathison, Ph.D. Neil R. Cooper, M.D. Ulla Gissi Knaus, Ph.D. Professor Emeritus Erica Ollmann Saphire, Ph.D. Anil Munshi, Ph.D. Associate Professor Assistant Professor Linda K. Curtiss, Ph.D. Rafal Pawlinski, Ph.D. Dwight Kono, M.D. Professor Nora Sarvetnick, Ph.D. Associate Professor Professor Ramona Petrovan, Ph.D. Edward A. Dennis, Ph.D. Jiing-Dwan Lee, Ph.D. Adjunct Professor David Schlaepfer, Ph.D. M. Germana Sanna, Ph.D. Associate Professor Associate Professor Henrik Ditzel, M.D., Ph.D. Laura Solforosi, Ph.D. Adjunct Professor Erguang Li, Ph.D. Linda A. Sherman, Ph.D. Assistant Professor Professor Deborah Witherden, Ph.D. Frank J. Dixon, M.D. Professor Emeritus Cheng Liu, M.D., Ph.D. Jonathan Sprent, M.D., Ph.D. Director Emeritus, Scripps Assistant Professor Adjunct Professor Research SENIOR RESEARCH Nigel Mackman, Ph.D. ASSOCIATES Charles D. Surh, Ph.D. Thomas S. Edgington, M.D. Associate Professor Professor Associate Professor Jasimuddin Ahamed, Ph.D. Michael McHeyzer-Williams, Ann J. Feeney, Ph.D. Ph.D. Luc Teyton, M.D., Ph.D. Gourab Bhattarcharjee, Ph.D. Associate Professor Associate Professor Associate Professor Joao da Silva Correia, Ph.D. Philippe Gallay, Ph.D. Dianne McKay, M.D. Argyrios N. Theofilopoulos, Associate Professor Assistant Professor M.D. Amr Abdelhamid El Sheikh, Professor Ph.D Nicholas R.J. Gascoigne, Donald E. Mosier, M.D., Ph.D. Ph.D. Peter S. Tobias, Ph.D. Kasper Hoebe, Ph.D. Professor Professor Associate Professor Eujing Jo, Ph.D. Amanda Gavin, Ph.D. Kerri A. Mowen, Ph.D. Susan R. Webb, Ph.D. Assistant Professor Assistant Professor Associate Professor Hyun-ku Lee, Ph.D. 100 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Rui Lin, Ph.D. Eleuterio De La Camara, Emma Hamilton-Williams, Carsten Kreig, Ph.D. Ph.D. *** Ph.D. Qilin Pan, Ph.D. Centro Nacional de Joerge Krueger, M.D.*** Investigaciones Lars Hangartner, Ph.D. Charité Children’s Hospital Andrew Saphire, Ph.D. Cardiovasculares Berlin, Germany Madrid, Spain Masaaki Hayashi, M.D., Yan Wu, Ph.D.**** Ph.D. Toru Kurokawa, Ph.D. Violane Delorme, Ph.D. Natasha Hill, Ph.D. Yumi Kurokawa, Ph.D. Celine Der Mardirossian, RESEARCH ASSOCIATES Ph.D. Neil John Hime, Ph.D. Young Back Kwon, Ph.D.**** Djemel Ait-Azzouzene, Ph.D. Benoit Besnues, Ph.D. Shihe Hou, Ph.D.*** Cheng Yu Lai, Ph.D. Abraxis BioScience Inc. Christopher Alfonso, Ph.D.*** Anthony Don, Ph.D. Los Angeles, California Jennifer Lamoureux, Ph.D. BD PharMingen San Diego, California Helen Donners, Ph.D.*** Hong Hua, Ph.D. Elise Landais, Ph.D. Institute of Tropical Medicine Sandrine Arnaud-Dabernat, Timothy Huang, Ph.D. Mansun Law, Ph.D. Antwerp, Belgium Ph.D. Christoph Huber, Ph.D. Jeff Lee, Ph.D. Caroline Aylott, Ph.D.**** Celine Eidenschenk, Ph.D. Milena Iacobelli, Ph.D. Sang-Un Lee, Ph.D.*** Celia Espinoza, Ph.D.**** Ann Bellon, Ph.D. Cummings School of Hassan Issafras, Ph.D. Veterinary Medicine at Nicolas Fazilleau, Ph.D. Michael Berger, Ph.D. Tufts University Zhengfan Jiang, Ph.D. North Grafton, Clemens Feistritzer, M.D. Dafang Bian, Ph.D.**** Massachusetts Wong Soon Justin, Ph.D. Christofer Flood, Ph.D. Joerge Birkenfeld, Ph.D. Sung-Hyung Lee, Ph.D.*** Young Jun Kang, Ph.D. Baylor College of Medicine Onur Boyman, Ph.D.*** Linda Frederick, Ph.D.*** Houston,Texas Centre Hospitalier Favrille, Inc. Yu-Ya Kao, Ph.D. Universitaire Vaudois San Diego, California Cheng Li, Ph.D.**** Immunology and Allergology Charles Kaplan, Ph.D.*** Lausanne, Switzerland Stefan Freigang, Ph.D. Genentech Corporation Jiali Li, Ph.D. San Francisco, California Carlos Cantu, Ph.D.*** Guo Fu, Ph.D. Xiang Li, Ph.D. Digital Gene Technologies Linda Kidd, Ph.D. La Jolla, California Michelle Fung, Ph.D. Yang Mi Li, Ph.D. Chang-Hoon Kim, Ph.D. John B. Carey, Ph.D. Philippe Georgel, Ph.D.*** Yilei Li, Ph.D.*** Laboratoire Hee Ok Kim, Ph.D.**** South China Medical Jianming Chen, Ph.D. d’Immunogénétique University Moléculaire Humaine Jun-Sub Kim, Ph.D. Guangzhou, China Marie Cherrier, Ph.D. Strasbourg, France Sungwoo Kim, Ph.D.*** Ssang-Taek Lim, Ph.D. Roshni Chintalapati, Ph.D. Davide Gianni, Ph.D. Assistant Professor University of Calgary Yang Mi Lim, Ph.D. Jae Ho Cho, Ph.D.**** Cristina Gil-Lamaignere, Calgary, Alberta, Canada Ph.D.*** Ting-Kun Lin, M.D., Ph.D.*** David Chodniewicz, Ph.D.**** Department of Cell Biology Rachel Kohler, Ph.D. Kaiser Permanente Medical Scripps Research Center Ben Croker, Ph.D. Edguardo Kolkowski, Ph. D. San Francisco, California Pedro Gonzalez-Cabrera, Aimee de Catherlineu, Ph.D. Ph.D. Marek Kovar, Ph.D.**** Guoxun Liu, Ph.D.****

Shrimati Datta, Ph.D. Fang Guo, Ph.D. Jirina Kovarova, Ph.D.**** Yuan Liu, Ph.D. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 101

Carina Lotz, Ph.D.*** Jared Purton, Ph.D. Joie Trifilo, Ph.D.**** Sun-Hee Yoon, Ph.D.**** Netherlands Cancer Institute Amsterdam, the Netherlands Christopher Ramsey, Ph.D. Marina Tsatmali, Ph.D. Kenji Yoshida, Ph.D.

Christine Louis dit Sully, M. Rachel Richards, Ph.D. David Valenta, Ph.D. Jiangiang Yu, Ph.D. Ph.D. Elise Romeo, Ph.D. Sebastian Vallee, Ph.D. Hui Zhang, Ph.D. Yunping Luo, M.D., Ph.D. Mark Rubenstein, Ph.D.*** Ester Van Leeuwen, Ph.D. You Qing Zhang, Ph.D. James P. Luyendyk, Ph.D. University of California San Diego, California Laurent Verkoczy, Ph.D.*** Tieming Zhao, Ph.D. Michael Lyman, Ph.D. Duke University Monica Ruse, Ph.D. Durham, North Carolina He Zhou, Ph.D. Chitladda Mahanivong, Ph.D.

Sophie Rutschmann, Ph.D. Hendrik Versteeg, Ph.D. Huamin Zhou, Ph.D.*** Laurent Malherbe, Ph.D. Xiamen University Maria Manukyan, Ph.D. Janelle Salkowitz-Bokal, Nicole Von Allmen-Zurcher, Xiamen, China Ph.D. Ph.D. Annette Marleau, Ph.D. Prabhakar Salunkhe, Katharina Von Lohneysen, SCIENTIFIC Beatriz Maroto, Ph.D.*** Ph.D.*** Ph.D. ASSOCIATES Centro Nacional de Burnham Institute Investigaciones Oncológicas La Jolla, California Meng Wang, M.D. Rosana Gonzales-Quintal, Madrid, Spain Ph.D. Jorge Luis Schettini, Ph.D. Yingchun Wang, Ph.D.*** David Marsolais, Ph.D. Moores Cancer Center Marcie Rose Kritzik, Ph.D. Nicolas Schrantz, Ph.D. San Diego, California Javier Martinez, Ph.D. Nora Leaf Reto Andreas Schupbach, Zhao Wang, Ph.D. Terrence Meehan, Ph.D. Ph.D. Ralph Pantophlet, Ph.D. Megumi Watanabe, Ph.D. Satyajit Mitra, Ph.D. Alim Seit-Nebi, Ph.D. *** Dongyuan Xia, Ph.D. Department of Molecular Chenghong Wei, Ph.D. Johann Mols, Ph.D. Biology Scripps Research Christopher Wiethoff, Adam Mullick, Ph.D. * Joint appointment in the Ph.D.*** Department of Cell Biology Suganya Selvarajah, Ph.D. Loyola University Perihan Nalbant, Ph.D. ** Joint appointment in the Chicago, Illinois Department of Molecular Biology Ron Nepomuceno, Ph.D. Shigeki Shimada, Ph.D. Justin Soon Boon Wong, *** Appointment completed, new location shown Frank Karl Niessen, Ph.D. Jason Smith, Ph.D. Ph.D. **** Appointment complete

Miyo Ota, Ph.D. Michelle Solomon, Ph.D.**** Chia Cheng Wu, Ph.D.

Takayuki Ota, Ph.D. Gabriel Sternik, Ph.D. Wenyuan Wu, Ph.D.

Motoyuki Otsuka, Ph.D. Konstantin Stoletov, Ph.D.*** Chengran Xu, Ph.D. Moores Cancer Center Sandrine Pacquelet, Ph.D. San Diego, California Yue Xu, Ph.D.

Nadige Pelletier, Ph.D. Joyce Tan, Ph.D.*** Pia Yachi, Ph.D. Anadys Pharmceuticals Olivier Pertz, Ph.D.*** San Diego, California Deepak Yadav, Ph.D. University of California San Diego, California Rachel Tilley, Ph.D. Michael Ye, Ph.D.

Helle Petersen, Ph.D. Antoine Toulon, Ph.D. Sook Wah Yee, Ph.D. 102 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

In 2006, he received the Excellence in Mentoring Award from the American Association of Immunologists for “exemplary career contributions to a future generation of scientists.” Many of Dr. Klinman’s more than 50 former trainees attended the award ceremony, which was held in Boston in May. We wish Dr. Klinman all the best in his future activities. We will always welcome his presence at Scripps Research. The end of 2005 also marked the departure of Jonathan Sprent, who returned to his native Australia after 30 years in the United States—first at the University of Pennsylvania and then at Scripps Research. Dr. Sprent is currently professor at the Garvan Institute of Medical Research in Sydney, where he will continue his seminal work on T-cell function combining his unique knowledge of the immune response in whole animals with molecular models using cell-based systems. We look forward to reading about Dr. Sprent’s new findings as they impact immunologic memory and tolerance, transplantation immunity, and cancer immunotherapy. His longstanding collaborations with Charlie Surh at Scripps Research will

Richard Ulevitch, Ph.D. undoubtedly continue, and we will welcome his visits to our campus. Coinciding with these departures is the recent recruit- Chairman’s Overview ment of Karsten Sauer, who joined us after a number of years at the Genomics Institute of the Novartis Research rogress in biomedical research demands innovation Foundation. Dr. Sauer specializes in studies of lympho- and depends on our ability to identify the leading cyte signal transduction and will bring additional strength P edges of science. The approach to science that to our department in this important field. Defects in lym- looks backward instead of forward stifles originality and phocyte development or function underlie various immune perpetuates conventional thinking. I am always impressed disorders, including immunodeficiencies, inflammatory by the ability of our department members to develop new and autoimmune diseases, and allergy. Dr. Sauer’s stud- paradigms to advance scientific knowledge. The environ- ies promise to significantly expand our understanding of ment at Scripps Research encourages our scientists to these key processes in health and disease. Specifically, apply the very latest approaches to solving core problems his laboratory will combine state-of-the-art genomic of immunology. This often involves taking risks, especially and proteomic profiling techniques, functional genomics, in today’s world of peer-reviewed funding where out-of- high-throughput screening, and imaging technologies as the-box thinking is not always rewarded. However, I am well as classical biochemistry, molecular biology, and pleased to report that our investigators are continuing to cell biology to explore the functions of novel signaling take appropriate risks with their science and, as a result, modules. This effort will result in a new understanding are being widely recognized for their accomplishments. of how antigen-receptor signaling directs diverse cellular This is evidenced by the numerous scientific papers pub- responses and how its malfunction leads to immunologic lished by our department members and by the prestigious disease. We look forward to Dr. Sauer’s contributions awards and other forms of recognition bestowed upon as an active faculty member in our department. them. Some of these are highlighted below. Highlights of scientific publications provide clear But first I would like to acknowledge some key transi- evidence of the important place our department holds tions within our faculty. During the past year, Norman within the national and international community of scien- Klinman retired after an incredibly productive career of tists interested in immunology. Bruce Beutler has provided more than 30 years in the field of immunology research. Scripps Research with a unique resource in the form of IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 103 a program of random germline mutagenesis. During the Williams and members of his laboratory, whose work past year, he and his colleagues have provided remark- on B-cell function was recently described in an article able new insights into mechanisms of innate and adap- published in Immunity. tive immunity. Key findings include those of Koichi Tabeta In both the United States and worldwide, HIV and and his colleagues, published in Nature Immunology, in hepatitis C infection remain significant problems for which which an unanticipated function has been assigned to there are still no appropriate therapies. Dennis Burton the Unc93b1 gene product. These investigators showed and his group continue their important research in these that a mutation in Unc93b1, known as the 3d muta- two areas. This past year, they published articles in tion, disrupts signaling via Toll-like receptors 3,7, and 9, Science, Nature Immunology, and Proceedings of the and also alters antigen presentation. Building on these National Academy of Sciences that provided insights data are findings from Kasper Hoebe and colleagues, into key issues for advancement of new treatments. These published this year in Immunity, have identified a novel recent publications represent the important collabora- pathway of T-cell activation that appears to be indepen- tions established by Dennis Burton and his colleagues dent of Toll-like receptor signaling. Taken together, these both inside and outside of Scripps Research. two publications promise to shape our thinking about Work from the laboratory of Hugh Rosen combines mechanisms involved in promoting adaptive immunity chemical and genetic approaches to proof-of-concept through the innate immune networks. studies that will allow a better understanding of human In addition to uncovering new biology, the germline disease mechanisms. This work paves the way for the mutagenesis approach provides new insights into protein development of new small-molecule therapeutics for use structure as revealed in Proceedings of the National in immunologic and inflammatory diseases in humans. Academy of Sciences by Zhengfan Jiang and his col- Specifically, publications in Nature Immunology, Nature leagues, who describe molecular details of interactions Chemical Biology, and other top journals illustrate the between MyD88 and Toll-like receptors. We also look importance of the approaches implemented by Hugh forward to continued productivity and seminal advances Rosen and his group. Further illustration of the power from Bruce Beutler and his group during the coming year. of genetics is found in work published in Nature Medi- Adding to the department’s strengths in innate immu- cine by members of the J-D Lee’s laboratory. Here they nity is the work done in the laboratory of Jiahuai Han. used conditional knock-out of the HSP40 gene to prove DaSilva Correia and his group provided an unexpected a role for this protein in a serious human syndrome, insight into a mechanism whereby Nod1 regulates the cardiomyopathy. This work may well lead to the devel- function of the estrogen receptor. Nod1 provides a brake opment of drugs that can be used to selectively inter- on the estrogen receptor, and the absence of Nod1 results vene in this serious medical problem. in enhanced sensitivity to estrogen and promotes tumor These examples illustrate the productivity of the mem- growth in a xenograft model. This work was published bers of our department. More details and a complete list in Proceedings of the National Academy of Sciences. of accomplishments can found in the individual reports. Members of Luc Teyton’s laboratory combine struc- I apologize in advance for not including comments about ture-function studies with both cell-based and animal each publication from the 2005-2006 period and urge models to probe the function of the natural killer T-cell readers to examine the reports for more details. As always, receptor and its ligand. This work, which was published it is a great pleasure for me to read the material from in Nature and Nature Immunology, includes a longstanding each of our faculty members and to reflect on the prog- collaboration with Albert Bendelac at the University of ress of the past year. Chicago. Following on studies of immune regulatory pathways is work done by Charlie Surh and his colleagues, which was published in Science. This report documents an unexpected finding showing how an immune complex of anti-IL2 and IL2 act to stimulate T-cell subsets. These data not only provide new insights into uses of therapeutic antibodies but also alert us to concerns about unanticipated side effects. Another advance in understanding adaptive immunity comes from Michael McHeyzer- 104 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

INVESTIGATORS’REPORTS Analyzing Host Resistance: Phenomenon to Phenotype to Gene

M. Barnes, K. Benson, M. Berger, B. Croker, K. Crozat, X. Du, C. Eidenschenk, K. Hoebe, Z. Jiang, B. Layton, N. Nelson, B. Ortiz, T. Palmer, S. Rutschmann, S. Sovath, H. Uy, K. Whitley, Y. Xia, B. Beutler

ecause microbes are an enormous threat to most Fig. 1. Map locations of mutations with immunologic effects vertebrate species, mammalian evolution has produced to date. Shaded ovals indicate that the mutation has been shaped by the microbial environment, and B been identified by using positional cloning. many examples of protein modification were driven by the need to evade infection. On an evolutionary time- tion of proteins, which in the final analysis often act scale, a fairly large fraction of the genome in mammals as minute “machines.” We have seen each of these has been appropriated chiefly to create resistance to outcomes in the course of our research. infection. Those proteins with nonredundant function A phenotype called 3d (to connote a “triple defect” of in resistance to a particular organism are components nucleic acid sensing) was tracked to a multispanning of the host “resistome.” membrane protein with no previously known function. To identify key resistance proteins, we use random This protein, known as UNC-93B, is required for the germ-line mutagenesis. In this process, a chemical muta- integrity of a specific class of endosomes, small bodies gen, N-ethyl-N-nitrosourea, is used to induce a distinctive within cells within which sensing of nucleic acids and phenotype (e.g., inadequate defense against infection). processing of antigens for activation of the immune sys- The phenotype is then genetically mapped, and the tem occur. The 3d mutation causes susceptibility to a causal mutation is found by using DNA sequencing. Once broad range of infections because microbial nucleic cause and effect have been established, we can deter- acids are an important clue to the presence of infection, mine precisely how a given protein contributes to defense and when the host remains unaware of these nucleic against infection. To date, we have created scores of acids, infections may grow out of control. In addition, mutations that impair resistance to various pathogens; the 3d mutation prevents antigen-presenting cells from we have mapped a total of 32 of these mutations to delivering foreign proteins to T lymphocytes, particu- chromosomal intervals. Most of the mapped mutations larly CD8+ cells, which are required to directly attack have been solved at a molecular level (Fig. 1). and eliminate infection within tissues of the host. Many of the proteins identified in this way are previ- Some mutations create subtle changes within pro- ously unknown components of the resistome. Some of teins, revealing functions that were previously hidden the proteins serve signaling by the Toll-like receptors, because total disruption of the protein is lethal. One which we previously showed are key sensors by which such mutation, called woodrat, causes graying of the mammals detect infection. Other identified proteins are fur and a moderate, generalized immunodeficiency important components of the cytokine response appa- (e.g., failure to contain infection caused by mouse ratus, required for the production or activity of TNF or cytomegalovirus). This mutation affects an enzyme type I interferons. Remarkably, many of the proteins that is of central importance in cholesterol metabolism. do not yet fit into a cohesive picture. Rather, they are Known as the site 1 protease, the enzyme cleaves a puzzles waiting to be solved. Germ-line mutations can latent transcription factor required for activation of disclose the function of proteins where no function was genes that encode other enzymes required to synthe- previously assigned. The mutations can also reveal new size cholesterol. Although mice lacking the site 1 pro- functions of a protein, if some functions were previously tease cannot survive to term, woodrat mutants are known. And the mutations can shed light on the func- fully viable, have exceptionally low blood levels of cho- IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 105

lesterol, and have a diminished ability to cope with Beutler, B. Microbial pathogenesis and the discovery of Toll-like receptor function. In: Vaccine Adjuvants: Immunological and Clinical Principles. Hackett, C.J., Harn, infection. Hence, a new function of the site 1 protease D.A., Jr. (Eds.). Humana Press, Totowa, NJ, 2005, p. 1.

has come to light. Beutler, B. The Toll-like receptors. In: Genetic Susceptibility to Infection. Kaslow, The syndrome of shock that occurs during serious R.L., McNicholl, J., Hill, A.V.S. (Eds.). Oxford University Press, New York, in press.

infections of all kinds is mediated by signaling via Toll- Beutler, B. The Toll-like receptors: analysis by forward genetic methods. Immuno- like receptors, and for this reason, the exact structure genetics 57:385, 2005. of the transduction apparatus that alerts the host to Beutler, B., Casanova, J.-L. New frontiers in immunology. EMBO Rep. 6:620, 2005.

the presence of lipopolysaccharide, bacterial lipopep- Beutler, B., Georgel, P., Rutschmann, S., Jiang, Z., Croker, B., Crozat, K. Genetic tides, unmethylated DNA, and other signature molecules analysis of innate resistance to mouse cytomegalovirus (MCMV). Brief. Funct. Genomic Proteomic 4:203, 2005. has been the object of intense interest. The Toll-like receptors recruit specific adapter proteins, which in Beutler, B., Hoebe, K., Georgel, P., Tabeta, K., Du, X. Genetic analysis of innate immunity: identification and function of the TIR adapter proteins. Adv. Exp. Med. turn activate protein kinases that ultimately promote Biol. 560:29, 2005.

the transcription of hundreds of genes that shape the Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S., Du, X., inflammatory response. A mutation in one such adapter Hoebe, K. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annu. Rev. Immunol. 24:353, 2006. protein, MyD88, has helped us understand precisely how the initial signal is transmitted. Called Pococurante, Crozat, K., Georgel, P., Rutschmann, S., Mann, N., Du, X., Hoebe, K., Beutler, B. Analysis of the MCMV resistome by ENU mutagenesis. Mamm. Genome 17:398, this mutation has revealed the location of the recep- 2006.

tor-adapter interface, and molecular docking studies, Du, X., Tabeta, K., Mann, N., Crozat, K., Mudd, S., Beutler, B. An essential role performed in collaboration with I.A. Wilson and A.J. for Rxrα in development of Th2 responses. Eur. J. Immunol. 35:3414, 2005.

Olson, Department of Molecular Biology, have sug- Fischer, H., Yamamoto, M., Akira, S., Beutler, B., Svanborg, C. Mechanism of gested exactly how the receptor and adapter subunits pathogen-specific TLR4 activation in the mucosa: fimbriae, recognition receptors and adaptor protein selection. Eur. J. Immunol. 36:267, 2006. fit together (Fig. 2). Hoebe, K., Beutler, B. TLRs as bacterial sensors. In: Toll-like Receptors in Inflam- mation. O’Neil, L.A.J., Brint, E. (Eds.). Birkhauser, New York, 2006, p. 000. Prog- ress in Inflammation Research, Parnham, M.J. (Series Ed.).

Hoebe, K., Beutler, B. TRAF3: a new component of the TLR-signaling apparatus. Trends Mol. Med. 12:187, 2006.

Hoebe, K., Jiang, Z., Georgel, P., Tabeta, K., Janssen, E., Du, X., Beutler, B. TLR signaling pathways: opportunities for activation and blockade in pursuit of therapy. Curr. Pharm. Des., in press.

Hoebe, K., Jiang, Z., Tabeta, K., Du, X., Georgel, P., Crozat, K., Beutler, B. Genetic analysis of innate immunity. Adv. Immunol. 91:175, 2006.

Huber, M., Kalis, C., Keck, S., Jiang, Z., Georgel, P., Du, X., Shamel, L., Sovath, S., Mudd, S., Beutler, B., Galanos, C., Freudenberg, M.A. R-form LPS, the master key to the activation of TLR4/MD2 positive cells. Eur. J. Immunol. 36:701, 2006.

Janssen, E., Tabeta, K., Barnes, M.J., Rutschmann, S., McBride, S., Bahjat, K.S., Schoenberger, S.P., Theofilopoulos, A.N., Beutler, B., Hoebe, K. Efficient T cell activation via a Toll-Interleukin 1 receptor-independent pathway. Immunity. 24:787, 2006. Fig. 2. The junction between a Toll-like receptor and its cytoplasmic adapter protein. Only the so-called TIR domains, which are Jiang, Z., Georgel, P., Li, C., Choe, J., Crozat, K., Rutschmann, S., Du, X., Bigby, T., involved in binding, are shown. Mudd, S., Sovath, S., Wilson, I.A., Olson, A., Beutler, B. Details of Toll-like receptor:adapter interaction revealed by germ-line mutagenesis. Proc. Natl. Acad. Sci. U. S. A. 103:10961, 2006. These few examples illustrate the power of the Kim, K.I., Malakhova, O., Hoebe, K., Yan, M., Beutler, B., Zhang, D.-E. Enhanced classical genetic approach—which proceeds from phe- antibacterial potential in UBP43-deficient mice against Salmonella typhimurium nomenon to phenotype to gene—in analyzing immu- infection by up-regulating type I IFN signaling. J. Immunol. 175:847, 2005.

nity. However, the same approach also sheds light on Nemazee, D., Gavin, A.L., Hoebe, K., Beutler, B. Immunology: Toll-like receptors many other aspects of biology. Some of the mutations and antibody responses. Nature 441:E4, 2006. that we have produced answer questions in the realm Rutschmann, S., Hoebe, K., Zalevsky, J., Du, X., Mann, N., Dahiyat, B.I., Steed, P., Beutler, B. PanR1, a dominant negative missense allele of the gene encoding TNF- of behavioral neuroscience, metabolism, and develop- α (Tnf), does not impair lymphoid development. J. Immunol. 176:7525, 2006. ment and raise new questions in turn. Tabeta, K., Hoebe, K., Janssen, E.M., Du, X., Georgel, P., Crozat, K., Mudd, S., Mann, N., Sovath, S., Goode, J., Shamel, L., Herskovits, A.A., Portnoy, D.A., PUBLICATIONS Cooke, M., Tarantino, L.M., Wiltshire, T., Steinberg, B.E., Grinstein, S., Beutler, Beutler, B. Innate Immunity. In: Williams Hematology, 7th ed. Lichtman, M.A., et B. The Unc93b1 mutation 3d disrupts exogenous antigen presentation signaling al. (Eds.). McGraw-Hill, New York, 2005, p. 231. via Toll-like receptors 3, 7 and 9. Nat. Immunol. 7:156, 2006. 106 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Wieland, C.W., Florquin, S., Maris, N.A., Hoebe, K., Beutler, B., Takeda, K., Akira, S., van der Poll, T. The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable Haemophilus influenzae from the mouse lung. J. Immunol. 175:6042, 2005.

Xia, C.H., Liu, H., Cheung, D., Cheng, C., Wang, E., Du, X., Beutler, B., Lo, W.K. Gong, X. Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis. Development 133:2033, 2006.

Regulation of Cell Function by Rho GTPases

G.M. Bokoch, J. Birkenfeld, V. Delorme, C. DerMardirossian, A.M. DeCathelineau, D. Gianni, T. Huang, Y.-Y. Kao, J.-S. Kim,

P. Nalbant, K. Pestonjamasp, S.-H. Yoon, H. Zhang, T. Zhao, Fig. 1. Two-step activation mechanism for Rac GTPase–mediated B.P. Bohl, M. Crawford, B. Fowler, J.-Y. Seo, Z.-F. Chang* regulation of oxidant formation by the phagocyte NADPH oxidase. * National Taiwan University, Taipei, Taiwan poral localization of Rho GTPase activation. We are ho GTPases control the assembly of the actin beginning to determine the molecular signals that gov- and microtubule cytoskeletons, the production ern the chemotactic responses of human leukocytes. of reactive oxygen species (ROS), and the activ- R Recently, we described the ability of Rac1 signaling in ity of kinase cascades that mediate cell growth, death, neutrophils to stimulate RhoA activation at the rear of and motility. This spectrum of activities makes Rho cells. Such Rho GTPase cross talk promotes the devel- GTPases key components of such physiologic and patho- opment of the stable cell polarity necessary to main- logic processes as tumor growth and metastasis, wound tain directionality of chemotaxis during inflammatory healing, neuronal connectivity, inflammatory responses, responses. Studies of the dynamics of Cdc42 activation and development. We use cellular, molecular, biophys- during neutrophil chemotaxis are ongoing. ical, and biochemical approaches to understand how the activities of Rho GTPases are regulated, to identify REGULATION OF INNATE IMMUNITY BY ANTHRAX TOXINS the proteins they interact with to control cell function, Bacillis anthracis inhibits the function of immune and to ascertain how these regulatory processes are cells by generating lethal toxin and edema toxin. As abnormal in various disease states. part of a program grant funded by the Centers for Dis- RHO GTPases AND HUMAN LEUKOCYTES We previously established that the GTPase Rac2 ease Control and Prevention, we are investigating the regulates the formation of ROS that are used by human molecular basis for the suppressive effects of the anthrax phagocytic leukocytes for microbial killing and that result toxins on the function of human leukocytes. We have in inflammatory responses. Our discovery of a func- established that anthrax edema toxin and lethal toxin tional interaction between Rac2 and cytochrome b, a effectively block the ability of chemoattractant recep- component of the membrane-bound NADPH oxidase, tors to stimulate the production of ROS by human neu- independent of p67phox, led us to propose a 2-step trophils. The molecular basis for such inhibition is mechanism for regulation of electron transfer to form currently under investigation. A requirement for Rho superoxide (Fig. 1). We are mapping the binding site GTPases in the uptake and action of anthrax toxins in for Rac2 on cytochrome b to investigate the molecular macrophages is also under study (Fig. 2). basis for regulation of ROS production by Rac2. In addi- CYTOSKELETAL REGULATION BY RHO GTPases tion to their role in innate immunity, NADPH oxidases The p21-activated kinases (PAKs) are Rac and participate in intracellular signaling. Regulation of non- Cdc42 effectors that serve as important mediators of phagocytic NADPH oxidases is largely not understood, chemotaxis, wound healing, tumor metastasis, neurite but we are investigating their modulation by kinase outgrowth, antigen presentation, and other processes pathways that phosphorylate regulatory components of dependent on cytoskeletal polarization. In collabora- the oxidases. tive studies with G. Danuser and C. Waterman-Storer, We are using live-cell imaging in combination with Department of Cell Biology, we are using quantitative fluorescent methods to determine the spatial and tem- fluorescent speckle microscopy to investigate the regu- IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 107

GDP dissociation inhibitors are critical regulators of Rho GTPase function. They have been linked to kidney disease and to the ability of cancer cells to metastasize. We found that the interaction of GDP dissociation inhibitors with Rho GTPases is regulated by phosphorylations initiated through various signaling pathways. Indeed, tyrosine phosphorylation may disrupt the regulatory capability of the inhibitors to promote cell transformation and metastasis. Cell division also requires highly regulated actin- myosin-microtubule dynamics. We established that cross talk between the actin and microtubule cytoskeletons involving Rho regulation occurs via physical sequestration of the Rho guanine nucleotide exchange factor H1 (GEF-H1) by microtubules. GEF-H1 serves as a link Fig. 2. Rho GTPase regulation of the action of anthrax lethal between mitotic spindle microtubules and the initiation toxin. Anthrax toxin is a ternary complex consisting of 1 binding of Rho-dependent formation of cleavage furrows in divid- subunit, protective antigen (PA), and 2 enzymatic subunits, edema ing cells (Fig. 3). GEF-H1 activity is also controlled by factor (EF) or lethal factor (LF). Full-length PA (PA83) binds to receptors on the cell surface and is cleaved by a furinlike protease to its active form (PA63). Active PA oligimerizes, driving receptor aggregation and internalization by endocytosis. During normal maturation and acidification of the endosomes, PA forms a channel through which EF and LF are transported from the endosomal compartment and into the cytoplasm to act on their respective effectors. Rho GTPases may act to regulate endocytosis, endosomal maturation, and toxin escape or activity. Figure courtesy of Aimee DeCathelineau. Fig. 3. Immunofluorescent images show colocalization of endogenous GEF-H1 with microtubules (tubulin) in the mitotic spindle. lation of leading-edge actin dynamics by PAK1 downstream of Rac GTPase. We found that PAK1 plays an cell cycle–dependent kinases. Detailed analysis of the important role in coupling cell-edge protrusion mechan- function of GEF-H1 in cell division and motility is under ics to upstream signaling events and downstream motility. way. Of interest, GEF-H1 is abundant in blood cells The phosphorylation of cofilin, which depolymerizes and is downregulated by recently developed drugs that and severs actin, by PAK1 acting through LIM kinase inhibit chronic leukemias. is an important regulatory point in cell motility. Using a biochemical screen, we identified a unique cofilin PUBLICATIONS Belvindrah, R., Nalbant, P., Ding, S., Wu, C., Bokoch, G.M., Müller, U. Integrin- phosphatase, termed chronophin, that regulates stim- linked kinase regulates Bergmann glial differentiation during cerebellar develop- ulus-dependent activation of cofilin. Using small inter- ment. Mol. Cell. Neurosci. 33:109, 2006. fering RNA to reduce the expression of chronophin, we Birukova, A.A., Adyshev, D., Gorshkov, B., Bokoch, G.M., Birukov, K.G., Verin, A.D. GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier discovered that this phosphatase is involved in the con- dysfunction. Am. J. Physiol. Lung Cell Mol. Physiol. 290:L540, 2006. trol of cytokinesis during cell division. Chronophin is Bokoch, G.M., Zhao, T. Regulation of the phagocyte NADPH oxidase by Rac implicated in the formation of aneuploid cancers; it is GTPase. Antioxid. Redox. Signal. 8:1533, 2006. overexpressed in such tumors and is an autoantigen in Chang, Y.-C., Lee, H.-H., Chen, Y.-J., Bokoch, G.M., Chang, Z.-F. Contribution of patients with cancer. Our recent data indicate that this guanine exchange factor H1 in phorbol ester-induced apoptosis. Cell Death Differ., in press. unique regulatory phosphatase orchestrates actin dynam- Crawford, M., Aylott, C., Bourdeau, R.W., Bokoch, G.M. Bacillus anthracis toxins ics at the leading edge by modulating cofilin activity, inhibit human neutrophil NADPH oxidase activity. J. Immunol. 176:7557, 2006. thereby increasing cancer cell motility stimulated by DeCathelineau, A.M., Bokoch, G.M. Peptide inhibitors MAP the way towards fight- epidermal growth factor. We have also linked chronophin ing anthrax. Biochem. J. 395:e1, 2006. to cytoskeletal changes initiated during cellular energy DerMardirossian, C., Bokoch, G.M. Phosphorylation of RhoGDI by p21-activated (ATP) depletion induced by processes such as ischemia. kinase 1. Methods Enzymol. 406:80, 2006. 108 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

DerMardirossian, C., Rocklin, G., Seo, J.Y., Bokoch, G.M. Phosphorylation of of passive transfer studies in a number of animal model RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling. Mol. Biol. Cell, in press. systems, the answer is clearly yes. Complete protection is possible at serum titers of neutralizing antibody Dong, X., Mo, Z., Bokoch, G.M., Guo, C., Li, Z., Wu, D. P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils. Curr. Biol. 15:1874, greater than about 1:100, although lower titers can 2005. provide benefit in terms of lowered or delayed viremia. Huang, T.Y., DerMardirossian, C., Bokoch, G.M. Cofilin phosphatases and regula- We also showed that topically applied antibody can tion of actin dynamics. Curr. Opin. Cell Biol. 18:26, 2006. protect monkeys against vaginal challenge with virus. Pestonjamasp, K.N., Forster, C., Sun, C., Gardiner, E.M., Bohl, B., Weiner, O., In addition, passive transfer studies with engineered Bokoch, G.M., Glogauer, M. Rac1 links leading edge and uropod events through Rho and myosin activation during chemotaxis. Blood. 108:2814, 2006. antibodies in macaques suggest that antibody effector functions, as well as classical neutralization, may be Stofega, M., DerMardirossian, C., Bokoch, G.M. Affinity-based assay of Rho GTPase activation. Methods Mol. Biol. 332:269, 2006. important in protection against HIV. Another major issue is the best method for eliciting protective neutralizing antibodies. Accumulated Human Antibodies and Design evidence suggests that protective neutralizing antibodies are those antibodies that bind avidly to the enve- of a Vaccine to HIV Type 1 lope trimer on the surface of HIV-1 virions. However, such antibodies, particularly those to conserved regions M.B. Zwick, R.A. Pantophlet, R.O. Aguilar-Sino, of the envelope that are most important for vaccines, R.D. Astronomo, D.R. Bowley, H. Donners, A.K. Gakhal, are difficult to elicit. Apparently the envelope trimer, E. Giang, L. Hangartner, A.J. Hessell, R.C. Jensen, M. Law, which is composed of 2 glycoproteins, gp120 and J.D. Nelson, S. Pollock, E.M. Scherer, M. Wang, R.A. Dwek,* gp41, has low antigenicity and immunogenicity. Sev- D. Calarese, R.M. Cardoso,R.L. Stanfield, I.A. Wilson, eral strategies to circumvent these problems are being investigated. One strategy is to study the interaction of D.R. Burton the neutralizing antibodies with envelope glycoprotein * Oxford Glycobiology Institute, Oxford, England at the molecular level and then use the knowledge IV type 1 (HIV-1) is a scourge on humanity. gained to design antigens capable of eliciting the rele- Nearly 40 million persons are infected with the vant antibodies. In these studies, we are collaborating H virus, and about 20 million have died of AIDS. with I.A. Wilson, Department of Molecular Biology. We It is widely recognized that a vaccine most likely is the are also working closely with P. Dawson, Department best way to control HIV infection worldwide. All cur- of Cell Biology, to design peptide immunogens and with rent antiviral vaccines elicit antibody responses that C.-H.Wong, Department of Chemistry, and R. Dwek, are thought to be crucial to the efficacy of the vac- Oxford Glycobiology Institute, to design and select car- cines. We wish to understand antibody responses to bohydrate immunogens. HIV in humans and to design vaccines that will elicit Finally, we are exploring the specificities of anti- protective responses to the virus. bodies from those rare humans who make antibodies We used phage display technology to generate pan- that neutralize a broad array of different strains of HIV. els of human monoclonal antibodies to HIV. We are We have evidence that a number of specificities are examining human antibody responses to the virus and involved, and we are attempting to describe these. We the antiviral activities of these antibodies. In particu- are generating human monoclonal antibodies by using lar, we generated a human monoclonal antibody, b12, not only phage display but also yeast display and the that neutralizes a broad array of different strains of HIV. rescue of memory B cells. The existence of this antibody indicates that some features of HIV are conserved and are attractive targets PUBLICATIONS Braciale, T.J., Hahn, Y.S., Burton, D.R. Adaptive immune responses to viral infec- for vaccines. Further, b12 and a few monoclonal anti- tion. In: Fields Virology, 5th ed. Knipe, D.M., et al. (Eds.). Lippincott Williams & bodies with similar qualities are powerful tools for Wilkins, Philadelphia, in press. exploring antibody activity against HIV-1. Brunel, F.M., Zwick, M.B., Cardoso, R.M.F., Nelson, J.D., Wilson, I.A., Burton, D.R., Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutraliz- Among the first questions we have tackled were the ing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006. following: Can antibodies protect against HIV-1 infec- Burton, D.R., Stanfield, R.L.. Wilson, I.A. Antibody versus HIV in a clash of evolution, and, if so, under what conditions? On the basis tionary titans. Proc. Natl. Acad. Sci. U. S. A. 102:14943, 2005. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 109

Calarese, D.A., Lee, H.-K., Huang, C.-Y, Best, M.D., Astronomo, R.D., Stanfield, deadliest human pathogens. Results indicate strong R.L., Katinger, H., Burton, D.R., Wong, C.-H, Wilson, I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. cross-reactivity and immunogenicity of the different Natl. Acad. Sci. U. S. A. 102:13372, 2005. glycoproteins. This finding supports the hypothesis Calarese, D., Scanlan, C., Lee, H.-K., Rudd, P., Wong, C.-H., Dwek, R.A., Bur- that some of the soluble forms of the glycoproteins act ton, D.R., Wilson, I.A. Towards a carbohydrate-based HIV-1 vaccine. In: Carbohy- drate Drug Design. Klyosov, A.A., Witczak, Z.J., Platt, D. (Eds.). Oxford University as decoys. Press, New York, 2006, p. 161. American Chemical Society Symposium Series. We have also been investigating the effects of a

Delves, P.J., Martin, S.J., Burton, D.R., Roitt, I.M. Roitt’s Essential Immunology, human monoclonal antibody that neutralizes Ebola 11th ed. Blackwell Publishing, Oxford, England, in press. virus; the antibody was isolated from a patient who

Koff, W.C., Johnson, P.R., Watkins, D.I., Burton, D.R., Lifson, J.D., Hasenkrug, was infected with the virus in the Democratic Repub- K.J., McDermott, A.B., Schultz, A., Zamb, T.J., Boyle, R., Desrosiers, R.C. HIV lic of Congo and who recovered. Previously, we showed vaccine design: insights from live attenuated SIV vaccines. Nat. Immunol. 7:19, 2006. that the antibody protects guinea pigs against challenge with Ebola virus. However, studies done in col- Law, M., Sanna, P.P., Burton, D.R. Viral subversion of humoral immune responses. In: Microbial Subversion of Immunity: Current Topics. Lachmann, P.J., Oldstone, laboration with scientists at the National Institute of M.B.A. (Eds.). Caister Academic Press, Norfolk, England, 2006, p. 177. Allergy and Infectious Diseases, indicate that the anti- Lindenbach, B.D., Evans, M.J., Syder, A.J., Wolk, B., Tellinghuisen, T.L., Liu, body does not protect monkeys and indeed appears to C.C., Maruyama, T., Hynes, R.O., Burton, D.R., McKeating, J.A., Rice, C.M. Complete replication of hepatitis C virus in cell culture. Science 309:623, 2005. offer little benefit even when given at high doses. Sur- prisingly, viral replication apparently can proceed unhin- Moore, P.L., Crooks, E.T., Porter, L., Zhu, P., Cayanan, C.S., Grise, H., Corcoran, P., Zwick, M.B., Franti, M., Morris, L., Roux, K.H., Burton, D.R., Binley, J.M. dered in the tissues of the monkeys even in the presence Nature of nonfunctional envelope proteins on the surface of human immunodefi- of high serum concentrations of antibody.We are ciency virus type 1. J. Virol. 80:2515, 2006. attempting to understand this phenomenon and rec- O’Connor, D.H., Burton, D.R. Immune responses and HIV: a little order from the chaos. J. Exp. Med. 203:501, 2006. oncile it with the ability of certain vaccines to protect against challenge with Ebola virus. Pantophlet, R., Burton, D.R. GP120: target for neutralizing HIV-1 antibodies. Annu. Rev. Immunol. 24:739, 2006.

Selvarajah, S., Puffer, B., Pantophlet, R., Law, M., Doms, R.W., Burton, D.R. Comparing antigenicity and immunogenicity of engineered gp120. J. Virol. Toll-like Receptors and 79:12148, 2005.

Venturini, S., Allicotti, G., Zhao, Y., Simon, R., Burton, D.R., Pinilla. C., Poignard, P. Inflammation in Atherosclerosis Identification of peptides from human pathogens able to cross-activate an HIV-1- gag-specific CD4+ T cell clone. Eur. J. Immunol. 36:27, 2006. A.E. Mullick, C. Flood, N.J. Hime, M.R. Richards, Yuste, E., Sanford, H.B., Carmondy, J., Bixby, J., Little, S., Zwick, M.B., Gree- D.T.Valenta, R.J. Petrovan, P.S. Tobias, L.K. Curtiss nough, T., Burton, D.R., Richman, D.D., Desrosiers, R.C., Johnson, W.E. Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV- 1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive he ability of Toll-like receptors (TLRs) to detect a plasma. J. Virol. 80:3030, 2006. spectrum of pathogen-derived molecules defines Zhang, M.X., Bohlman, M.C., Itatani, C., Burton, D.R., Parren, P.W.H.I., St. Jeor, T the importance of the receptors in innate immu- S.C., Kozel, T.R. Human recombinant antimannan immunoglobulin G1 antibody nity and provides a mechanistic link between infection confers resistance to hematogenously disseminated candidiasis in mice. Infect. Immun. 74:362, 2006. and disease. Atherosclerosis is a chronic inflammatory disease in which immune and metabolic factors interact Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D.R., Wieland, S.F., Uprichard, S.L., Wakita, T., Chisari, F.V. Robust hepatitis C virus to initiate and propagate arterial lesions. An understand- infection in vitro.Proc. Natl. Acad. Sci. U. S. A. 102:9294, 2005. ing of TLRs in atherosclerosis could clarify the etiology of this complex process. Furthermore, the existence of host-derived endogenous ligands for TLRs may implicate Antibodies and Emerging Viruses involvement of the receptors in disease mechanisms beyond innate immunity, such as homeostatic mecha- W.B. Oswald, E.O. Saphire, N.L. Sullivan,* P.B. Jahrling,* nisms to resolve injury. Our studies of atherosclerosis- P.W.H.I. Parren,** D.R. Burton susceptible mouse models highlight TLR involvement * National Institute of Allergy and Infectious Diseases, Bethesda, Maryland in the process of this vascular disease. ** Genmab, B. V., Utrecht, the Netherlands Distinguishing between local and systemic factors e are interested in determining the immuno- that contribute to the development of atherosclerotic genicity of soluble vs surface glycoproteins lesions via activation of TLRs induced by endogenous W of Ebola virus, a filovirus that is one of the TLR ligands is formidable. However, bone marrow trans- 110 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE plantation allows alteration of gene expression for spe- of aortic sinus and en face aortic lesions were increased cific types of cells found in atherosclerotic lesions. Such 82% and more than 400%, respectively. In similarly chimeric mice can be produced to express or lack a sin- treated LDL–/– mice that also lacked the gene for TLR2, gle gene in key effector cells, including macrophage- no increase in lesion severity occurred. derived foam cells, involved in the development of To dissect the proatherosclerotic mechanism of atherosclerosis. Furthermore, by using reverse bone TLR2 activation, we used bone marrow transplanta- marrow transplantation, chimeric mice can be produced tion to produce TLR2-deficient chimeras. LDLr–/– mice that express the gene solely in bone marrow–derived were reconstituted with bone marrow cells from wild- cells. These procedures enable us to study the effects type or TLR2-deficient mice. Using the same method of gene expression specific to bone marrow cells dur- of TLR2 activation as before, we found a proathero- ing disease progression. sclerotic effect of Pam3 only in mice reconstituted with Using bone marrow transplantation, we generated wild-type bone marrow. These mice had increases in atherosclerosis-susceptible chimeric mice with bone lesion severity of 89% and 240% in the aortic sinus marrow cells that lacked the gene for either TLR2 or and the aorta, respectively. TLR4. Lesions containing macrophage-derived foam Hence, although both groups of mice had intact cells lacking TLR2 or TLR4 developed in these animals. TLR2 signaling in cells not derived from bone marrow, Surprisingly, when chimeric mice that lacked TLR2 or only those mice that expressed TLR2 in bone marrow– TLR4 solely in bone marrow–derived cells and that also derived cells, such as monocyte-derived macrophages, lacked receptors for low-density lipoprotein (LDLr–/–) had increased atherosclerosis when activated with the were fed an atherogenic diet for 4 months, no changes exogenous agonist. Additionally, the TLR2-induced accel- occurred in the size of lesions in either the aortic sinus eration of lesion development occurred with an unusual or the aorta. pattern of atherosclerosis, with profuse abdominal To rule out the possibility of a role for TLR2 expres- lesions. Subsequent experiments with intravenous sion by bone marrow–derived cells, we used reverse administration of Pam3 did not reproduce this peculiar bone marrow transplantation. Chimeras with TLR2 development of abdominal lesions, thereby implicating expression confined to bone marrow–derived cells a role of peritoneal inflammatory mediators in the effects –/– –/– were produced by using female LDLr TLR2 mice of Pam3 injected intraperitoneally. as recipients and wild-type or TLR2–/– mice as donors.

Again, no differences in the severity of atherosclerotic PUBLICATIONS lesions were observed. All LDLr–/–TLR2–/– mice that Boisvert, W.A., Rose, D.M., Boullier, A., Quehenberger, Q., Sydlaske, A., John- son, K.A., Curtiss, L.K., Terkeltaub, R. Leukocyte transglutaminase 2 expression received bone marrow transplants had smaller lesions limits atherosclerotic lesion size. Arterioscler. Thromb. Vasc. Biol. 26:563, 2006. than did LDLr–/– recipient mice, regardless of the donor Boisvert, W.A., Rose, D.M., Johnson, K.A., Fuentes, M.E., Lira, S.A., Curtiss, TLR2 genotype. Thus, a TLR2 deficiency in cells not L.K., Terkeltaub, R.A. Up-regulated expression of the CXCR2 ligand KC/GROα in derived from bone marrow led to less disease. Collec- atherosclerotic lesions plays a central role in macrophage accumulation and lesion progression. Am. J. Pathol. 168:1385 2006. tively, these data suggest that endogenous ligand signaling via TLR2 and/or TLR4 affected disease outcome Curtiss, L.K. Is two out of three enough for ABCG1? Arterioscler. Thromb. Vasc. Biol. 26:2175, 2006. and that TLR2 and/or TLR4 expression on cells not derived from bone marrow mediated the effects of TLR2 Curtiss, L.K., Valenta, D.T., Hime, N.J., Rye, K.-A. What is so special about apolipoprotein AI in reverse cholesterol transport? Arterioscler. Thromb. Vasc. Biol. and/or TLR4 activated by endogenous TLR ligands. 26:12, 2006. To determine the atherosclerotic effect of systemic Mullick, A.E., Tobias, P.S., Curtiss, L.K. Modulation of atherosclerosis in mice by activation of TLR2, we administered the exogenous Toll-like receptor 2. J. Clin. Invest. 115:3149, 2005. TLR2 ligand synthetic N-palmitoyl-S-[2,3-bis(palmitoy- Mullick, A.E., Tobias, P.S., Curtiss, L.K. Toll-like receptors and atherosclerosis: key loxy)-(2RS)-propyl]-(R)-cysteine (Pam3), a lipopeptide contributors in disease and health? Immunol. Res. 34:193, 2006. that induces signaling in cells of the immune system Tilley, R.E., Pedersen, B., Pawlinski, R., Sato, Y., Erlich, J.H., Shen, Y., Day, S., through TLR2, to hypercholesterolemic LDLr–/– mice. Huang, Y., Eitzman D.T., Boisvert, W.A., Curtiss, L.K., Fay, W.P., Mackman, N. After 10 weeks of high-fat feeding and weekly intra- Atherosclerosis in mice is not affected by a reduction in tissue factor expression. Arterioscler. Thromb. Vasc. Biol. 26:555, 2006. peritoneal injections of Pam3, the mice had a striking Valenta, D.T., Bulgrien, J.J., Banka, C.L., Curtiss, L.K. Overexpression of human dose-dependent increase in lesion severity. Compared apoAI transgene provides long-term atheroprotection in LDL receptor-deficient with lesions in control mice not given Pam3, the areas mice. Atherosclerosis, in press. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 111

Valenta, D.T., Ogier, N., Bradshaw, G., Black, A.S., Bonnet, D.J., Lagrost, L., Curtiss, L.K., Desrumaux, C.M. Atheroprotective potential of macrophage-derived Infarctive Eradication of Tumors phospholipid transfer protein in low-density lipoprotein receptor-deficient mice is overcome by apolipoprotein AI overexpression. Arterioscler. Thromb. Vasc. Biol. 26:1572, 2006. by Selective Tumor Vascular Thrombosis

Enhanced Tissue Factor A. EL-Sheikh, P. Borgstrom, G. Bhattacharjee, M. Belting, Initiation of the Coagulation T.S. Edgington hen fused to the extracellular domain of tis- and Thrombogenic Cascades sue factor (TFt), the exon-encoded polypep- by -Macroglobulin W tide of the heparin-binding domain (HBDt) α2 of vascular endothelial cell growth factor localizes selectively to endothelial surfaces of intratumoral microvas- G. Bhattacharjee, S. Arandjelovic, N. Mackman, W. Ruf, culature. Tissue factor is the initiating receptor and S.L. Gonias, T.S. Edgington requisite cofactor for initiation of the thrombogenic he broad spectrum poteinase inhibitor α2-Macro- cascade. In tumor-bearing mice, intravenous infusion globulin differs from all 4 mechanistic classes of of the fused domains, HBDt-TFt, results in selective T proteinase inhibitors. It inhibits coagulation pro- rapid occlusive thrombosis of tumor microvasculature. teinases such as thrombin and factor Xa and fibrino- We found that infusion of an optimal combination of lytic proteinases such as plasmin. a2-Macroglobulin is HBDt-TFt and its ligand factor VIIa in tumor-bearing also a carrier of growth factors and may regulate their animals results in infarctive eradication of tumors and function. Receptors for a2-macroglobulin include the often is curative. low-density lipoprotein receptor-related protein (LRP) Binding studies and confocal microscopy indicated and 78-kD glucose-regulated protein (Grp78), a high- that the target for HBDt-TFt is a trimolecular complex affinity receptor. In its native conformation, a2-macro- of chondroitin C sulfate proteoglycan, neuropilin-1, and globulin is present in plasma at high concentrations. vascular endothelial cell growth factor receptor 2, which Upon binding to serum proteinases or small primary is overexpressed in highly angiogenic sites of the tumor amines, a2-macroglobulin is converted to the receptor- microenvironment. HBDt-TFt also colocalized with the recognized form, which binds to LRP. Complexes com- trimolecular receptor complex in endothelial sprouts posed of LRP, a2-macroglobulin, and proteinases are from tumor tissues, and binding inhibited growth of internalized by cells. sprouts. In vitro, HBDt had the highest affinity for Previously, we found that Grp78 binds tissue fac- chondroitin 6 sulfate. We are evaluating the potential tor and inhibits its function. Recently, we discovered of HBDt-TFt as a therapeutic agent. that the receptor-recognized form of α2-macroglobulin enhances initiation of the coagulation and thrombotic cascades by tissue factor, generation of factor Xa, and Legumain Expression and expression of the gene for tissue factor. This enhancement depends on binding of the receptor-recognized Cell-Surface Translocation form of α2-macroglobulin to LRP, but not to Grp78, because receptor-associated protein blocks the as a Catalytic Target for response. The effects of α2-macroglobulin on tissue factor–dependent procoagulant activity are most pro- Prodrug Delivery and Therapy nounced in RAW 264.7 macrophage-like cells, but the W. Wu, Y. Luo, C. Sun, Y. Liu, P. Kuo, J. Varga, R. Xiang, effects also occur in THP-1 monocytic cells. A mutant R.A. Reisfeld, K.D. Janda, T.S. Edgington, C. Liu of the receptor-recognized form of α2-macroglobulin that does not bind LRP has no effect on tissue factor egumain, a novel and highly specific asparaginyl activity or gene expression. endopeptidase of the cysteine protease family, is L conserved as the only proteinase of this specificity from plant to humans. It is highly expressed by endo- 112 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE thelial, neoplastic, and stromal cells in most tumors, in PUBLICATIONS Bhattacharjee, G., Ahamed, J., Pedersen, B., EL-Sheikh, A., Mackman, N., Ruf, humans and animals. We developed a legumain-activated, W., Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the cell-impermeable preprodrug that incorporates doxorubi- coagulation cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol. 25:1737, 2005. cin as the cytotoxic element, although a wide variety of cytotoxic molecules can be incorporated to make the EL-Sheikh, A., Borgstrom, P., Bhattacharjee, G., Belting, M., Edgington, T.S. A selective tumor microvasculature thrombogen that targets a novel receptor complex molecules nontoxic. Designated LEG3, the preprodrug in the tumor angiogenic microenvironment. Cancer Res. 65:11109, 2005. targets extracellular legumain on tumor microvascu- Lin, R., Maeda, S., Liu, C., Karin, M., Edgington, T.S. A large noncoding RNA is a lature, tumor stroma, and neoplastic cells. Upon bind- marker for murine hepatocellular carcinoma and a spectrum of human carcinomas. Oncogene, in press. ing, it is catalytically converted to a cell-permeable prodrug, which is then converted to doxorubicin inside Wu, W., Luo, Y., Sun, C., Liu, Y., Kuo, P., Varga, J., Xiang, R., Reisfeld, R., Janda, K.D., Edgington, T.S., Liu, C. Targeting cell-impermeable prodrug activation the cells, where it results in tumor-specific killing. to tumor microenvironment eradicates multiple drug-resistant neoplasms. Cancer Pharmacokinetics and tissue distribution of LEG3 Res. 66:970, 2005. compared with doxorubicin alone confirmed selective local prodrug activation leading to 30- to 100-fold increases of doxorubicin in tumor cell nuclei but with Control of V(D)J Recombination 100-fold reductions of doxorubicin in normal tissues. Importantly, this prodrug completely inhibited growth and Formation of the Antibody of a variety of multidrug-resistant human tumors in Repertoire in Normal and mice, produced tumor eradication, and significantly extended survival without any evidence of myelosup- Autoimmune Mice pression or toxic cardiac effects. A.J. Feeney, C.R. Espinoza, J. Lamoureux, M. Cherrier, C.R. Xu, J. Carey, S. Salerno, L. Watson

Association of a Large main focus of our laboratory is the molecular Noncoding RNA Riboregulator analysis of factors that influence the composi- A tion of the antibody repertoire and elucidation With Neoplasia of the mechanisms that control the V(D)J rearrangement process. In each precursor B lymphocyte, a dif- R. Lin, S. Maeda, C. Liu, M. Karin, T.S. Edgington ferent set of V, D, and J genes recombine to form exons arge noncoding RNAs account for an unexpectedly for the light and heavy chains of the antibody mole- large proportion of the transcribed genome but cule. Each locus has many V, D, and J genes, but the L have been sparsely analyzed. We found a novel gene segments are not used equally. One of our goals murine gene encoding a 7000-base mRNA-like tran- is to understand the basis of this nonrandom use of script. This gene and mammalian counterparts lack gene segments. credible or conserved open reading frames, character- We previously showed that much of this bias occurs istics of large noncoding RNAs. because V genes undergo recombination with different In murine hepatocellular carcinomas induced with intrinsic frequencies due to differences in the recombi- a procarcinogen and in all samples of human hepato- nase signal sequence, the binding site for the recom- cellular carcinomas analyzed, expression of this gene binase, flanking each gene segment. The recombinase was enhanced. Compared with normal liver, hepato- signal sequence is composed of a relatively conserved cellular carcinomas had a 6- to 7-fold increase of this heptamer and nonamer flanking a “spacer” of conserved noncoding RNA. Thus, this large noncoding RNA is a new length but only modestly conserved sequence. Few genes marker for hepatocellular carcinoma. The RNA was also have consensus sequences, however, and changes in this significantly overexpressed in all 5 nonhepatocellular natural variation in the recombinase signal sequence human carcinomas analyzed. Expression was enhanced can greatly affect recombination frequency in vitro and in cells enriched at the mitotic stage, advancing this large in vivo. noncoding RNA as a generic marker for carcinomas In addition, other factors clearly influence recom- and suggesting a role for it as a riboregulator (regula- bination frequencies; currently we are focusing on the tory RNA) in the molecular cell biology of neoplasia. role of transcription factors and chromatin modifications IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 113 in controlling accessibility to V(D)J recombination and Zhang, Z., Espinoza, C.R., Yu, Z., Stephan, R., He, T., Williams, G.S., Burrows, P.D., Hagman, J., Feeney, A.J., Cooper, M.D. Transcription factor Pax5 (BSAP) recombination frequency. Genes in loci that are under- transactivates the RAG-mediated VH-to-DJH rearrangement of immunoglobulin going V(D)J recombination are often associated with genes. Nat. Immunol. 7:616, 2006. histones that are acetylated. We hypothesized that the extent of histone modification affects the frequency of recombination of individual genes, and indeed we Syndecans and HIV Type 1 observed a positive correlation between the relative rearrangement frequency of several individual genes Pathogenesis in vivo and the extent of acetylation of histones H3 and H4 associated with those genes, as assessed by M. Bobardt, U. Chatterji, S. Selvarajah, A. de Parseval,* chromatin immunoprecipitation. Other modifications J.H. Elder,* P.A. Gallay associated with repressed or activated genes are now * Department of Molecular Biology, Scripps Research being investigated. he syndecans belong to the heparan sulfate family We have also uncovered a novel role for the tran- of proteoglycans. In syndecans, the sulfation scription factor Pax5 in promoting V(D)J rearrangement. T pattern of the heparan sulfate chains dictates Pax5 is essential for B-cell development. In the absence the ligand specificity. HIV type 1 (HIV-1) has maximized of Pax5, V to DJ rearrangement is severely impaired. H H its use of syndecans. It uses them as receptors to facili- We found Pax5 binding sites in the coding regions of tate infection of macrophages. It uses them as recep- many V genes. Furthermore in collaboration with H tors on the endothelium to enhance endurance of the Z. Zhang and M. Cooper, University of Alabama, Birm- virus in the hostile environment and to trans infect ingham, we showed that Pax5 binds to the recombi- circulating T cells. HIV-1 also exploits syndecans to nase proteins RAG1 and RAG2. Hence, we propose facilitate transport of the virus through the blood-brain that Pax5 may recruit RAG1 or RAG2 to the recombi- barrier and the genital epithelium. Thus, the interplay nase signal sequence or may stabilize the interaction between HIV-1 and syndecans may profoundly affect of the RAG complex with its binding site. HIV-1 pathogenesis. In other studies, we are examining the breakdown Sexual transmission is the most common mode of of B-cell tolerance in autoimmunity. When precursor infection in the global HIV-1 epidemic. In the absence B cells successfully recombine both heavy- and light- of an effective vaccine, additional strategies to prevent chain gene segments, they express a B-cell receptor for new HIV-1 infections are urgently needed. Mucosal the first time. If the receptor is autoreactive, then the immature B cell normally continues to undergo light- dendritic and Langerhans cells are the first cells that chain V-J rearrangement until an innocuous receptor is HIV-1 encounters. These cells may thus play a crucial made. This process is termed receptor editing and is role in HIV-1 transmission. Interactions between HIV-1 an important checkpoint in B-cell tolerance. We have and dendritic or Langerhans cells are poorly understood. evidence that this process is not functioning as efficiently Although researchers initially proposed that C-type in lupus-prone mice as in nonautoimmune mice, and lectin receptors such as DC-SIGN, the mannose recep- we are investigating why this difference occurs. Such tor, and langerin mediated contact between HIV-1 and misregulation of this key checkpoint could lead to the dendritic or Langerhans cells, blocking of CD4, CCR5, release of autoreactive B cells into the periphery, where and C-type lectin receptors only partly prevents HIV-1 they can become activated to secrete autoantibodies capture by dendritic cells. This finding led researchers and cause autoimmune disease. to postulate the existence of unidentified HIV-1 receptors on dendritic cells. PUBLICATIONS We found that syndecan-3, which is normally poorly Espinoza, C.R., Feeney, A.J. The extent of histone acetylation correlates with differential rearrangement frequency of individual VH genes in pro-B cells. J. expressed or even absent on many cell types and tis- Immunol. 175:6668, 2005. sues, is highly expressed on both dendritic cells and Espinoza, C.R., Feeney, A.J. Quantifying chromatin accessibility of individual gene Langerhans cells. More importantly, we obtained evi- family members by combining ligation-mediated PCR with real-time PCR. Biotech- niques 41:404, 2006. dence that syndecan-3 plays a key role in HIV-1 cap-

Watson, L.C., Moffatt-Blue, C.S., MacDonald, R.Z., Kompfner, E., Aït-Azzouzene, ture and trans infection of T cells by dendritic cells. D., Nemazee, D., Theofilopoulos, A.N., Kono, D.H., Feeney, A.J. Paucity of V-D- Because dendritic and Langerhans cells may be the D-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT–/– and TdT+/+ mice. J. Immunol. 172:1120, 2006. first Trojan horses that HIV-1 exploits to colonize 114 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE humans, understanding the contribution of syndecan-3 capsids represent abortive infectious events. Further in the capture and transfer of HIV-1 by dendritic cells work is required to determine whether TRIM5α-medi- and Langerhans cells to T cells is imperative to develop ated capsid degradation and capsid core uncoating new therapies to prevent HIV-1 infections. represent linked or distinct events. An understanding of the nature of restrictions to PUBLICATIONS Binley, J.M., Ngo-Abdalla, S., Moore, P., Bobardt, M., Chatterji, U., Gallay, P., HIV-1 infection after the virus enters cells is critical Burton, D.R., Wilson, I.A., Elder, J.H., de Parseval, A. Inhibition of HIV Env bind- for several reasons. First, information on the viral and ing to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120. Retrovirology. 3:39, 2006. cellular factors that modulate these processes will shed light on the poorly understood series of events that Bobardt, M.D., Chatterji, U., Selvarajah, S., Van der Schueren, B., David, G., Kahn, B., Gallay, P.A. Cell-free HIV-1 transcytosis through primary genital epithe- govern the fate of capsids after entry. Second, species- lial cells. J. Virol., in press. specific barriers to HIV-1 infection present obstacles de Parseval, A., Bobardt, M.D., Chatterji, A., Chatterji, U., Elder, J.H., David, G., to the development of animal models for the study of Zolla-Pazner, S., Farzan, M., Lee, T.H., Gallay, P.A. A highly conserved arginine in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs. J. HIV-1 pathogenesis and treatment. Finally, an under- Biol. Chem. 280:39493, 2005. standing of this critical part of the HIV-1 life cycle

Saphire, A.C., Gallay, P.A., Bark, S.J. Proteomic analysis of human immunodefi- may suggest approaches to intervene in transmission ciency virus using liquid chromatography/tandem mass spectrometry effectively dis- of the virus or its spread within the host. tinguishes specific incorporated host proteins. J. Proteome Res. 5:530, 2006.

PUBLICATIONS Barry, S.M., Melar, M., Gallay, P., Hope, T.J. Review of the twelfth West Coast Innate Intracellular Immunity Retrovirus Meeting. Retrovirology 2:72, 2005. Chatterji, U., Bobardt, M.D., Gaskill, P., Sheeter, D., Fox, H., Gallay, P.A. TRIM5α and Infection With HIV Type 1 accelerates degradation of cytosolic capsid associated with productive HIV-1 entry. J. Biol. Chem., in press.

U. Chatterji, M. Bobardt, S. Selvarajah, P.A. Gallay Chatterji, U., Bobardt, M.D., Stanfield, R., Ptak, R.G., Pallansch, L.A., Ward, P.A., Jones, M.J., Stoddart, C.A., Scalfaro, P., Dumont, J.M., Besseghir, K., onhuman cells contain intracellular innate factors Rosenwirth, B., Gallay, P.A. Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl that inhibit infection by HIV type 1 (HIV-1) by monkey cells. J. Biol. Chem. 280:40293, 2005. N targeting the incoming viral capsid core, which makes up the shell that surrounds the viral genome. The first intracellular primate restriction factor identified, Imaging Molecular Interactions TRIM5α, is a member of the tripartite motif (TRIM) family of proteins. However, the mechanism by which in T-Cell Development and TRIM5α blocks HIV-1 infection is unknown. We asked if TRIM5α blocks HIV-1 by diverting the Activation incoming capsid core into an abortive degradation pathway. We compared the degradation status of incoming N.R.J. Gascoigne, J. Ampudia, G. Fu, C. Goergen, cores in restrictive and nonrestrictive cells. We found K. Holmberg, H.-C. Hung, H.-O. Kim, C. Lotz, A. Munshi, that monkey, but not human, TRIM5α provokes destruc- G. Sternik, S. Vallee, P.Yachi, M.A. Zal. T. Zal, N. Bosco,* tion of capsids immediately after HIV-1 entry. The R. Ceredig,* M. Daniels,** E. Palmer** capsid degradation is specific because the integrity of * U548 INSERM, Grenoble, France other abundant viral proteins is preserved. TRIM5α ** University Hospital, Basel, Switzerland directs capsids to a nonproteasome degradation path- IMAGING OF INTERACTIONS BETWEEN T-CELL way. Capsids delivered into the vesicular compartment RECEPTORS AND CORECEPTORS IN T-CELL undergo similar degradation in restrictive and nonre- ACTIVATION strictive cells. In contrast, capsids delivered into the luorescence resonance energy transfer (FRET) is cytosol of nonrestrictive cells remain intact, whereas a physical phenomenon that occurs at distances capsids delivered into the cytosol of restrictive cells F less than 10 nm. Thus, FRET between fluores- are destroyed. cent proteins, for example, between cyan and yellow Our finding that TRIM5α specifically degrades fluorescent proteins, attached to proteins of interest cytosolic capsids suggests that cytosolic capsids rep- can be used to investigate interactions between pro- resent authentic infectious events, whereas vesicular teins in living cells. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 115

Using FRET, we showed that the coreceptor CD8 absence of PKCθ, PKCη is expressed at an earlier stage and the T-cell receptor (TCR) signal-transducing proof thymocyte development, where it functions in place of tein CD3ζ are recruited to the “immunologic synapse,” PKCθ. Inhibition of PKCη expression during thymocyte where they interact when antigenic MHC-peptide com- development by short hairpin RNAs results in inhibition plexes are presented to T cells. No FRET occurs when of T-cell development. We are now using short hairpin weaker (e.g., TCR antagonist) ligands are used. We RNA and mice lacking the gene for PKCη to determine showed previously that endogenous MHC-peptides aid the roles of PKCη in T-cell activation and development. in recognizing antigenic MHC-peptide complexes. The IDENTIFICATION OF A NOVEL PROTEIN IMPOR- interaction between CD8 and endogenous MHC-pep- T ANT IN T-CELL DIFFERENTIATION tides improves TCR recognition of antigenic MHC-pep- We identified a novel protein that is expressed pri- tides, including the ability to associate with CD8. This marily during thymocyte differentiation. It is expressed surprising finding suggests how T cells can respond to during the stages at which TCR genes undergo rearrange- small amounts of antigens in a “sea” of nonstimula- ment, and it interacts with the cell-cycle and DNA tory MHC-peptides. damage-repair enzyme ATM. Transgenic expression of We are now testing the relative importance of TCR the gene that encodes the protein provides some pro- and CD8 interactions with the endogenous MHC-pep- tection against DNA double-strand breaks induced by tides in helping stimulation by antigenic MHC-peptide ionizing radiation, suggesting that the novel protein complexes. We also compared the formation of immu- acts in synergy with ATM. The protein also interacts nologic synapses and the TCR-CD8 interaction in a with phospholipase C γ1, important in T-cell signaling. system in which the affinity of the interaction between We have produced a strain of mice that lack the gene TCRs and MHC-peptides is known. The strength of weak for this novel protein. Mice that lack the gene have agonists is more closely related to the speed at which defects in thymic positive selection and reduced phos- they recruit TCRs to the synapse and start to induce phorylation of phospholipase C γ1 in response to TCR FRET than it is to the affinity of the interaction between stimulation. We intend to identify the role of this pro- TCRs and MHC-peptides. tein in T-cell signaling and development. In collaboration with E. Palmer, University Hospi- TCR ENDOCYTOSIS, RECYCLING, AND tal, Basel, Switzerland, we investigated new signaling UBIQUITINATION induced through the TCR that differs between positive Because allelic exclusion of the TCR α-chain is and negative selection. The induction of FRET appears maintained after translation, many mature T cells to explain why some agonists are stronger or weaker express 2 α-chain proteins. However, expression of than would be predicted on the basis of their affini- 2 α-chains on the cell surface is rare. We previously ties, and the kinetics of FRET differ between positive- showed that functional allelic exclusion is attained in and negative-selecting stimuli. We are directly testing the thymus through TCR signaling involving the kinase the potentially different roles of CD8αα and CD8αβ in Lck and the ubiquitin ligase Cbl, which controls degra- formation of the immunologic synapse and the dynam- dation of endocytosed TCRs. We are developing a ics of association of the kinase Lck with CD8 before transgenic minigene system to analyze the effects of and during antigenic stimulation. expression of 2 α-chains on the cell surface. We are ROLE OF THE PROTEIN KINASE C η ISOFORM IN using FRET between ubiquitin monomers and TCR THE IMMUNOLOGIC SYNAPSE subunits labeled with fluorescent proteins to analyze We previously showed that the η isoform of protein ubiquitination of TCRs after endocytosis. In collabora- kinase C (PKC) is upregulated during positive selection tion with R. Ceredig, INSERM, Grenoble, France, we of developing thymocytes. Of the PKC isoforms, only examined the TCR α-chain repertoire of specialized PKCθ is known to have a special role in T cells, where CD25+CD4+ regulatory T cells. We found that the reper- it is recruited to the immunologic synapse during anti- toire is as diverse as that of mainstream CD4+ T cells. gen recognition. The finding that mice deficient in PKCθ We discovered a new property of the dye FM4-64 that have normal thymic selection suggested that PKCη could enables us to selectively image the nuclear membrane be replacing PKCθ in the developing thymocytes. in living cells. This discovery should be useful to cell We found that PKCη is also naturally recruited to biologists studying nuclear trafficking of proteins and the synapse in mature thymocytes and T cells. In the diseases of the nuclear envelope. 116 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

PUBLICATIONS MEDIATION OF TLR4 SIGNALING BY THE 4-1BB Bosco, N., Hung, H.-C., Pasqual, N., Jouvin-Marche, E., Marche, P.N., Gascoigne, LIGAND N.R.J., Ceredig, R. Role of the T cell receptor α-chain in the development and phenotype of naturally arising CD4+CD25+ T cells. Mol. Immunol. 43:246, 2006. TLRs are an important first contact between host

Daniels, M.A., Teixeiro, E., Gill, J., Hausmann, B., Roubaty, D., Holmberg, K., and microorganisms after infection. Once activated, the Werlen, G., Holländer, G., Gascoigne, N.R.J., Palmer, E. Thymic selection thresh- receptors generate downstream signaling pathways old defined by compartmentalization of Ras/MAK signalling. Nature, in press. leading to gene expression. The 4-1BB ligand is highly Yachi, P.P., Ampudia, J., Zal, T., Gascoigne, N.R. Altered peptide ligands induce induced and expressed at the cell surface of macro- delayed CD8-T cell receptor interaction: a role for CD8 in distinguishing antigen quality. Immunity. 25:203, 2006. phages after inflammatory stimulation. We showed that once expressed, the ligand can interact with TLRs at Zal, T., Zal, M.A., Lotz, C., Goergen, C.J., Gascoigne, N.R. Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living the cell surface of macrophages, but mostly activation cells. Traffic, in press. of the TLR4 signaling pathway leads to p38 activa- Zambricki, E., Zal, T., Yachi, P., Shigeoka, A., Sprent, J., Gascoigne, N., McKay, tion. These results revealed a new mechanism in which D. In vivo anergized T cells form altered immunological synapses in vitro. Am. J. macrophages undergo a 2-step activation process, Transplant. 6:2572. 2006. involving expression and translocation of 4-1BB ligand to the cell surface and afterward interaction with TLRs, sustaining the inflammatory responses.

Signaling Pathways in the ANTIVIRAL RESPONSES MEDIATED BY MICRORNA Innate Immune System We found that some microRNAs interfere with propagation of vesicular stomatitis virus (VSV) and VSV- induced cell death. MicroRNAs are single-stranded J. Chen, R.T. Cook, Y. Kang, S. Lee, J. Mols, M. Otsuka, RNA molecules of 19–23 nucleotides that originate S. Shimada, C.C. Wu, C. Xie, Y. Xu, J. Han from longer and imperfectly matching hairpin precur- he p38 MAP kinase pathway plays a crucial func- sors processed by several ribonucleoprotein complexes. tion in the cellular response after infection by The cytosolic enzyme Dicer converts hairpin precur- T pathogens or inflammatory stimulation. Our inter- sors into 19- to 23-nucleotide RNA duplexes and ests are the functions of p38 in innate immunity in fruit therefore is of key importance for the maturation of flies, in Toll-like receptor (TLR) signaling, and in control microRNAs. of the cell cycle. We are also interested in the involve- We generated Dicer-deficient mice and investigated ment of microRNAs in the innate immune system. macrophage sensitivity to VSV infection. Macrophages A NEW PATHWAY IN THE IMMUNE RESPONSE TO from Dicer-deficient mice had greater VSV propagation BACTERIAL INFECTION IN DROSOPHILA and VSV-induced cell death than did control macro- Using the P-element insertion-excision technique, phages from wild-type mice. Furthermore, we found we generated mutant fruit flies that lacked individual that the anti-VSV functions of Dicer are at least partly p38 isoforms and a double mutant that lacked both mediated by endogenous microRNAs that target viral p38 isoforms. As widely described previously, the Toll RNA genes encoding the RNA-directed RNA polymer- and Imd pathways are essential in Drosophila for pro- ase complex. Therefore, microRNAs may be a broadly tection against microbial infection. In contrast, we important element of the innate immune response to showed that the p38 pathway mediates another defense viral infection in mammals. mechanism independent of those 2 pathways and that REGULATION OF RNA INTERFERENCE BY TNF the deficiency of either Toll or Imd does not impair p38 STIMULATION activation after infection. Regulation of the mRNA stability of cytokines by In the p38 double mutants, melanization of the inflammatory stimuli is widely documented and is hindgut, which is related to bacterial infection, devel- linked to p38 activity. Previously, we showed that ade- ops during the larval stage. Furthermore, p38 regu- nine-uridine–rich elements (AREs) in the 3′ untranslated lates some of the functions of heat-shock factor, and region of cytokine mRNAs dictate mRNA degradation expression of heat-shock proteins mediated by heat- in a microRNA-dependent manner. Recently, we found shock factor is, at least in part, responsible for the that exogenously added short interfering RNAs and the p38 anti-infectious functions, revealing a new innate endogenous ARE-microRNA systems act as regulators immune system in Drosophila. of mRNA stability and that both systems are regulated IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 117

by inflammatory stimulation. Indeed, TNF-α stimulation Zhuang, S., Yan, Y., Han, J., Schnellmann, R.G. p38 Kinase-mediated transactivation of the epidermal growth factor receptor is required for dedifferentiation of renal blocks both short interfering RNA– and ARE-dependent epithelial cells after oxidant injury. J. Biol. Chem. 280:21036, 2005. mRNA degradation. The mechanism of action involves lengthening of the poly (A) tail and a partial decrease of the activity of the RNA-induced silencing complex Specificity and Function of that does not impair the synthesis and maturation of short interfering RNAs and microRNAs. We conclude Intraepithelial γδ T Cells that RNA interference is based on mechanisms regulated by inflammatory stimulation, linking the ARE-depen- W.L. Havran, M. Haynes, J.M. Jameson, C.H. Kim, dant mRNA stabilization to the function of short inter- E. Kolkowski, H.K. Komori, T. Meehan, R. Mills, A. Toulon, fering RNAs. M. Watanabe, J. Whitelock, D. Witherden e have a long-term interest in interactions PUBLICATIONS Al Sarraj, J., Vinson, C., Han, J., Thiel, C. Regulation of GTP cyclohydrolase I between intraepithelial γδ T cells and their gene transcription by basic region leucine zipper transcription factors. J. Cell. neighboring epithelial cells. We focus on Biochem. 96:1003, 2005. W interactions in the thymus, skin, and intestine. We da Silva Correia, J., Miranda, Y., Austin-Brown, N., Hsu, J., Mathison, J., Xiang, R., Zhou, H., Li, Q., Han, J., Ulevitch, R.J. Nod1-dependent control of tumor are investigating the development, specificity, and growth. Proc. Natl. Acad. Sci. U. S. A. 103:1840, 2006. function of these γδ T cells. Our results have defined

Fu, J., Yang, Z., Wei, J., Gu, J. Nuclear protein NP60 regulates p38 MAPK activity. unique properties of these cells and support a spe- J. Cell Sci. 119(Pt. 1):115, 2006 cialized role for intraepithelial γδ T cells in immune

Han, J. MyD88 beyond Toll. Nat. Immunol. 7:370, 2006. surveillance, wound repair, inflammation, and protection from malignant tumors. Han, J., Ulevitch, R.J. Limiting inflammatory responses during activation of innate immunity. Nat. Immunol. 6:1198, 2005. MOLECULES REQUIRED FOR γ δ T CELL ACTIVATION Liu, W., Rui, H., Wang, J., Lin, S., He, Y., Chen, M., Li, Q., Ye, Z., Zhang, S., Chan, S.C., Chen, Y.G., Han, J., Lin, S.C. Axin is a scaffold protein in TGF-β sig- In murine skin, γδ T cells express an invariant naling that promotes degradation of Smad7 by Arkadia. EMBO J. 25:1646, 2006. γδ T cell receptor that recognizes an unknown antigen

Lu, G., Kang, Y.K., Han, J., Herschman, H.R., Stefani, E., Wang, Y. TAB-1 modu- expressed by damaged or malignant neighboring ker- lates intracellular localization of p38 MAP kinase and downstream signaling. J. atinocytes. We have now produced soluble skin γδ T cell Biol. Chem. 281:6087, 2006. receptor molecules for use as a tool to detect expres- Meng, F., Yamagiwa, Y., Taffetani, S., Han, J., Patel, T. IL-6 activates serum and sion and facilitate isolation and characterization of this glucocorticoid kinase via a p38α mitogen-activated protein kinase pathway. Am. J. Physiol. Cell. Physiol. 289:C971, 2005. unidentified antigen. Future structural studies will determine how these T-cell receptors interact with antigen. Shi, G.X., Han, J., Andres, D.A. Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation. J. Biol. Chem. We propose that in addition to antigen, damaged 280:37599, 2005. keratinocytes express molecules that participate in Tang, J., Qi, X., Mercola, D., Han, J., Chen, G. Essential role of p38β in K-Ras activation of skin γδ T cells by binding to coreceptors transformation independent of phosphorylation. J. Biol. Chem. 280:23910, 2005. or costimulatory molecules on the T-cell surface. Skin Xie, C., Zhang, N., Zhou, H., Li, J., Li, Q., Zarubin, T., Lin, S., Han, J. Distinct γδ T cells do not express classical molecules, includ- roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced death in L929 cells. Mol. Cell. Biol. 25:6673, 2005. ing CD4, CD8, and CD28, known to affect activation of αβ T cells. Xu, Y., Huang, S., Liu, Z.G., Han, J. Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activa- We recently identified several molecules expressed tion. J. Biol. Chem. 281:8788, 2006. by the skin γδ T cells and keratinocytes that provide

Zarubin, T., Han, J. Activation and signaling of the p38 MAP kinase pathway. Cell important costimulatory signals for activation of γδ Res. 15:11, 2005. T cells. One such molecule, JAML, is uniquely costimu-

Zhou, H., Zarubin, T., Ji, Z., Min, Z., Zhu, W., Downey, J.S., Lin, S., Han, J. Fre- latory for intraepithelial γδ T cells. We have identified quency and distribution of AP-1 sites in the human genome. DNA Res. 12:139, another JAM family member, the coxsackievirus-adeno- 2005. virus receptor, as a ligand for JAML that is expressed on Zhou H., Zheng, M., Chen, J., Xie, C., Kolatkar, A.R., Zarubin, T., Ye, Z., Akella, epithelial cells in the skin and intestine. Interactions R., Lin, S., Goldsmith, E.J., Han, J. Determinants that control the specific interactions between TAB1 and p38β. Mol. Cell. Biol. 26:3824, 2006. between JAML and the coxsackievirus-adenovirus recep-

Zhu, X., Mei, M., Lee, H.G., Wang, Y., Han, J., Perry, G., Smith, M.A. p38 Acti- tor may play important roles in γδ T cell responses dur- vation mediates amyloid-β cytotoxicity. Neurochem. Res. 30:791, 2005. ing wound repair and other epithelial challenges. 118 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

We also found that the semaphorin Sema4D (CD100) inflammatory disorders and may be useful in designing is expressed by skin and intestinal γδ T cells upon or testing new therapies. activation. CD100 binds to a new member of the plexin superfamily of semaphorin receptors, plexin-B2, PUBLICATIONS Havran, W.L., Jameson, J.M., Witherden, D.A. Epithelial cells and their neigh- expressed on epithelial cells. We found that interac- bors, III: interactions between intraepithelial lymphocytes and neighboring epithe- tions between CD100 and plexin-B2 deliver signals lial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 289:G627, 2005. to both the intraepithelial γδ T cells and epithelial Komori, H.K., Meehan, T.F., Havran, W.L. Epithelial and mucosal γδ T cells. Curr. Opin. Immunol. 18:534, 2006. cells in the skin and intestine.

A ROLE FOR INTRAEPITHELIAL γ δ T CELLS IN EPITHELIAL TISSUE REPAIR Mechanisms of γδ T-Cell We recently showed a role for skin γδ T cells in the reepithelialization stage of wound repair. The γδ T cells Dysfunction in Nonhealing are activated at wound sites and produce cytokines, including the epithelial growth factors KGF-1 and KGF-2. Wounds In the absence of skin γδ T cells, keratinocyte proliferation and tissue reepithelialization after wounding are J.M. Jameson, R. Mills, K. Taylor defective. Recent results indicated that a keratinocyte- kin γδ T cells are activated by stressed or dam- responsive γδ T-cell receptor is necessary for activation aged keratinocytes to produce molecules such of the T cells by damaged keratinocytes during wound S as insulin-like growth factor 1 (IGF-1) and ker- healing and is also required for the maintenance of atinocyte growth factors that are important for the T cells in the epidermis. In addition, we found that maintenance of skin homeostasis and wound repair. the skin γδ T cells are necessary for the recruitment We are examining skin γδ T cells in nonhealing wounds of inflammatory cells into the wound site. In a novel to investigate the mechanisms that inhibit normal mechanism, γδ T cell–produced KGFs stimulate pro- wound healing. duction of hyaluronan by epidermal cells, which then CROSS TALK OF γ δ T CELLS AND KERATINOCYTES controls migration of macrophages into wounds. IN THE SKIN IN DIABETES Skin γδ T cells play roles not only in the repair of Treatment of nonhealing wounds is a considerable damaged tissue but also in the normal maintenance of problem for patients and healthcare services. In murine the epidermis. Insulin-like growth factor 1 is required models of diabetes, levels of IGF-1 and keratinocyte by keratinocytes in the skin for maintenance and dur- growth factors in wounds contribute to defective wound ing wound healing. We determined that after activation repair. When these growth factors are replenished, the skin γδ T cells produce this growth factor that affects markedly delayed wound repair is reversed. Previously, wound healing and apoptosis in the skin. Together we showed that skin γδ T cells play roles in wound these results indicate a role for skin γδ T cells in multi- reepithelialization via the production of growth factors. ple aspects of wound repair and for homeostasis of Now we are investigating whether decreased levels of the epithelium. growth factors and impaired proliferation/migration of In previous studies, we showed that intestinal intra- keratinocytes in nonhealing wounds in mice with dia- epithelial γδ T cells play a similar role in responding betes are due to dysregulation of skin γδ T cells. Ini- to tissue damage in a model of colitis. Our recent results tially, we are focusing on how the cross talk between indicate that in the absence of CD100-mediated signals, the key growth factors produced by skin γδ T cells and increased damage and delayed repair occur, indicating the factors’ receptors expressed on keratinocytes is an important role for costimulation through these mol- defective in mice with diabetes. ecules in γδ T-cell functions in the gut. Results in both In addition, we are examining mechanisms that may models support our hypothesis that intraepithelial γδ T contribute to skin γδ T-cell dysfunction in diabetes, cells respond to epithelial damage or disease and play including changes in insulin or IGF-1 receptor signaling important roles in tissue repair and epithelial homeo- and inhibition by glucocorticoids. The decreased levels stasis. Future studies should provide information that of IGF-1 in the epidermal compartment may result in will further define the role of γδ T cells in epithelial reduced IGF-1 receptor signaling in skin γδ T cells, a IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 119 situation that may exacerbate dysfunction of skin γδ ago. Members of the HMG box protein superfamily share T cells. one or more copies of a sequence-related and structurally γ δ T-CELL FUNCTION IN RESPONSE TO related DNA-binding domain that can recognize distorted RAPAMYCIN DNA structures and modify chromatin by bending DNA. Rapamycin is approved by the Food and Drug In general, HMG box proteins function as architectural Administration for prophylaxis of acute rejection of trans- factors that regulate gene expression by promoting for- planted organs. Patients who receive rapamycin have an mation of transcriptional complexes or by acting as com- increased incidence of complications of wound healing. ponents of chromatin remodeling complexes. Because these patients have difficulties with wound We found that TOX belongs to a small subfamily of healing, rapamycin may not only be targeting allograft- evolutionarily conserved proteins whose members share specific αβ T lymphocytes but also be suppressing skin almost identical HMG box sequences. The HMG box γδ T cells. In preliminary experiments, we observed sequence in TOX can recognize distorted DNA but is a defects in the proliferation of skin γδ T cells and the pro- relatively poor bender of DNA, because of the lack of duction of IGF-1 in the presence of rapamycin. We are a critical internal wedge residue. investigating the mechanism of this suppression. Expression of TOX in the thymus is tightly regulated. Once we better understand the role of T cells in Signaling through the serine/threonine phosphatase tissue repair, it may be possible to design therapies calcineurin is required for positive selection of thymo- that enhance the ability of these immune cells to heal cytes, and the gene for TOX is a target of this signaling ulcers and chronic wounds. Our objective is to deter- pathway. TOX is expressed in early thymocyte progeni- mine the mechanisms by which skin γδ T cells, nor- tors and then is transiently upregulated during β-selec- mally important in wound repair of healthy wild-type tion and positive selection. To analyze the function of mice, are not functioning properly in chronic wounds. this nuclear factor, we produced transgenic mice that express either wild-type or a mutant TOX and gene- targeted mice that lack TOX. Regulators of T-Lymphocyte Our data indicate that expression of TOX is sufficient to initiate the differentiation of immature thymo- Development and Function cytes to the CD8+ T-cell lineage, even in the absence of signals mediated by T-cell antigen receptors. Both the J. Kaye, P. Aliahmad, M. Fung, O. Goularte, C. Krieg, DNA-binding domain and the N-terminal domain of TOX N. Sanathara are required for this in vivo activity. Although TOX is not recursor cells in the thymus undergo a complex a T cell–specific protein, mice that lack the gene for developmental program before seeding periph- TOX are grossly normal but have a specific block in an P eral lymphoid organs as mature T lymphocytes. early stage of thymic positive selection. We are using Developmental checkpoints in the thymus, termed these tools to delineate the mechanism of action of this β-selection, positive selection, and negative selection, critical regulator of the fate of T cells in the thymus. narrow the repertoire of T-cell antigen specificities to NEGATIVE REGULATION OF T-CELL RESPONSES those that are not overtly autoreactive but maintain The functional outcome of engagement of the T-cell weak reactivity against self-MHC-peptide complexes. antigen receptor is modulated by secondary signals, We are interested in the mechanisms that determine which can have costimulatory or coinhibitory functions. the fate of developing T cells and the control of gene We isolated a gene that encodes a cell-surface protein expression during these processes. Our identification of the immunoglobulin superfamily, now designated of a cell-surface protein that is first expressed in the BTLA (B- and T-lymphocyte attenuator), that is upreg- T-cell lineage during positive selection led to studies ulated during positive selection and that is expressed on regulation of the immune response and the poten- by mature lymphocytes and antigen-presenting cells. tial of this protein as a novel therapeutic target. Evidence indicates that this protein can act as a nega- REGULATION OF THYMOCYTE SELECTION BY A tive regulator of lymphocyte activation. We produced NUCLEAR ARCHITECTURAL PROTEIN mice that lack the gene for BTLA and panels of mono- We identified thymocyte selection–associated high clonal antibodies specific for BTLA to analyze the in mobility group (HMG) box protein (TOX) several years vivo function of this protein. 120 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

One of our monoclonal antibodies acts as an agonist and Rho, which are essential regulators for various for this inhibitory molecule, thereby inhibiting facets of leukocyte functions ranging from production of ROS to T-cell activation. In vivo studies indicated that BTLA is chemotaxis and phagocytosis. Generation of superox- a negative regulator of homeostatic expansion of T cells ide is accomplished by a Rac-dependent NADPH oxi- and production of CD8+ memory T cells. For vaccines dase (Nox) upon stimulation with chemotactic factors against intracellular infections and tumors, development or phagocytic stimuli. of methods to regulate CD8+ T-cell responses and mem- GTPases of the Rho family are also involved in sig- ory formation is paramount, and thus BTLA may be a naling cascades, which originate from pathogen-activated useful therapeutic target in this regard. Preliminary data Toll-like receptors. Toll-like receptors 2, 3, and 4 stim- also suggest that one of our monoclonal antibodies to ulated by microbial products derived from bacteria and BTLA prolongs allograft survival in mice, and another viruses activate Rac1, Cdc42, and RhoA, which regu- antibody may ameliorate disease in a murine model of late pathways required for activation of gene transcrip- inflammatory bowel disease. tion. We are studying different aspects of signaling by Toll-like receptors in several primary human cell types, PUBLICATIONS including macrophages and neutrophils, and in geneti- Aliahmad, P., Kaye, J. Commitment issues: linking positive selection signals and lineage diversification in the thymus. Immunol. Rev. 209:253, 2006. cally altered mouse models. We are also examining the impact of this signaling on the functions, such as Krieg, C., Boyman, O., Fu, Y.-X., Kaye, J. T cell intrinsic regulation of homeostasis and CD8+ memory cell generation by BTLA. Nat. Immunol., in press. apoptosis and upregulation of proinflammatory mediators, of innate immune cells. Krieg, C., Han, P., Stone R., Goularte, O.D., Kaye, J. Functional analysis of B and T lymphocyte attenuator engagement on CD4+ and CD8+ T cells. J. Immunol. Another area of research is the interaction and 175:6420, 2005. communication between innate immune cells and the pulmonary epithelium. To this end, we established an in vitro reconstitution system for lung epithelium that Regulation of the Innate we use to examine signaling mechanisms initiated by pathogens. The differentiated and fully functional lung Immune Response in epithelium also serves as a model for studies of lung Inflammation and Infection barrier function and the influence of bacteria-derived ligands and toxins on transmigration of neutrophils. In U.G. Knaus, B. Desnues, M. Lehmann, K. von Loehneysen, addition, we are investigating processes that lead to S. Luxen, M. Manukyan, S. Pacquelet, M. Ruse, M. Tsatmali, uptake of pathogens or environmental particles and M. Valo, M. Ye the impact of these pathogens or particles on airway epithelial functions. nnate immune cells are the first line of defense in Recently, ROS-generating Nox proteins have been the fight against invading pathogens. We focus pri- identified in epithelial cells, and work is in progress to I marily on understanding molecular mechanisms that study the molecular basis for ROS generation by these phagocytes and the pulmonary epithelium use to protect novel proteins. Nox proteins may serve as compartmen- the host from the injury and how some responses wind talized signaling modules, thereby activating or inhibiting up damaging the host. For example, second messengers signaling cascades via superoxide, or as an epithelial such as reactive oxygen species (ROS) or nitric oxide host defense mechanism via hydrogen peroxide–gener- that are produced during infection can have beneficial ating Nox/Duox isoforms. Because of their tissue-specific as well as detrimental effects. The overall outcome distribution and distinct localization patterns, Nox pro- depends on precise spatial and temporal regulation of teins might have highly specialized functions and undergo these second messengers by the affected cell popula- isoform-dependent regulation. For example, Nox4, an tions. The intracellular signaling pathways that control oxidase expressed in colon tissue and melanomas, is these turn on–turn off mechanisms are an ideal target constitutively active in certain conditions and does not for intervention in disease. require any of the known oxidase components for super- Almost all processes connected to pathogen uptake, oxide generation. Elucidating physiologic stimuli and pathogen elimination, and sustained inflammation are control mechanisms for these Nox proteins combined governed by small GTPases of the Ras superfamily. Our with structure-function studies will help define the research centers on the Rho GTPases Rac, Cdc42, biological functions of Nox in health and disease. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 121

PUBLICATIONS Tid1 in breast cancer cells overexpressing ErbB2 facil- Chan, A.Y., Coniglio, S.J., Chuang, Y.Y., Michaelson, D., Knaus, U.G., Philips, M.R., Symons, M. Roles of the Rac1 and Rac3 GTPases in human tumor cell inva- itated the ubiquitination and degradation of ErbB2 sion. Oncogene 24:7821, 2005. and growth inhibition of the cells. Moreover, using

Martyn, K.D., Frederick, L.M., von Loehneysen, K., Dinauer, M.C., Knaus, U.G. RNA interference to deplete the physiologic levels of Functional analysis of Nox4 reveals unique characteristics compared to other Tid1 in breast cancer cells, we discovered that the NADPH oxidases. Cell. Signal. 18:69, 2006. metastatic potential of Tid1-depleted cells was sub- Martyn, K.D., Kim, M.J., Quinn, M.T., Dinauer, M.C., Knaus, U.G. p21-Activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils. Blood stantially enhanced. This enhancement was due to 106:3962, 2005. increased production of IL-8 through upregulation of

Ruse, M., Knaus, U.G. New players in TLR-mediated immunity: PI3K and small the nuclear transcription factor NF-κB in the Tid1- Rho GTPases. Immunol. Res. 34:33, 2006. depleted cells. Thus, because Tid1 attenuates signals generated from the ErbB2 receptor and negatively regulates the Regulatory Mechanisms for activity of NF-κB, we hypothesize that Tid1, like its Drosophila counterpart, may be an important tumor Tumor Carcinogenesis suppressor, especially in breast carcinogenesis and metastasis. To evaluate this hypothesis in an animal M. Hayashi, J.-F. Lo, S.-W. Kim, J.-D. Lee model, we have established a mouse model in which THE FOURTH MAP KINASE PATHWAY the gene for Tid1 can be deleted specifically in mam- ig mitogen-activated kinase 1 (BMK1), also mary epithelial cells. We are investigating and evaluat- called extracellular signal–regulated kinase 5, a ing the mechanistic role of Tid1 as a tumor suppressor B newer member of the mammalian MAP kinase in the regulation of tumorigenesis and metastasis of family, is activated by angiogenic growth factors. Using breast cancer not only at the molecular and cellular a mouse model in which expression of the gene for levels but also in an organismal context. BMK1 can be deleted, we showed that the BMK1 path- PUBLICATIONS way is required for tumor-associated angiogenesis and Abbasi, S., Lee, J.-D., Su, B., Chang, X., Alcon, J.L., Yang, J., Kellems, R.E., Xia, Y. consequent tumor growth through the BMK1/ribosomal Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1. J. Biol. Chem. 281:7717, 2006. S6 kinase/ribosomal protein S6 pathway. To investigate and define the function and molecular actions of the Hayashi, M., Fearns, C., Eliceiri, B., Yang, Y., Lee, J.-D. Big mitogen-activated protein kinase 1/extracellular signal-regulated kinase 5 signaling pathway is essen- BMK1 pathway in endothelial cells and in angiogene- tial for tumor-associated angiogenesis. Cancer Res. 65:7699, 2005. sis, we are investigating the signaling pathways that Hayashi, M., Imanaka-Yoshida, K., Yoshida, T., Wood, M., Fearns, C., Tatake, interact with or are regulated by the BMK1 cascade in R.J., Lee, J.-D. A critical role of mitochondrial Hsp40 in preventing dilated cardiomyopathy. Nat. Med. 12:128, 2006. endothelial cells and are exploring the mechanism of Kim, S.W., Hayashi, M.l., Lo, J.F., Fearns, C., Xiang, R., Lazennec, G., Yang, Y., action of the BMK1 cascade during angiogenesis. These Lee, J.-D. Tid1 negatively regulates the migratory potential of cancer cells by studies should provide new information on the regula- inhibiting the production of interleukin-8. Cancer Res. 65:8784, 2005. tory mechanisms of neovascularization. We hope to Zhou, H., Luo, Y., Lo, J.-F., Kaplan, C.D., Mizutani, M., Mizutani, N., Lee, J.-D., use the results to identify novel and important targets Primus, F.J., Becker, J.C., Xiang, R., Reisfeld, R.A. DNA-based vaccines activate innate and adaptive antitumor immunity by engaging the NKG2D receptor. Proc. for a more effective and specific therapeutic interven- Natl. Acad. Sci. U. S. A. 102:10846, 2005. tion for human cancer by inhibition of tumor-associated angiogenesis. THE TUMOR SUPPRESSOR/PROTEIN CHAPERONE Host-Pathogen Interactions: TID1 Tid1 is the human counterpart of the Drosophila Mechanisms and Applications tumor suppressor Tid56. Mutations that cause loss of function of the gene for Tid56 result in tumorous imagi- E. Li, S.P. Lad, J. Li nal discs due to continuous cell proliferation without icrobial pathogens can be classified into 2 differentiation. Using yeast 2-hybrid screening, we broad categories: those that infect the host found that Tid1 interacts with the signaling domain of M accidentally and those that do so for growth. the receptor protein-tyrosine kinase ErbB2/Her2. Sub- The outcome of an infection by “accidental” pathogens sequent studies indicated that increased expression of is commonly associated with severe host inflammatory 122 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE responses and is often lethal. In contrast, obligate intra- The Cysteine Protease Network cellular pathogens such as chlamydiae and rickettsiae have developed efficient yet poorly defined mechanisms in Tumor Progression to evade host immune surveillance and secure a favorable habitat. We focus on host responses to viral and and Therapy bacterial infections. We use infections with adenovirus and the obligate intracellular bacterial pathogen Chlamy- C. Liu, W. Wu, Y. Liu, F. Guo, K. Shumilak, Q. Fang dia trachomatis as models systems. eoplasms in humans arise via a multistage Chlamydia trachomatis infection affects 140 million process that involves the functions of a pro- persons worldwide. It is also the most notifiable disease N tease network. We first reported that legumain, in the United States; it leads to 50,000–100,000 new a lysosomal asparaginyl endopeptidase with a caspase- cases of pelvic inflammatory diseases and infertility like catalytic site, is highly expressed in a majority of each year. Although an inflammatory cellular response rodent and human solid tumors. Compared with adja- and chronic inflammation are the underlying mechanisms cent normal tissues, both tumor cells and tumor stro- of chlamydial diseases, a hallmark of a chlamydial infec- mal cells must survive under substantially lower oxygen tion is its asymptomatic nature. We found that Chlamy- tension within the tumor microenvironment. We found dia can downregulate host inflammatory responses by that legumain expression is induced by hypoxia and converting a regulatory molecule of the inflammation occurs early during tumor development. We showed pathway to a negative inhibitor of the same pathway. that legumain enhances tumor cell metastasis and The host responds to viral and bacterial infections invasion and protects cells from apoptosis through a by using the nuclear transcription factor NF- B to mod- κ complex and precise regulation of a cathepsin and ulate genes involved in inflammation and innate immu- caspase network (Fig. 1). nity. NF-κB consists of a heterodimeric complex composed of 2 subunits, commonly p50/NF-κB1 and p65/RelA, which are sequestered in the cytoplasm and are made inactive through their association with inhibitory molecules, including IκBα. Bacterial and viral infections, proinflammatory cytokines, and stimulation with lipopolysaccharide all can induce rapid degradation of IκBα, resulting in the release and nuclear translocation of the NF-κB complex for gene regulation. In addition to protecting IκBα from degradation induced by TNF-α, chlamydial infections promote p65/RelA cleavage. The N-terminal cleavage product functions as a dominant negative inhibitor of the NF-κB pathway and hence can block NF-κB–modulated gene expression associated with inflammatory responses. We are identifying and characterizing the bacterial protease that causes the cleavage. We are also designing and modifying adenovirus for targeted gene delivery. Although widely used for gene therapy studies, adenovirus-based vectors cannot target specific tissues. We generated modified adenoviruses that are equipped with a tumor-targeting antibody, enabling the selective delivery of therapeutic genes Fig. 1. Legumain promotes tumor cell invasion and metastasis to tumor cells by antibody-guided “missiles.” by binding to cell-surface integrins and activates both matrix metalloproteinase 2 (MMP2) and cathepsin L. It also protects cells from PUBLICATIONS programmed cell death by catalytically inactivating caspase 9. It Li, J., Lad, S., Yang, G., Lou, Y., Iacobelli-Martinez, M., Primus, F.J., Reisfeld, R.A., Li, E. The adenovirus fiber shaft contains a trimerization element supporting prevents Bid activation by cathepsin B by binding to and modulat- peptide fusion for targeted gene delivery. J. Virol., in press. ing the activity of the cathepsin. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 123

SUPPRESSION OF TUMOR INVASION, METASTASIS, Wu, W., Luo, Y., Sun, C., Liu, Y., Kuo, P., Varga, J., Xiang, R., Reisfeld, R., Janda, K.D., Edgington, T.S., Liu, C. Targeting cell-impermeable prodrug activation AND ANGIOGENESIS BY INHIBITION OF LEGUMAIN to tumor microenvironment eradicates multiple drug-resistant neoplasms. Cancer We showed that legumain is present in the tumor Res. 66:97, 2006. microenvironment, where it binds to cell-surface integrins such as αvβ3 and α5β1. Complexes consisting of legumain and αvβ3 are predominantly at the front of Role of the Tissue migrating cells. Binding of legumain to αvβ3 significantly enhances the activity of legumain toward physi- Factor–Thrombin ologic substrates such as pro–matrix metalloproteinase Pathway in Thrombosis, 2 and procathepsin L. Therefore, integrins are both receptors and cofactors of legumain. Inflammation, and Cardiac Inhibition of legumain activity by a high-affinity asparaginyl endopeptidase inhibitor suppressed angio- Ischemia-Reperfusion Injury genesis and tumor cell invasion in vitro and in vivo. R. Pawlinski, R.E. Tilley, J.P. Luyendyk, L. Kidd, E. Romeo, Systemic administration of the inhibitor in mice N. Mackman reduced tumor growth, vascular density, and tumor invasiveness in breast cancer models. Importantly, this e are interested in the role of tissue factor (TF) treatment inhibited both spontaneous and experimen- in hemostasis, thrombosis, inflammation, tal lung metastasis. These data indicate that legumain W myocardial infarction, and cardiac remodel- is a critical modulator of pericellular proteolytic cas- ing. TF is the primary initiator of blood coagulation and cades and an effective target for cancer therapy. plays an essential role in hemostasis by activating blood coagulation after vessel injury. However, aberrant TF LEGUMAIN PROTECTION AGAINST MULTIPLE expression within the vasculature induces thrombosis PROGRAMMED CELL DEATH PATHWAYS in a variety of diseases, including sepsis (Fig. 1). In Caspases do not occur in plants, and legumain is the effector protease for plant cell apoptosis. However, we found that in mammals legumain evolved to have an antiapoptotic activity. Overexpression of legumain protects cells from multiple programmed cell death pathways. We showed that during TNF-induced cell death, lysosomal proteases diffuse into cytoplasm. Legumain exerts its antiapoptosis activity by depleting procaspase 9 and inactivating caspase 9 in apopto- somes. Prolegumain can be activated by caspase 3. Therefore, the presence of legumain serves as a brake Fig. 1. Role of the tissue factor–thrombin pathway in thrombo- for the caspase cascade. In addition, legumain binds sis and cardiac remodeling. Abbreviations: LPS, lipopolysaccharide; I/R, ischemia-reperfusion. directly to cathepsin B and suppresses autoactivation of the cathepsin and cathepsin B–mediated activation addition, we have shown that TF-dependent generation of the proapoptotic protein Bid. of thrombin contributes to the size of infarcts and cardiac These findings indicate that legumain plays a criti- remodeling after cardiac ischemia-reperfusion injury. cal role in mitochondria and in apoptosis mediated by THROMBOSIS death receptors. Inhibition of legumain activity and In sepsis, bacterial lipopolysaccharide released by expression sensitizes tumor cells to natural death cues gram-negative bacteria induces TF expression by intra- and chemotherapeutic agents, providing a novel tech- vascular cells. This expression leads to disseminated nique for cancer intervention. intravascular coagulation, tissue ischemia, and inflammation. Notably, inhibition of TF reduces both coagu- PUBLICATIONS lation and inflammation in animal models of sepsis. We Bhattacharjee, G., Ahamed, J., Pedersen, B., El-Sheikh, A., Mackman, N., Ruf, W., Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the coagula- are interested in the relative contribution of TF expres- tion cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol. 25:1737, 2005. sion by monocytes, endothelial cells, and platelets to 124 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE lipopolysaccharide-induced coagulation. In collabora- remodeling and improved left ventricular function 2 tion with A. Weyrich, University of Utah, Salt Lake City, weeks after ischemia-reperfusion injury. These results we recently found that lipopolysaccharide-stimulated suggest that PAR1 contributes to cardiac remodeling human platelets express TF. after ischemia-reperfusion injury. To delete the gene for TF in the different cell types, In parallel studies, we determined the effect of over- we generated floxed TF mice. These mice have the TF expressing PAR1 in cardiomyocytes on heart morphology gene flanked by specific sequences (loxP sites) that can and function. Cardiac hypertrophy developed in a trans- be used for tissue-specific expression of TF via the Cre genic mouse model, and heart function was impaired. recombinase system. In the first experiments, floxed These data indicate that PAR1 signaling in cardiomy- TF mice were crossed with mice that express the Cre ocytes induces hypertrophy and cardiac remodeling. recombinase in myeloid cells. These mice had reduced Future studies will determine the role of TF-dependent levels of TF expression in lipopolysaccharide-stimulated generation of thrombin in the activation of PAR1 in macrophages in a murine endotoxemia model, indicating the heart. that monocytes are a major source of intravascular TF in endotoxemia. Future studies will determine how delet- PUBLICATIONS Bhattacharjee, G., Ahamed, J., Pedersen, B., El-Sheikh, A., Mackman, N., Ruf, W., ing the TF gene in either endothelial cells or platelets Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the coagula- affects lipopolysaccharide-induced coagulation. We plan tion cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol. 25:1737, 2005. to design novel strategies targeting intravascular TF that Hayashi, M., Matsushita, T., Mackman, N., Ito, M., Adachi, T., Katsumi, A., Yamamoto, K., Takeshita, K., Kojima, T., Saito, H., Murohara, T., Naoe, T. Fatal will reduce lipopolysaccharide-induced coagulation with- thrombosis of antithrombin-deficient mice is rescued differently in the heart and liver out increasing the risk of bleeding. by intercrossing with low tissue factor mice. J. Thromb. Haemost. 4:177, 2006.

INFLAMMATION Kamimura, M., Viedt, C., Dalpke, A., Rosenfeld, M.E., Mackman, N., Cohen, We are interested in the role of the phosphatidylinos- D.M., Blessing, E., Preusch, M., Weber, C.M., Kreuzer, J., Katus, H.A., Bea, F. Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimulated itol-3′-kinase (PI3K)–Akt intracellular signaling pathway macrophages via inhibition of Egr-1 and a serum response element/MEK-ERK1/2 in lipopolysaccharide-induced inflammation and coag- pathway. Circ. Res. 97:305, 2005. ulation. We have shown that activation of the PI3K-Akt Luyendyk, J.P., Tilley, R.E., Mackman, N. Genetic susceptibility to thrombosis. pathway inhibits lipopolysaccharide induction of inflam- Curr. Atheroscler. Rep. 8:193, 2006. matory mediators and TF in human monocytic and Mackman, N. Role of tissue factor in hemostasis and thrombosis. Blood Cells Mol. endothelial cells and in a murine endotoxemia model. Dis. 36:104, 2006. Interestingly, several compounds that improved survival Mackman, N. Tissue-specific hemostasis in mice. Arterioscler. Thromb. Vasc. Biol. 25:2273, 2005. in sepsis also activated the PI3K-Akt pathway. For example, we found that a low dose of insulin, which Motton, D.D., Mackman, N., Tilley, R.E., Rutledge, J.C. Postprandial elevation of tissue factor antigen in the blood of healthy adults. Thromb. Haemost. 94:504, 2005. did not affect blood glucose levels, reduced inflammation and improved survival in a murine endotoxemia model Rushworth, S.A., Chen, X.-L., Mackman, N., Ogborne, R.M., O’Connell, M.A. Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic in a PI3K-dependent manner. More recently, we found cells is mediated via Nrf2 and protein kinase C. J. Immunol. 175:4408, 2005. that simvastatin, a widely used cholesterol-lowering Schabbauer, G., Mackman, N. Tissue factor expression by the endothelium. In: drug, inhibits lipopolysaccharide induction of inflamma- The Endothelium: A Comprehensive Reference. Aird, W. (Ed.). Cambridge Univer- tion and coagulation in mice. Importantly, the protective sity Press, New York, in press. effect of simvastatin was abolished by inhibition of PI3K. Tilley, R.E., Mackman, N. Tissue factor in hemostasis and thrombosis. Semin. Future studies will determine how insulin and simvas- Thromb. Haemost. 32:5, 2006. tatin reduce lipopolysaccharide-induced TF and cyto- Tilley, R.E., Pedersen, B., Pawlinski, R., Sato, Y., Erlich, J.H., Shen, Y., Day, S., Huang, Y., Eitzman, D.T., Boisvert, W.A., Curtiss, L.K., Fay, W.P., Mackman, N. kine expression in vitro and in vivo. Atherosclerosis in mice is not affected by a reduction in tissue factor expression. MYOCARDIAL INFARCTION AND CARDIAC Arterioscler. Thromb. Vasc. Biol. 26:555, 2006. REMODELING We have shown that the TF-thrombin pathway contributes to infarction after cardiac ischemia-reperfusion injury. More recently, we found that a deficiency of protease-activated receptor 1 (PAR1), a receptor for thrombin, did not affect the size of infarcts. However, PAR1 deficiency was associated with reduced cardiac IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 125

ANTIGEN-EXPERIENCED DENDRITIC CELLS Regulating Adaptive Immunity Dendritic cells are the most efficient innate initiators of adaptive immunity. Immune adjuvants substantially M.G. McHeyzer-Williams, L.J. McHeyzer-Williams, affect the developmental response of subsets of dendritic L.P. Malherbe, N.R. Fazilleau, N. Pelletier cells that in turn regulate separable functions in the REGULATION OF B-CELL IMMUNITY BY HELPER helper T cell and B-cell compartments in vivo. Using T CELLS antibodies that reveal specific peptide–MHC II com- elper T cells are the master regulators of adap- plexes on dendritic cells, we can directly isolate anti- tive immunity that control the development of gen-experienced dendritic cells to evaluate the effect of H antigen-specific B-cell immunity. We seek to immune adjuvants on priming of the cells in vivo. define the cellular and molecular details of the major developmental checkpoints that regulate the fate of PUBLICATIONS Alfonso, C., McHeyzer-Williams, M.G., Rosen, H. CD69 down-modulation and these helper T cells and B cells in vivo. We recently inhibition of thymic egress by short- and long-term selective chemical agonism of extended our studies to the earliest innate immune sphingosine 1-phosphate receptors. Eur J. Immunol. 36:149, 2006. events that initiate and shape this adaptive immune McHeyzer-Williams, L.J., Malherbe, L.P., McHeyzer-Williams, M.G. Checkpoints response. If we can understand the rules that control in memory B-cell evolution. Immunol. Rev. 211:255, 2006. adaptive immunity, we can design safe and effective McHeyzer-Williams, L.J., Malherbe, L.P., McHeyzer-Williams, M.G. Helper T cell protein subunit vaccines. regulated B cell immunity. Curr. Trends Microbiol. Immunol. 311:59, 2005.

SELECTION OF ANTIGEN-SPECIFIC HELPER T CELLS McHeyzer-Williams, L.J., McHeyzer-Williams, M.G. Memory B cell development. We recently described an affinity threshold model for In: The Autoimmune Diseases, 4th ed. Rose, N.R., Mackay, I.R. (Eds.). Elsevier, St. Louis, 2006, p. 157. selecting antigen-specific helper T cells in the response to protein antigens. The unique characteristic of this McHeyzer-Williams, M.G., McHeyzer-Williams, L.J., Malherbe, L.P. B cells dis- criminate the rules of engagement. Immunity 24:125, 2006. model is how clonal diversity is achieved and maintained through the progressive loss of lower affinity cells rather than through the preferential expansion of the high-affinity clonotypes. Our evidences suggests that Adaptive and Innate Responses selection is not due to interclonal competition but is a to Alloantigens more intrinsic threshold with a “set point” for selection. Hence, in order to manipulate antigen-specific clonal D.B. McKay, A. Shigeoka diversity, this affinity threshold must be altered. We are examining multiple variables of vaccination to urgical and medical advances have provided an define these rules, with emphasis on the impact of opportunity for life to patients with end-stage immune adjuvant on the priming of adaptive immunity. S organ disease. One of the most remarkable EVOLUTION OF MEMORY B CELLS advances has been the replacement of organs through The rapid evolution of memory B cells occurs at the transplantation. Despite remarkable technological cellular level and is controlled by multiple cognate advances, the host immune system must be suppressed checkpoints during the development of adaptive immu- to prevent rejection of the allogeneic transplanted organ. nity. Although clonal selection is the fundamental pro- The suppression of host immunity requires the life-long cess that underpins adaptive immunity, surprisingly little use of toxic nonspecific immunosuppressive medica- is understood about the mechanism of its action across tions. One experimental method has allowed survival multiple junctures of development of antigen-specific of transplanted organs without the use of immunosup- memory B cells in vivo. Our recent studies focus on the pressive medications: intravenous exposure of the organ consolidation of B-cell memory upon antigen recall in recipient to donor antigens before transplantation. In the boost phase of vaccination with protein antigens. several animal models and human clinical trials, expo- Cellular and molecular analysis of the memory response sure to donor antigens before transplantation downregu- reveals the developmental staging of memory B cells lated the T-cell responses of recipients to donor antigens. that express different antibody subtypes. These studies One focus of our research is the intracellular signal- highlight the importance of antibody isotype as a fun- ing events that lead to the induction of peripheral T-cell damental cellular division within antigen-specific B- tolerance by exposure to donor antigens. We found that cell memory. intravenous infusion of semiallogeneic donor cells into 126 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE recipient mice leads to a series of events that culmi- receptors CD4 and CCR5. Because CCR5 binding occurs nate in acquired unresponsiveness to donor antigens after CD4 binding, CCR5 is defined as a coreceptor. The and tolerance to allografts. We discovered that several importance of CCR5 in HIV-1 infection was first appre- proximal T-cell receptor–coupled signaling molecules are ciated because some persons have a natural mutation altered in peripheral T cells of the recipient mice. We that prevents expression of CCR5. These persons are are investigating how these proximal molecules may be resistant to HIV-1 infection, and they have few clinical regulated in T cells from recipients that do not reject consequences of lacking CCR5. These observations led their transplanted organs. to the development of CCR5-blocking agents to prevent In addition, we are interested in the initial events HIV-1 infection. that regulate activation of recipient T cells, namely, the However, HIV-1 can undergo mutations that allow events that regulate activation of the cells that present a second chemokine receptor, CXCR4, to replace the donor antigens. Several recent studies in animals have binding function of CCR5. We have studied the costs suggested that the family of evolutionarily conserved to viral fitness of those mutations and their importance cell-surface Toll-like receptors might respond to ligands in resistance to CCR5 inhibitors. released by apoptotic and necrotic tissue and that such ANTIVIRAL COMPOUNDS THAT TARGET CCR5 ligands might provide an important molecular trigger The normal function of CCR5 is to bind chemokines for adaptive responses to ischemic injury. More recently, and signal cell migration. RANTES (CCL5) is the CCR5- we have been investigating how binding of Toll-like binding chemokine with the most potent activity against receptors leads to activation of responses to donor HIV-1, but its activity is limited. We prepared synthetic antigens and how to target these mechanisms to pre- modifications of the N-terminal domain of RANTES. vent allograft recognition and rejection. We found that the most potent of these compounds, PSC-RANTES, is 1000 times more effective than PUBLICATIONS Josephson, M.A., McKay, D.B. Management of pregnancy in the transplant recipi- native RANTES at inhibiting HIV-1 infection. A single ent. Adv. Chronic Kidney Dis., in press. injection of PSC-RANTES before inoculation of virus McKay, D.B., Adams, P., Bumgardner, G.L., Davis, C.L., Fine, R.N., Krams, S.M., prevented HIV-1 infection in 100% of mice with severe Martinez, O.M., Murphy, B., Pavlakis, M., Tolkoff-Rubin, N., Sherman, M.S., Josephson, M. Reproduction and pregnancy in transplant recipients: current prac- combined immunodeficiency repopulated with human tices. Prog. Transplant. 16:127, 2006. peripheral blood leukocytes. Brief exposure of human McKay, D.B., Josephson, M. Pregnancy in transplant recipients of solid organs: cells to PSC-RANTES leads to prolonged internaliza- effects on mother and child. N. Engl. J. Med. 354:1281, 2006. tion of CCR5. McKay, D., Shigeoka, A., Rubinstein, M., Surh, C., Sprent, J. Simultaneous dele- These properties led to the formulation of PSC- tion of MyD88 and Trif delays major histocompatibility and minor antigen mismatch allograft rejection. Eur. J. Immunol. 36:1994, 2006. RANTES as a topical microbicide to prevent sexual transmission of HIV-1. Treatment with PSC-RANTES Zambricki, E., Zal, T., Yachi, P., Shigeoka, A., Sprent, J., Gascoigne, N., McKay, D. In vivo anergized T cells form altered immunological synapses in vitro. Am. J. can prevent vaginal transmission of a chimeric virus Transplant. 6:2572, 2006. consisting of simian immunodeficiency virus and HIV in the rhesus macaque model. To ensure that PSC- RANTES will be effective against HIV-1 variants Blocking CCR5 to Inhibit HIV present in Africa, we examined the sensitivity of a Type 1 Infection number of recently transmitted isolates from Kenya. Fortunately, all of the isolates were more sensitive to D.E. Mosier, R. Nedellec, C. Pastore, A. Ramos, PSC-RANTES inhibition than were laboratory HIV-1 J. Salkowitz-Bokal, S. Pontow,* L. Ratner,* O. Hartley,** isolates from the United States. R. Offord,** J. Overbaugh,*** M. Lederman**** MUTATIONAL COSTS OF CORECEPTOR SWITCHING * Washington University School of Medicine, St. Louis, Missouri One concern about CCR5-blocking agents such as ** Centre Médical Universitaire, Geneva, Switzerland PSC-RANTES is that they might select for resistant *** University of Washington, Seattle, Washington viruses that can infect via other chemokine receptors, **** Case Western Reserve University, Cleveland, Ohio such as CXCR4. Although previously we showed that IV type 1 (HIV-1), the cause of the AIDS pan- such “coreceptor switch” mutants can arise during treat- demic, infects human cells by sequential binding ment, a detailed analysis revealed that most mutants H of the viral envelope protein to the cell-surface have a loss of fitness during coreceptor switching that IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 127 coincides with a period when neither CCR5 nor CXCR4 Control of Cytokine Expression supports efficient virus infection. The mutations in the HIV-1 envelope that drive core- by Arginine Methylation ceptor switching occur mainly in the exposed variable loops (V1/V2 and V3), and different HIV-1 isolates K.A. Mowen, D.M. Hill, S. Hemmers, K. Bonham require as few as 1 mutation or as many as 7 mutations to switch from use of CCR5 to use of CXCR4. Poor elper T cells can be divided into 2 distinct pop- replication on both CCR5- and CXCR4-expressing tar- ulations on the basis of their immune specific- get cells and increased sensitivity to both CCR5 and H ity and cytokine profiles. Type 1 helper T cells CXCR4 inhibitors were common features of viruses that produce IFN-γ and are responsible for cell-mediated were switching coreceptors. immunity; type 2 helper T cells secrete IL-4 and are To more fully understand the cost of each mutation associated with the humoral immune response. These associated with changing coreceptor binding from CCR5 2 types of cells have been associated with susceptibil- to CXCR4, we reconstructed all possible mutational ity to malignant, infectious, allergic, and autoimmune pathways between 4 parental CCR5-using viruses and diseases. The improper development of type 2 helper their CXCR4-using descendants separated by 3–7 muta- T cells can lead to allergy and asthma, and an overac- tions. We used site-directed mutagenesis to introduce all tive response by type 1 helper T cells can lead to auto- possible combinations of single and multiple mutations immune diseases such as type 1 diabetes. in the HIV-1 envelope gene. These mutated envelopes Because of the opposing roles of the 2 types in were combined with an envelope-deficient reporter immune function, the development and migration of virus to make HIV-1 particles capable of only a single helper T cells must be tightly regulated. Indeed, the cycle of infection. discrete subsets, type 1 and type 2, reciprocally antago- Mutations in variable loops 1 and 2 of the envelope nize the maturation and behavior of each other in the improved the use of CCR5 but did not permit infection immune response, resulting in a population of helper via CXCR4. Mutations in variable loop 3 led to use of T cells that is primarily type 1 or type 2. Thus, manipu- CXCR4 for viral entry, but only poorly. Combinations of lating the ratio of type 1 to type 2 T helper cells pro- mutations in all 3 variable loops improved the ability vides an intriguing avenue of therapy, and understanding of the virus to use CXCR4. The sequence in which the molecular events that control lineage-specific cyto- mutations were introduced was critical. The probabil- kine expression may provide useful tools for modulat- ity of coreceptor switching is thus constrained by having the helper T cell response. ing to make the right mutation at the right place at the Although several lineage-specific and nonspecific right time. transcription factors are required for the development Using chimeric coreceptors with the 4 extracellular and function of type 1 and type 2 helper T cells, less domains derived from either CCR5 or CXCR4, we also is known about the events that occur after the reacti- mapped the domains of CCR5 or CXCR4 required for vation of type 1 and type 2 effector populations and infection by each of the mutated envelopes. The initial result in the disparate cytokine profiles of the 2 types stage of coreceptor switching favored use of the second of helper T cells. Signal transduction pathways use post- extracellular domain of CXCR4, and 2 mutants required translational modifications to translate changes in the only this domain for infection. This result allows the extracellular milieu into environment-sensitive gene mapping of mutations near the tip of variable region 3 expression in a timely and efficient fashion. of the envelope that must confer binding to extracellu- Phosphorylation of serine, threonine, and tyrosine lar loop 2 of CXCR4. The results from these mapping residues and protein ubiquitination has been widely studies of envelope interactions with coreceptors will studied. Although methylation of arginine residues was enable us to predict the likelihood of resistance-con- discovered more than 30 years ago, it has only recently ferring mutations in patients during clinical trials of aroused renewed interest. Arginine methylation of CCR5 inhibitors. proteins by members of the protein arginine methyl-

PUBLICATIONS transferase (PRMT) family regulates the subcellular Pastore, C., Nedellec, R., Ramos, A., Pontow, S., Ratner, L., Mosier, D.E. Human localization of the methylated proteins and modulates immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. J. Virol. 80:750, 2006. protein-protein interactions. 128 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

We discovered a unique contribution of arginine resulting in “receptor editing,” a process in which pre- methylation to cytokine gene expression downstream of viously expressed genes for antibody light chains are signaling by T-cell receptors. Our goal is to investigate inactivated and replaced by secondary DNA recombi- more broadly the role for arginine methylation in immune nation. More recent data indicated that editing can function, including further study of helper T cells and also play an important role in inactivating and replac- other immune cell types. We also plan to examine the ing receptor genes that are underexpressed at the pro- upstream regulation of PRMT expression and activity tein level. In this situation, subnormal expression of and to characterize the effects of ablation or suppres- unligated surface immunoglobulin does not provide a sion of PRMT expression. Understanding the role of needed signal. posttranslational modifications, such as arginine methy- These recent results suggest that quality control of lation, of proteins that are key in regulating cytokine newly formed B lymphocytes is surprisingly stringent production will give us novel targets in diseases induced and that through recombinase regulation B cells are or exacerbated by the cytokine environment, such as often able to “repair” unacceptable light-chain genes inflammatory arthritis. by replacing the unacceptable genes with new genes. Because of the apparent efficiency of the editing process, we suspect that we have uncovered a major cel- Analysis of Immune Learning lular “proofreading” pathway. A key question of current interest is how signaling in B Lymphocytes through the antigen receptor regulates editing. A major nuclear end point is the regulation of RAG transcription. D. Nemazee, A. Gavin, D. Aït-Azzouzene, C. Huber, T. Ota, We are assessing the biochemical signaling pathways by J. Vela, B. Duong, P. Skog, M. Lim which the signal from antigen receptors regulate RAG he main goal of our research is to understand transcription. Recent studies suggested that NF-κB and how lymphocytes distinguish between self and rel transcription factors may be involved in both positive T nonself antigens. Because antigen receptors on and negative regulation of the RAG genes. We have also lymphocytes are assembled from component parts made progress in our understanding of the triggering through an essentially random mechanism, many lym- involved in B-cell positive selection, in which innocuous phocytes have self-reactive receptors. Regulation of such B-cell receptors, via tonic signaling, activate a signaling autoreactive specificities may be important to prevent cascade that involves the activity of phosphatidylinositol- autoimmune disease and to ensure efficient response 3′-kinase and recruited effectors, including phospholi- to microbes. pase C γ2 and Akt. Tonic signaling refers to the weak The development of B lymphocytes is a multistep pro- signal generated by the unoccupied B-cell receptor for cess punctuated by the somatic generation of antibody antigen. This pathway appears to be inactivated in heavy and light chain genes through DNA recombination, autoreactive immature B cells, a finding that probably which is catalyzed by the products of recombinase explains why the time frame of editing is limited. activator gene 1 (RAG-1) and RAG-2. Because V(D)J To assess the role of receptor editing in preventing recombination is imperfect and error prone, pre-B and unwanted autoreactivity, we generated mice that have B cells are endowed with sensing mechanisms to detect a defect in receptor editing. These mutant mice lack a protein expression of heavy chains and assembled heavy functional recombining sequence/κ light chain–deleting and light chains (i.e., intact surface IgM). A major func- element, which is involved in destructive editing of loci tion of the expression of immunoglobulin in immature for κ light chains in cells that go on to rearrange either B cells is signaling to downregulate recombinase activ- a second allele for κ light chains or genes for λ light ity and to stimulate developmental progression. Newly chains. These mice are being assessed for their ability formed B-cell receptors are also screened for autoreac- to produce autoantibodies and to accelerate autoim- tivity. These quality control mechanisms rely on signal- mune disease when crossbred with mice that are prone ing by antigen receptors. to autoimmunity. Previously, we showed that B cells with autoreactive In other studies, we focused on the cues that mature receptors do not downregulate recombination because B cells use to distinguish self from nonself. Fully mature of excessive signaling through the antigen receptor, recirculating B cells can be rapidly inactivated and IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 129 induced to apoptosis when confronted with tissue anti- In recent studies, we analyzed different types of aden- gen, whereas the same cells are able to respond to oviruses that use coxsackievirus-adenovirus receptors antigens expressed by microbes. We are investigating or CD46 as their primary receptors for the ability of the both the death pathway involved in self-tolerance and viruses to trigger innate immune responses as measured the nature of the signals that prevent this pathway in by production of type I interferon in human peripheral responses to nonself antigens. Recently, we found that blood mononuclear cells. the ability of B cells to distinguish self from nonself in We found that adenoviruses that use CD46 preferen- this setting is independent of T lymphocytes and instead tially induced production of IFN-α and that this finding most likely involves a novel pathway of self-recognition. most likely was due to activation of Toll-like receptor 9, We are testing the hypothesis that signaling by Toll-like because an oligonucleotide antagonist of the receptor receptors is not required for this discrimination. We inhibited cytokine expression. Moreover, empty/imma- are also exploring the idea that immune tolerance in ture adenovirus particles that lacked double-stranded mature B cells depends on specific costimulation by DNA did not induce interferon production even though self-tissue, a mode of signaling akin to missing self- they were fully capable of binding to and entering host recognition by natural killer cells. cells. In further studies, we found that epithelial cells that supported equivalent levels of infection by aden- PUBLICATIONS oviruses that used coxsackievirus-adenovirus receptors Aït-Azzouzene, D., Gavin, A.L., Skog, P., Duong, B., Nemazee, D. Effect of cell:cell competition and BAFF expression on peripheral B cell tolerance and B-1 cell sur- and by adenoviruses that used CD46 produced signifi- vival in transgenic mice expressing a low level of Igκ-reactive macroself antigen. cantly higher amounts of interferon upon infection by Eur. J. Immunol. 36:985, 2006. the viruses that used CD46. Aït-Azzouzene, D., Verkoczy, L., Duong, B., Skog, P., Gavin, A.L., Nemazee, D. Split tolerance in peripheral B cell subsets in mice expressing a low level of Igκ- These findings indicate that distinct receptor-medi- reactive ligand. J. Immunol. 176:939, 2006. ated entry pathways may play a pivotal role in activation

Collins, C.E., Gavin, A.L., Migone, T.S., Hilbert, D.M., Nemazee, D., Stohl, W. B of Toll-like receptor 9 by adenovirus. The findings also lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease have implications for the development of safer adeno- activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels. Arthritis Res. Ther. 8:R6, 2005. virus vectors for clinical applications.

Gavin, A.L., Hoebe, K., Duong, B., Ota, T., Martin, C., Beutler, B., Nemazee, D. Minor role for toll-like receptor signaling in adjuvant-enhanced antibody responses. Science, in press. Improving Adenovirus Nemazee, D. Receptor editing in lymphocyte development and central tolerance. Nat. Rev. Immunol. 6:728, 2006. Transduction of Human Nemazee, D., Gavin, A., Hoebe, K., Beutler, B. Immunology: Toll-like receptors and antibody responses. Nature 441:E4, 2006. Myeloid Cells

Watson, L.C., Moffatt-Blue, C.S., McDonald, R.Z., Kompfner, E., Aït-Azzouzene, D., Nemazee, D., Theofilopoulos, A.N., Kono, D.H., Feeney, A.J. Paucity of V-D- R. Nepomuceno, L. Pache, G.R. Nemerow D-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT-/- and Tdt+/+ mice. J. Immunol. 177:1120, 2006. endritic cells are ideal targets for immunomodulatory regimens to treat genetic or acquired D diseases. However the lack of coxsackievirus- Toll-like Receptor 9 Signaling by adenovirus receptors on dendritic cells has stymied transfer of genes to these cell types via type 5 adeno- Adenoviruses That Use CD46 viruses. In recent studies, we found that the fiber knob derived from adenovirus type 37 (37FK) could signifi- Receptors cantly enhance adenovirus-mediated gene transfer to primary human monocytes and to dendritic cells but M. Martinez-Iacobelli, G.R. Nemerow not to T and B lymphocytes. Fiber knobs derived from uman adenoviruses are potent activators of the other adenovirus strains, including type 5 and type 16, innate immune response, a feature that limits did not have this ability. Enhancement of gene transfer H their use for in vivo gene transfer. However, the by 37FK depended on sialic acid, because removal of precise mechanisms by which different adenoviruses sialic acid residues by treatment with neuraminidase trigger innate immunity have not been fully elucidated. abrogated gene transfer. Moreover, lectins with speci- 130 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE ficity for α2,6-linked sialic acid residues, but not lectins protein VI molecules can be incorporated into naturally with specificity for α2,3-linked sialic acid residues, could occurring nanoparticles in an attempt to improve gene inhibit gene transfer by 37FK. In further investigations, delivery to host cells. we found that 37FK bound directly to adenovirus particles and thereby increased virus binding to monocytic cells. Structure Analyses of Adenovirus We concluded that an electrostatic interaction between the positively charged 37FK and the nega- via Electron Cryomicroscopy and tively charged virus capsid and cell-surface sialic acid X-ray Diffraction residues results in the formation of a ternary complex that potentiates adenovirus infection and gene trans- S. Saban,* P. Stewart,* V. Reddy, G.R. Nemerow fer. These findings may point the way for improving * Vanderbilt University School of Medicine, Nashville, Tennessee gene delivery to dendritic cells. denovirus is one of the largest macromolecular complexes whose structure has been analyzed by Adenovirus-Mediated Disruption A using electron cryomicroscopy or x-ray diffraction. In ongoing studies, we are using electron cryomi- of Endosomes and Development croscopy and images of approximately 2000 particles to determine the adenovirus structure to 6–7 Å. At this level of Nanoparticle Delivery Methods of resolution, we can clearly resolve multiple α-helices present in the penton base, protein VI, hexon, and pro- J.G. Smith, C. Wiethoff,* S. Police,** C.Y. Lai, tein IIIa. These studies have also revealed the location V. Kickhoefer,*** L. Rome,*** G.R. Nemerow and potential associations of the adenovirus proteins * Loyola University Medical Center, Maywood, Illinois located on the inner capsid surface that help stabilize ** Cell Genesys, Inc., San Francisco, California the virus before its disassembly in the early endosome. *** University of California, Los Angeles, California The pseudoatomic model generated from the electron he mechanisms by which nonenveloped viruses, cryomicroscopy data was used to facilitate structural including adenovirus, penetrate the barrier of the analyses of the virus by x-ray diffraction. We found that T host cell endosomal membrane are not well under- large single crystals of adenovirus diffracted to about stood. In previous studies, we identified an internal 5-Å resolution at different synchrotron beam lines. capsid protein, designated protein VI, that may mediate Recent studies indicated that the adenovirus crystals the disruption of the early endosome upon partial dis- can be frozen, thereby allowing the collection of nearly assembly of the virion at low pH. To test this hypothe- complete electron cryomicroscopy data sets from single sis, we recently examined the infectivity of a panel of crystals. Generation of electron density maps from x-ray mutant adenoviral particles that contain single amino diffraction data revealed distinct features of the inner acid substitutions in the putative membrane-reactive core of the virus. This new information may provide domain of protein VI. further insights into the assembly and disassembly of Using a quantitative fluorescence imaging device, adenovirus as well as the mode of viral DNA uncoat- we found that several of the particles with mutant pro- ing and recognition by Toll-like receptor 9. tein VI molecules had mildly reduced infectivity compared with that of wild-type virions. Further biochemical PUBLICATIONS assays revealed that the reduced infectivity was not due Maginnis, M.S., Forrest, J.C., Kopecky-Bromberg, S.A., Dickeson, S.K., Santoro, S.A., Zutter, M.M., Nemerow, G.R., Bergelson, J.M., Dermody, T.S. β1 Integrin to defects in virion assembly or disassembly. Currently, mediates internalization of mammalian reovirus. J. Virol. 80:2760, 2006. we are evaluating the membrane lytic and endosome- Nepomucena, R., Pache, I., Nemerow, G.R. Enhancement of gene transfer to disrupting properties of the mutant adenoviruses; we human myeloid cells by adenovirus-fiber complexes. Mol. Ther., in press. expect that some of the mutants will have defects in Nicklin, S.A., Wu, E., Nemerow, G.R., Baker, A.H. The influence of adenovirus these activities. In keeping with this expectation, we fiber structure and function on vector development for gene therapy. Mol. Ther. 12:384, 2005. found that mutations in protein VI that affect virus infection also reduce binding of protein VI to liposomes. Saban, S.D., Silvestry, M., Nemerow, G.R., Stewart, P.L. Visualization of α-helices in a 6 Å resolution cryoEM structure of adenovirus allows refinement of capsid protein Finally, efforts are under way to determine if wild-type assignments. J. Virol., in press. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 131

Wodrich, H., Cassany, A., D‚Angelo, M.A., Guan, T., Nemerow, G., Gerace, L. of dendritic cells, indicating the positive helper function Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways. J. Virol. 80:9608, 2006. of these cells in the activation of dendritic cells in vivo. We also found that depletion of CD8+ T cells or natural killer cells during the priming, but not the effector Enhancement of Lymphocyte phase, led to reduced activity of natural killer or cytotoxic T cells, respectively. Thus, indirect cross talk exists Cross Talk by Engagement of between these cells and is mediated through dendritic cells or by direct interactions between natural killer the NKG2D Receptor cells and CD8+ T cells. In contrast to natural killer and CD8+ T cells, CD4+ H. Zhou, Y. Luo, C.D. Kaplan, J.A. Krueger, S.H. Lee, T cells appear to negatively regulate these effector cells. R. Xiang, R.A. Reisfeld Depletion of CD4+ cells during the priming phase led to cells targeting tumor-associated antigens are the activation of dendritic cells, natural killer cells, and readily detectable in cancer patients who have CD8+ T cells and to enhanced activity of natural killer T received cancer vaccines, but often the cells do cells, suggesting the existence of CD4+CD25+ T regula- not eradicate tumors. Thus, established tumors can tory cells. Because CD4+CD25+ cells lack expression of apparently induce immune tolerance through as yet the NKG2D receptor, even after activation, this finding poorly defined mechanisms. We hypothesized that could explain the lack of enhancement of activation and immunization strategies that target different areas of activity of T regulatory cells despite the profound acti- the immune system could overcome this immune tol- vation of dendritic cells, natural killer cells, and CD8+ erance. We used interactions between the NKG2D T cells. Thus, by engaging the NKG2D receptor, our receptor and its ligand to test this hypothesis, because vaccine preferentially activated natural killer and CD8+ the receptor occurs at the crossroad between innate T cells, a situation that might have tilted the balance and adaptive immunity. toward immune surveillance and breakage of peripheral NKG2D, a stimulatory lectinlike receptor, is tolerance to tumor antigens mediated by T regulatory expressed on natural killer cells, activated CD8+ T cells. Our finding that depletion of CD4+ T cells during cells, γζ T cells, and activated macrophages and the effector phase also reduced the activity of cyto- mediates costimulatory signals for CD8+ T cells and toxic T cells suggests that CD4+ T-cell help is required stimulatory signals for natural killer cells and macro- to maintain antigen-specific CD8+ T cells in vivo. phages. In addition, NKG2D ligands are related to In our experimental model in which attenuated Sal- MHC class I molecules, which in mice include products monella typhimurium specifically delivers the DNA of the H60 ligand. Importantly, in syngeneic mice, encoding the pH60/survivin vaccine to Peyer’s patches, ectopic expression of NKG2D ligands causes natural these secondary lymphoid tissues most likely are the killer cell–mediated rejection of transfected tumor cells location for T-cell priming. The ability of the vaccine and primes cytotoxic T lymphocytes, which are to increase homing of dendritic cells and natural killer responsible for rejecting subsequent challenges by cells to Peyer’s patches but decrease the homing of tumor cells that do not express the NKG2D ligand. CD4+ T cells could presumably be due to changes in In previous studies, we showed that engagement the homing receptor profile of the cells. The upregula- of the NKG2D receptor markedly improved the antitu- tion of the chemokine receptor CCR7 we observed espe- mor efficacy of a DNA vaccine encoding both the cially on CD8+ T cells could occur if a greater percentage NKG2D ligand H60 and the inhibitor of apoptosis proof naive CD8+ T cells home to Peyer’s patches, because tein survivin. This combination vaccine activated both CCR7 is highly expressed on naive T cells and mediates innate and adaptive antitumor immunity and resulted their homing to these secondary lymphoid tissues. In in improved protection against tumors of different ori- this situation, these findings may indicate that the gins and with different levels of expression of NKG2D. pH60/survivin vaccine also induced changes in stro- More recently, we found that this combination vaccine mal cells in Peyer’s patches, especially because such induces intense cross talk between dendritic cells, cells are the source of ligands of CCR7, such as che- natural killer cells, and T cells. Depletion of natural mokines CCL19 and CCL21. Because lymphocytes killer or CD8+ T cells led to a decrease in activation activated in Peyer’s patches express homing receptors, 132 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE the cells most likely home to the periphery, where they protective effects of APC is incompletely understood. can combat tumor cells or become quiescent in the Protein C is activated to APC by the key procoagulant absence of antigen. Currently, determining their exact enzyme thrombin bound to thrombomodulin on the fate is difficult because such lymphocytes lack specific surface of endothelial cells. APC in turn downregulates and stable markers. generation of thrombin in a negative feedback loop. Taken together, we showed that by preferentially Previously, we showed that APC signaling in endo- activating and attracting positive regulators and reducing thelial cells requires binding to endothelial protein C negative regulators in Peyer’s patches, our pH60/survivin receptor and activation of protease-activated receptor 1 DNA vaccine induced increased lymphocyte cross talk (PAR1), the thrombin receptor. Thrombin-PAR1 signal- and thereby established a microenvironment more suit- ing has well-established proinflammatory effects, includ- able for activation of natural killer cells and T-cell priming disruption of endothelial barrier function, raising ing. The success of this vaccine in combating tumors the question of how the same receptor can also medi- of different origins and with different levels of NKG2D ate protective effects of APC. Large-scale gene expres- expression in prophylactic and therapeutic models and sion profiling indicated that APC-PAR1 downregulated in inducing long-lived immune memory indicates that transcript levels of proapoptotic proteins and that some activation of the innate and adaptive arms of the immune of these transcripts were upregulated by thrombin-PAR1. system is an attractive strategy to overcome tumor- Furthermore, APC-PAR1 had powerful endothelial bar- induced peripheral tolerance. rier protective effects through cross-activation of the sphingosine 1-phosphate (S1P) signaling pathway PUBLICATIONS Abdollahi, A., Griggs, D.W., Zieher, H., Roth, A., Lipson, K.E., Saffrich, R., (Fig. 1). Incubation of an endothelial monolayer with Grone, H.J., Hallahan, D.E., Reisfeld, R.A., Debus, J., Niethammer, A.G., Huber, P.E. Inhibition of αvβ3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy. Clin. Cancer Res. 11:6270, 2005.

Allen, B.J., Raja, C., Rizvi, S., Li, Y., Tsui, W., Graham, P., Thompson, J.F., Reis- feld, R.A., Kearsley, J. Intralesional targeted alpha therapy for metastatic melanoma. Cancer Biol. Ther. 4:1318, 2005.

Mahanivong, C., Krueger, J.A., Bian, D., Reisfeld, R.A., Huang, S. A simplified closing strategy for the generation of an endothelial cell selective recombinant adenovirus vector. J. Virol Methods 135:127, 2006.

Osenga, K.L., Hank, J.A., Albertini, M.R., Gan, J., Sternberg, A.G., Eickhoff, J., Seeger, R.C., Matthay, K.K., Reynolds, C.P., Twist, C., Krailo, M., Adamson, P.C., Reisfeld, R.A., Gillies, S.D., Sondel, P.M. A phase 1 clinical trial of the hu14.18- IL2 (EMD 273063) as a treatment for children with refractory or recurrent neuroblastoma and melanoma: a study of the Children’s Oncology Group. Clin. Cancer Res. 12:1750, 2006.

Schrama, D., Reisfeld, R.A., Becker, J.C. Antibody targeted drugs as cancer therapeutics. Nat. Rev. Drug Discov. 5:147, 2006. Fig. 1. Inflammatory disorders such as sepsis are associated with increased permeability of the endothelial cell monolayer at the Schrama, D., Voigt, H., Eggert, A.O., Xiang, R., Reisfeld, R.A., Becker, J.C. Ther- apeutic efficacy of tumor-targeted IL2 in Ltα–/– mice depends on conditioned T blood-tissue interface. APC-mediated enhancement of endothelial cells. Cancer Immunol. Immunother. 55:861, 2006. barrier integrity depends on binding of APC to endothelial protein C Zhou, H., Luo, Y., Kaplan, C.D., Krueger, J.A., Lee, S.H., Xiang, R., Reisfeld, R.A. receptor and activation of PAR1, cellular sphingosine kinase-1 (SK1), A DNA-based cancer vaccine enhances lymphocyte cross talk by engaging the and S1P receptor-1 (S1P1). Thrombin can affect barrier integrity in NKG2D receptor. Blood 107:3251, 2006. at least 3 ways: proinflammatory signaling by higher concentrations can disrupt endothelial barrier integrity, incubation with low concentrations has a barrier-enhancing effect, and activation of protein Protective Protease-Activated C by thrombin on the endothelial cell surface is linked to powerful autocrine protective signaling by the generated APC. Receptor 1 Signaling by the low concentrations of thrombin had a similar barrier Protein C Pathway protective effect. Taken together, these results establish that PAR1 M. Riewald, C. Feistritzer, R.A. Schuepbach, R. Lenta can mediate opposite effects on gene expression and ctivated protein C (APC), an anticoagulant ser- barrier integrity, and they reveal an unexpected role for ine protease, has been approved for treatment cross-communication between the prototypical barrier- A of severe sepsis, but the molecular basis for the protective S1P and barrier-disruptive PAR1 pathway. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 133

Using the anticoagulant double mutant thrombin Using small synthetic druglike organic molecules, W215A/E217A, we recently showed that activation of we elucidated specific molecular gatekeepers that con- protein C by thrombin on the endothelial cell surface trol the numbers of recirculating lymphocytes. These is mechanistically linked to highly efficient PAR1-depen- compounds alter lymphocyte trafficking and induce dent autocrine protective signaling by the generated clinically useful immunosuppression by activating a APC. These results suggest that W215A/E217A may single sphingosine 1-phosphate (S1P) receptor sub- have powerful protective effects in systemic inflamma- type, S1P1. Using 2-photon fluorescence and selective tion through signaling by the generated APC. agonists and antagonists of this receptor, we directly To dissect how signaling by the same receptor leads imaged the control of lymphocyte egress from lymph to different biological outcomes, we are using a panel nodes in living systems. of monoclonal antibodies to analyze how specific pop- MOLECULAR CONTROL OF LYMPHOCYTE MIGRATION Molecular control of the migration of lymphocyte ulations of PAR1 are affected by APC and thrombin on subsets within the recirculation pathway is a fundamen- the endothelial surface. Genetically modified mouse tal issue of therapeutic importance. Although transplan- strains that express in endothelial cells PAR1 variants tation involves the sensitization of an immunologically that are efficiently activated by APC but not by throm- naive host, treatment of most autoimmune diseases bin are currently used in models of systemic inflamma- requires intervention in a sensitized host that already tion to dissect the in vivo role of PAR1 signaling by has autoreactive effector T cells in the periphery. We exogenous and endogenously generated APC. approached this problem by examining the role of

PUBLICATIONS the S1P system in the control of lymphocyte egress Feistritzer, C., Lenta, R., Riewald, M. Protease-activated receptors-1 and -2 can from lymph nodes and thymus, and using chemical mediate endothelial barrier protection: role in factor Xa signaling. J. Thromb. Haemost. 3:2798, 2005. approaches, we revealed differences between intrinsic lymphocyte and barrier mechanisms that alter lympho- Feistritzer, C., Mosheimer, B.A., Sturn, D.H., Riewald, M., Patsch, J.R., Wieder- mann, C.J. Endothelial protein C receptor-dependent inhibition of migration of cyte migration. The rapid reversibility of agonist-medi- human lymphocytes by protein C involves epidermal growth factor receptor. J. ated lymphocyte arrest coupled with its competitive Immunol. 176:1019, 2006. reversal by molar excess of antagonist strongly support an endothelial barrier mechanism. ROLE OF SIGNALING LIPIDS IN THE CONTROL OF Chemical and Genetic LUNG INTEGRITY Approaches to Disease Pulmonary abnormalities, including acute respiratory distress syndrome, are characterized by disruption H. Rosen, G. Sanna, E. Jo, P. Gonzalez-Cabrera, A. Don, of pulmonary integrity and edema that compromise res- S. Cahalan, D. Marsolais, S. Brown, M.-T. Schaeffer, piratory function. S1P is a lipid mediator synthesized J. Chapman and/or stored in mast cells, platelets, and epithelial cells, and its production is upregulated by the proinflamma- ymphocytes develop in the thymus (T cells) and tory cytokines IL-1 and TNF. We used agonists and bone marrow (B cells) and upon maturation egress antagonists of receptors to examine this system. from their sites of development to enter the blood- L S1P1, found on lung capillaries, tightens capillary stream. Because the numbers of lymphocytes with spe- junctions and protects from leakage. Antagonists of cific receptors for antigen are limited, the probability of S1P1 therefore promote lung leakage from the vascular random productive collision of specific lymphocyte, anti- side. We found that changes in signaling-lipid regula- gen, and antigen-presenting cell in a permissive envi- tion of lung barrier function from either vascular or ronment for an efficient immune response is low. In endothelial interfaces induce acute pulmonary edema. the immune system, this probability is enhanced by The S1P-receptor axis may therefore be an important rapid recirculation of lymphocytes through secondary independent variable in the control of lung barrier func- lymphoid organs, so that each lymphocyte has many tion, and its activation and modulation in serious human opportunities to respond to its specific antigen. A suf- diseases such as acute respiratory distress syndrome ficient number of blood lymphocytes are therefore essen- are under study. tial for the development of efficient immune responses STRATEGIC OUTLOOK and are maintained by the recirculation of lymphocytes The S1P system thus regulates adaptive immunity through the secondary lymphoid organs. in at least 3 discrete ways: egress of naive cells from 134 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE lymph nodes, sequestration of effector T cells in lymph PUBLICATIONS Alfonso, C., McHeyzer-Williams, M.G., Rosen, H. CD69 down-modulation and nodes, and egress of mature medullary T cells from the inhibition of thymic egress by short- and long-term selective chemical agonism of thymus. The system can therefore alter both the periph- sphingosine 1-phosphate receptors. Eur. J. Immunol. 36:149, 2006. eral diversity of lymphocyte responses and the efficiency Chun, J., Rosen, H. Lysophospholipid receptors as potential drug targets in tissue of T-cell activation by misdirecting T cells to the wrong transplantation and autoimmune diseases. Curr. Pharm. Des. 12:161, 2006. lymph nodes and by inhibiting the egress of antigen- Gong, Q., Ou, Q., Ye, S., Lee, W.P., Cornelius, J., Diehl, L., Lin, W.Y., Hu, Z., Lu, Y., Chen, Y., Wu, Y., Meng, Y.G., Gribling, P., Lin, Z., Nguyen, K., Tran, T., Zhang, specific effector T cells from lymph nodes after anti- Y., Rosen, H., Martin, F., Chan, A.C. Importance of cellular microenvironment and gen activation and clonal proliferation. circulatory dynamics in B cell immunotherapy. J. Immunol. 174:817, 2005. These effects can alter adaptive immune responses Li, Z., Chen, W., Hale, J.J., Lynch, C.L., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.J., Milligan, J.A., Shei, G.J., Chrebet, G., Parent, S.A., Bergstrom, and the expression of tissue damage while providing J., Card, D., Forrest, M., Quackenbush, E.J., Wickham, L.A., Vargas, H., Evans, potentially significant advantages to patients by sparing R.M., Rosen, H., Mandala, S. Discovery of potent 3,5-diphenyl-1,2,4-oxadiazole sphingosine-1-phosphate-1 (S1P1) receptor agonists with exceptional selectivity innate host defenses to bacteria and pathogenic fungi. against S1P2 and S1P3. J. Med. Chem. 48:6169, 2005. The fine molecular control of this system and its effect Martinez, X., Kreuwel, H.T., Redmond, W.L., Trenney, R., Hunter, K., Rosen, H., on immune responses as a fundamental approach to Sarvetnick, N., Wicker, L.S., Sherman, L.A. CD8+ T cell tolerance in nonobese diabetic mice is restored by insulin-dependent diabetes resistance alleles. J. organization of the immune system and potential ther- Immunol. 175:1677, 2005. apeutic agents will remain our primary focus. Rosen, H., Goetzl, E.J. Sphingosine 1-phosphate and its receptors: an autocrine The recent discovery of a critical role for chemically and paracrine network. Nat. Rev. Immunol. 5:560, 2005. tractable S1P receptors in the innate immune system is Sanna, M.G., Wang, S.K., Gonzalez-Cabrera, P.J., Don, A., Marsolais, D., Matheu, a new focus in molecular pathogenesis of inflammatory M.P., Wei, S.H., Parker, I., Jo, E., Cheng, W.C., Cahalan, M.D., Wong, C.H., lung disease that is of long-term interest to us. Rosen, H. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo. Nat. Chem. Biol. 2:434, 2006. THE SCRIPPS RESEARCH INSTITUTE MOLECULAR Wei, S.H., Rosen, H., Matheu, M.P., Sanna, M.G., Wang, S.K., Jo, E., Wong, C.-H., SCREENING CENTER Parker, I., Cahalan, M.D. Sphingosine 1-phosphate type 1 receptor agonism The Scripps Research Institute Molecular Screen- inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat. Immunol. 6:1228, 2005. ing Center is a national center for small-molecule screening and is part of the National Institutes of Health (NIH) Molecular Libraries Screening Centers Network of the Protease Pathways in NIH Roadmap. The Scripps center is distributed between the La Jolla and the Florida campuses; its component Inflammation, Angiogenesis, parts are assay development, chemistry, assay implementation, and pharmacokinetics. These 4 cores are unified and Cancer in a single data environment by the Informatics Core. J. Ahamed, M. Kerver, T. Kurokawa, Y. Kurokawa, F. Niessen, The mission of the center is to use the NIH library H. Petersen, H. Versteeg, P.J. Hogg,* M. Friedlander, of more than 60,000 individual compounds to screen B.M. Mueller,** W. Ruf molecular and cell-based targets, which are accepted through an NIH-wide peer-reviewed application process, * University of New South Wales, Sydney, Australia for proof-of-concept small-molecule probes. Researchers ** La Jolla Institute for Molecular Medicine, San Diego, California at the Scripps center have successfully identified and DISULFIDE/THIOL EXCHANGE AS A REGULATORY published proof-of-concept molecules in the center’s SWITCH FOR RECEPTOR FUNCTION first year of operations. Compounds discovered by this ctivation of coagulation by the cell-surface recep- process are public information that can be accessed tor tissue factor (TF) is induced by binding of its by all scientists through the PUBCHEM database of the A ligand, the serine protease factor VIIa. In addition, National Center for Biotechnology Information. the TF-VIIa complex triggers cell signaling by cleaving The Scripps center joins human excellence with and activating the G protein–coupled protease-activated state-of-the-art robotics and informatics. The combina- receptor 2 (PAR2), a highly relevant promigratory recep- tion can provide new insights into the basic science of tor in tumor biology. However, the physiologic signifi- small-molecule probes of physiologic and pathologic cance of direct TF-VIIa signaling remained unclear, function, move scientific fields forward, and, over time, because activation of coagulation generates a number provide new, significant insights into therapies for of proteases that may override direct TF signaling path- human diseases. ways. We identified a surprisingly simple mechanism IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 135 by which TF-dependent coagulation is disabled while Koizume, S., Jin, M.S., Miyagi, E., Hirahara, F., Nakamura, Y., Piao, J.H., Asai, A., Yoshida, A., Tsuchiya, E., Ruf, W., Miyagi, Y. Activation of cancer cell migra- preserving TF-VIIa signaling. tion and invasion by ectopic synthesis of coagulation factor VII. Cancer Res. An extracellular disulfide (Cys186-Cys209) in TF fits 66:9453, 2006. the criteria for an “allosteric” disulfide; these disulfides Ruf, W. Flow perturbation is linked to endothelial PAR signaling. Arterioscler. are typically labile because of their bond geometry. Thromb. Vasc. Biol. 26:962, 2006.

Activation of coagulation requires this disulfide, but Ruf, W. Is APC activation of endothelial cell PAR1 important in severe sepsis? Yes. mutational breaking of the disulfide did not impair J. Thromb. Haemost. 3:1912, 2005. TF-VIIa signaling. Protein disulfide isomerase (PDI) Ruf, W., Mueller, B.M. Thrombin generation and the pathogenesis of cancer. targets this disulfide to disable coagulation. PDI is Semin. Thromb. Hemost. 32(Suppl. 1):61, 2006. an abundant intracellular chaperone and thiol/disul- Versteeg, H.H., Ruf, W. Emerging insights in tissue factor-dependent signaling fide exchange catalyst required for protein folding, events. Semin. Thromb. Hemost. 32:24, 2006. but TF-VIIa is regulated by extracellular PDI. PDI suppresses TF coagulant activity by nitric oxide–dependent redox pathways and protein S nitrosylation, indicating Structural Analysis of the an unexpected link between oxidative cardiovascular stress and thrombosis. These data also exemplify a new Host-Pathogen Interface role for PDI as an extracellular switch for the functional specificity of a receptor. E. Ollmann Saphire, D.M. Abelson, M.L. Fusco, PROTEASE SIGNALING PATHWAYS IN ANGIOGENE- C.R. Kimberlin, J.E. Lee, D.R. Burton, M.K. Hart* SIS AND CANCER * U.S. Army Medical Research Institute for Infectious Diseases, Frederick,

We are interested in protease pathways in cancer Maryland and angiogenesis, with a particular focus on signaling by e are crystallizing proteins that play key roles PARs. Previously, in ex vivo models, we showed that mice in the pathogenesis and lethality of viruses lacking the cytoplasmic domain of TF have enhanced that cause hemorrhagic fever. The resulting angiogenesis. Recently, we analyzed the role of PARs W in angiogenesis in vivo and found that PAR2, but not the crystal structures will provide (1) information for the thrombin receptor PAR1, plays a central role in angio- design of vaccines and inhibitors against the viruses genesis. We further found that blockade of TF-VIIa as the microbes exist naturally and (2) structural tem- suppresses angiogenesis in vivo and thus established plates that will enable us to anticipate and rapidly that TF-VIIa signaling through PAR2 is the major pathway respond to newly emerging and synthetic versions of in angiogenesis. We plan to further analyze the respec- the viruses and viral proteins. tive roles of PARs in tumor progression and angiogenesis. EBOLA VIRUS We have also characterized species-specific anti- At least 10 recognized outbreaks of Ebola virus in bodies that block TF-VIIa signaling through PAR2 on humans have occurred; in each outbreak, 50%–90% of tumor cells. We found that these antibodies suppress those infected died. Survival depends on the ability of tumor growth in xenograft models, providing evidence the host to mount early and strong immune responses. that PARs not only regulate angiogenesis in host cells However, filoviruses have evolved mechanisms by which but are an important determinant in tumor progression. the host immune system is suppressed. For example, the We will evaluate the role of PARs in transgenic mouse viral nucleocapsid proteins VP35 and VP24 block inter- models to provide additional genetic evidence for the feron-mediated activation of immunomodulatory genes. specific role of protease pathways in tumor progression. Structural analysis of these proteins, alone and in complex with the human proteins that they bind, will provide PUBLICATIONS Ahamed, J., Versteeg, H.H., Kerver, M., Chen, V.M., Mueller, B.M., Hogg, P.J., insights into viral replication and immunosuppression Ruf, W. Disulfide isomerization switches tissue factor from coagulation to cell sig- and will provide the structural basis for the design of naling. Proc. Natl. Acad. Sci. U. S. A. 103:13932, 2006. antiviral compounds and attenuated viral strains. Bhattacharjee, G., Ahamed, J., Pedersen, B., El-Sheikh, A., Mackman, N., Ruf, W., Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the An additional, unusual feature of the Ebola viral coagulation cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol. genome is its ability to encode 2 different glycoproteins, 25:1737, 2005. sGP and GP, from the same gene. These 2 glycopro- Chen, V.M., Ahamed, J., Versteeg, H.H., Berndt, M.C., Ruf, W., Hogg, P.J. Evi- teins share 295 amino acids of N-terminal sequence, dence for activation of tissue factor by an allosteric disulfide bond. Biochemistry 45:12020, 2006. but a transcriptional editing event causes them to have 136 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE different C-terminal sequences that result in unique determine structural features of epitopes associated patterns of disulfide bonding, structures, and roles in with neutralization and enhancement (Fig. 2). pathogenesis. Comparative structural analysis of sGP and GP should explain how 2 structures arise from the same sequence, provide templates for the design of vaccines that elicit antibodies that target the virus rather than the secreted proteins, and illustrate structural mechanisms by which the virus escapes immune surveillance. Additional crystal structures of these proteins in complex with rare human antibodies derived from sur- vivors of Ebola virus infection or with immunotherapeutic agents under development by the U.S. Army will assist in vaccine design. We recently determined the crystal structure at 2.0-Å resolution of a potential immunotherapeutic agent, 13F6-1-2, in complex with its GP epitope (Fig. 1). 13F6-1-2 contains a rare Vλx

Fig. 2. Crystals of envelope protein E of dengue virus serotype 1.

PUBLICATIONS Cárdenas, W.B., Loo, Y.-M., Gale, M., Jr., Hartman, A.L., Kimberlin, C.R., Fig. 1. Crystal structure of Fab 13F6-1-2 in complex with its Martínez-Sobrido, L., Saphire, E.O., Nichol, S.N., Basler, C.F. Ebola virus VP35 Ebola virus glycoprotein epitope. The antibody light chain is black, protein binds double-stranded RNA and inhibits α/β interferon production induced the heavy chain is gray, and the GP peptide is illustrated in ball- by RIG-I signaling. J. Virol. 80:5168, 2006. and-stick form. light chain and has several unusual structural features Autoimmune Mechanisms and in its combining site. DENGUE VIRUS Compensatory Responses Dengue virus is a mosquito-borne flavivirus that causes up to 100 million infections each year. Infec- N. Sarvetnick, M. Cleary, S. Dabernat, S. Datta, D. Dietz, tion with dengue virus results in either dengue fever or C. Fine, N. Hill, H. Hua, M. Kritzik, A. Marleau, P. Secrest, the much more severe disease dengue hemorrhagic A. Stotland, D. Yadav, Y.Q. Zhang fever. Dengue hemorrhagic fever usually occurs upon ype 1 diabetes occurs when self-reactive T cells secondary infection with a different viral subtype or in destroy the insulin-producing beta cells in the infants born to dengue virus–immune mothers. This T islets in the pancreas. The assumption has been potential antibody-mediated enhancement of infection that the fault lies exclusively in the immune system, is a major concern in the testing and use of vaccines but increasingly findings suggest that the targets of against dengue virus because antibodies elicited by the autoimmunity, the islets, may also be defective. Genetic vaccines could trigger severe disease. To aid in vac- linkage analysis of nonobese diabetic mice has led to cine design, we are determining crystal structures of the identification of critical intervals that confer sus- envelope proteins of contemporary field isolates of ceptibility to diabetes. One of these regions, Idd9, is dengue virus, alone and in complex with antibodies, to associated with strong protection from disease when it IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 137 is replaced with the B10 allele. Interestingly, we found Horwitz, M.S., Ilic, A., Fine, C., Sarvetnick, N. Induction of antigen specific peripheral humoral tolerance to cardiac myosin does not prevent CB3-mediated that genes at the Idd9 locus associated with susceptibility autoimmune myocarditis. J. Autoimmun. 25:102, 2005. to diabetes control islet resilience to CD8+ T cell–medi- Hua, H., Zhang, Y.Q., Dabernat, S., Kritzik, M.N., Dietz, D., Sterling, L., Sarvet- ated autoimmunity. nick, N. BMP4 regulates pancreatic progenitor cell expansion through Id2. J. Biol. Susceptible islets are hyperresponsive to the cyto- Chem. 281:13574, 2006. kines TNF and IFN-γ, resulting in increased expression Kayali, A.G., Stotland, A., Gunst, K.V., Kritzik, M., Liu, G., Dabernat, S., Zhang, of the death receptor Fas. Fas upregulation in beta cells Y.Q., Wu, W., Sarvetnick, N. Growth factor-induced signaling of the pancreatic epithelium. J. Endocrinol. 185:45, 2005. is mediated by TNF receptor 2 (TNFR2), and in nonobese diabetic mice, colocalization of the receptor with the Kim, S.H., Gunst, K.V., Sarvetnick, N. STAT4/6-dependent differential regulation of chemokine receptors. Clin. Immunol. 118:250, 2006. adaptor TNF receptor–associated factor 2 in beta cells is altered. The gene for TNFR2 lies within the candidate Marleau, A.M., Sarvetnick, N. T cell homeostasis in tolerance and immunity. J. Leukoc. Biol. 78:575, 2005. Idd9 interval, and the diabetes-associated variant contains a mutation adjacent to the binding site for TNF receptor– Martinez, X., Kreuwel, H.T., Redmond, W.L., Trenney, R., Hunter, K., Rosen, H., Sarvetnick, N., Wicker, L.S., Sherman, L.A. CD8+ T cell tolerance in nonobese associated factor 2. A component of diabetes suscepti- diabetic mice is restored by insulin-dependent diabetes resistance alleles. J. bility is therefore determined by the target of the auto- Immunol. 175:1677, 2005. immune response, and protective TNFR2 signaling in Solomon, M., Flodstrom-Tullberg, M., Sarvetnick, N. Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c islets may inhibit early cytokine-induced damage required and spontaneously-diabetic NOD recipient mice. Transplantation 15:1104, 2005. for the development of destructive autoimmunity. Zhang, Y.Q., Kritzik, M., Sarvetnick, N. Identification and expansion of pancreatic Because insulin-dependent diabetes mellitus is due stem/progenitor cells. J. Cell. Mol. Med. 9:331, 2005. to selective destruction of insulin-producing cells, strategies that promote growth of beta cells provide a means to prevent or reverse this type of diabetes. One approach is to replace insulin-producing cells by using genetic Promotion of Cell Migration engineering or by guiding stem cells (pancreas progen- and Invasion by Tyrosine itors) to differentiate into beta cells. The progression of pancreatic progenitor cells to beta cells is governed by Kinase Signaling basic helix-loop-helix transcription factors, which are regulated by inhibitor of differentiation proteins that D.D. Schlaepfer, J.A. Bernard-Trifilo, X.L. Chen, A. Chi, bind to and inhibit the function of the factors. Transcrip- D.A. Hanson, S. Hou, S.T. Lim, Y.M. Lim, S.K. Mitra, tion of inhibitor of differentiation proteins is induced by J.E. Molina, S. Uryu, A. Wang bone morphogenetic proteins (BMPs). e wish to understand how intracellular sig- We showed that BMP signaling is necessary and sufficient for proliferation of pancreatic progenitor cells naling networks promote complex biological and that this signaling is correlated with an increase W processes such as cell motility, cell invasion, in the expression of inhibitor of differentiation proteins. and tumor metastasis. We hypothesize that critical intra- Using a mouse model of regenerating pancreas, we cellular signaling proteins exist within cells that act as found that injection of an antibody that inhibits BMP4 signal “integrators” to process environmental stimuli that significantly reduced cell proliferation and caused an control cell movement. These proteins should be acti- increase in NeuroD, a basic helix-loop-helix factor vated by various extracellular inputs and act to regulate required for the differentiation of pancreatic islet cells. multiple downstream signaling pathways. One such Therefore, our results indicate that stimulation by BMP4 integrator is focal adhesion kinase (FAK), an intracel- blocks the differentiation of endocrine progenitor cells lular protein-tyrosine kinase that is associated with both and instead promotes their expansion, thereby reveal- transmembrane integrin and growth factor receptors. ing a novel model of signaling that explains the balance CONNECTIONS TO GROWTH FACTOR RECEPTORS between expansion and differentiation of pancreatic The ErbB family of protein-tyrosine kinase receptors duct epithelial progenitors. includes epidermal growth factor receptor (ErbB-1), ErbB-2, ErB-3, and ErB-4. Overexpression of ErbB-2 PUBLICATIONS Flodstrom-Tullberg, M., Hultcrantz, M., Stotland, A., Maday, A., Tsai, D., Fine, is associated with poor prognosis and invasiveness in C., Williams, B., Silverman, R., Sarvetnick, N. RNase L and double-stranded cancer in humans. An important early event implicated RNA-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection. J. Immunol. 174:1171, 2005. in controlling cell migration induced by growth factors 138 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE is FAK activation. Using cells that lacked the gene for fibroblasts lacking the gene for FAK in which point FAK, FAK-reconstituted fibroblasts, and human breast mutations affecting FAK catalytic activity or phosphor- carcinoma cells in which FAK expression was inhibited ylation disrupted the ability of FAK at tyrosine 925 to by short inhibitory RNA, we dissected the function of promote tumor growth–associated MAP kinase and FAK in ErbB-2/ErbB-3 oncogenic transformation and VEGF expression. Inhibition of FAK in breast, prostate, cell invasion. We found that these processes depend and neuroblastoma cells also resulted in reduced VEGF on FAK. In many cells, FAK activation promotes bind- expression. These studies provide the first biological ing of the protein-tyrosine kinase c-Src to FAK, thereby support for Y925 FAK phosphorylation and define a novel generating a dual FAK-Src signaling complex. In these role for FAK activity in promoting a MAP kinase–asso- studies, ErbB-2/ErbB-3–induced oncogenic transforma- ciated angiogenic switch during tumor progression. tion depended on a FAK-Src and MAP kinase activation, INTEGRIN SIGNALING INDEPENDENT OF FAK whereas ErbB-2/ErbB-3–induced cell invasion was In cell culture, fibroblasts that lack the gene for FAK FAK-Src dependent and independent of MAP kinase. have defects in motility but not in proliferation. The CONNECTIONS TO INTEGRINS fibronectin-binding integrins α5β1 and α4β1 generate Overexpression of FRNK, the FAK C-terminal domain, signals pivotal for cell migration through distinct yet can inhibit FAK activity, in part by disrupting FAK asso- undefined mechanisms. For α5β1, β1-mediated activa- ciation with integrins. Analyses of breast tumor samples tion of FAK promotes c-Src recruitment to FAK and the revealed that elevated FAK expression occurs with the formation of a FAK-Src signaling complex that promotes development of benign ductal hyperplasia into invasive motility. Interestingly, expression of human α4 integrin carcinomas. We inhibited FAK activity or FAK expres- in fibroblasts that lack the gene for FAK forms a func- sion in murine 4T1 breast carcinoma cells via transient tional α4β1 receptor that promotes motility of the cells, expression of FRNK or stable expression of anti-FAK equal to that of wild-type FAK-containing fibroblasts short hairpin RNA, respectively. stimulated with α5β1. This α4β1-stimulated signaling Expression of anti-FAK short hairpin RNA resulted connection was initiated by the cytoplasmic domain of in the inhibition of 4T1 cell invasion in vitro and sponta- α4 integrin and involved the activation of Src-family neous 4T1 metastasis after implantation of the cells in protein-tyrosine kinases in the absence of FAK. Currently, Balb/c mice.* Transient reexpression of wild-type but we are elucidating the molecular linkage of α4 integrin to Src and how this signaling pathway promotes neurob- not kinase-inactive FAK in 4T1 cells with the anti-FAK lastoma motility, invasion, and tumor progression. short hairpin RNA promoted in vivo lung metastasis, and this finding was associated with increased expres- PUBLICATIONS sion of urokinase plasminogen activator. Because the Benlimame, N., He, Q., Jie, S., Xiao, D., Xu, Y.J., Loignon, M., Schlaepfer, D.D., Alaoui-Jamali, M.A. FAK signaling is critical for ErbB-2/ErbB-3 receptor coopera- inhibition of FAK within 4T1 cells was also associated tion for oncogenic transformation and invasion. J. Cell Biol. 171:505, 2005. with increased host survival after tumor cell implanta- Bernard-Trifilo, J.A., Lim, S.T., Hou, S., Schlaepfer, D.D., Ilic, D. Analyzing FAK tion, our results support the pharmacologic targeting and Pyk2 in early integrin signaling events. In: Current Protocols in Cell Biology. Bonifacino, J.S., et al. (Eds.). Wiley, New York, 2006, Chap. 14.7.1. of FAK activity as a means to inhibit tumor spread. Hsia, D.A., Lim, S.T., Bernard-Trifilo, J.A., Mitra, S.K., Tanaka, S., den Hertog, TARGETS OF FAK ACTIVITY PROMOTING TUMOR J., Streblow, D.N., Ilic, D., Ginsberg, M.H., Schlaepfer, D.D. Integrin α4β1 pro- PROGRESSION motes focal adhesion kinase-independent cell motility via α4 cytoplasmic domain- specific activation of c-Src. Mol. Cell. Biol. 25:9700, 2005. Using stable FRNK overexpression to inhibit FAK Hu, B., Jarzynka, M.J., Guo, P., Imanishi, Y., Schlaepfer, D.D., Cheng, S.Y. in 4T1 breast carcinoma cells, we found that FRNK Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2 overexpression was not associated with alterations in expression through the αvβ1 integrin and focal adhesion kinase signaling pathway. Cancer Res. 66:775, 2006. cell proliferation or anchorage-independent cell survival Mitra, S.K., Lim, S.T., Chi, A., Schlaepfer, D.D. Intrinsic focal adhesion kinase in vitro. Instead, FRNK-expressing 4T1 cells secreted activity controls orthotopic breast carcinoma metastasis via the regulation of uroki- less vascular endothelial cell growth factor (VEGF) and nase expression in a syngeneic tumor model system. Oncogene 25:4429, 2006. formed small avascular tumors, findings associated with Mitra, S.K., Mikolon, D., Molina, J., Hsia, D.A., Hanson, D.A., Chi, A., Lim, S.T., the inhibition of a signaling linkage involving FAK, phos- Bernard-Trifilo, J.T., Ilic, D., Stupack, D.G., Cheresh, D.A., Schlaepfer, D.D. Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in phorylation of FAK at tyrosine 925, and MAP kinase that tumors. Oncogene 25:5969, 2006. regulates expression of VEGF. The biological importance Urbinati, C., Bugatti, A., Giacca, M., Schlaepfer, D., Presta, M., Rusnati, M. αvβ3 of this FAK signaling pathway was confirmed through integrin-dependent activation of focal adhesion kinase mediates NF-κB activation and motogenic activity by HIV-1 Tat in endothelial cells. J. Cell Sci. 118(Pt. reconstitution experiments with Src transformation of 17):3949, 2005. IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 139 The Consequences of T-Cell lymphoid tissue (central memory cells) or blood and parenchymal tissue (tissue-resident memory cells). Recognition of Self-Antigens and Compartmentalization is based on cell-surface expression of CD62L, an adhesion molecule required for migra- Tumor Antigens tion of T cells across high endothelial venules into secondary lymphoid tissue. Tissue-resident memory L.A. Sherman, X. Martinez, C.-H. Wei, J. Wong, cells express low levels of CD62L and are therefore E. Hamilton-Williams, J.A. Biggs, K.L. Marquardt, R.L. Trenney excluded from secondary lymphoid tissue. he consequence of antigen recognition by naive Previously, we showed that central memory CD8+ + CD8 T cells can be either tolerance or immunity, T cells are efficiently tolerized by either soluble peptide T depending on the activation status of the antigen- or tissue-derived antigen that is cross-presented in + presenting dendritic cells. If a CD8 T cell recognizes draining lymph nodes. More recently, we determined antigen on a quiescent dendritic cell that has relatively whether tissue-resident memory cells are also toler- low levels of expression of costimulatory molecules, then ized by these 2 forms of antigen. activation of the T cell results in deletion and tolerance. Soluble peptide was highly efficient at tolerizing Inflammatory signals, such as those due to the presence tissue-resident memory cells in all tissues tested except of foreign pathogens and activated lymphocytes, activate the brain. This finding may be due to the inability of dendritic cells to express cell-surface costimulatory soluble peptide to cross the blood-brain barrier. Cross- + molecules and cytokines. If CD8 T cells recognize presented antigen was able to tolerize central memory antigen on activated dendritic cells, the costimulatory cells but not tissue-resident memory cells. This differ- molecules and cytokines prevent deletion and promote ence in tolerance may occur because the cells are the clonal expansion of the T cells and the development excluded from entry into secondary lymphoid tissue, of effector functions. the site at which dendritic cells cross-present antigen Understanding the signals that result in either T-cell derived from tissue. Our results are also consistent deletion or immunity is of importance in preventing auto- with the possibility that some tissue-resident memory immunity, which represents a failure to control self- cells may not circulate out of tissue. destructive T lymphocytes. This understanding is also MECHANISMS OF PROTECTION FROM TYPE 1 important in promoting tumor immunity, in which the DIABETES BY GENETIC POLYMORPHISMS goal is to promote the autoimmune destruction of tumor The spontaneous diabetes that develops in NOD cells. We are comparing the consequence of the inter- mice is similar to type 1 diabetes in humans. The dis- action of naive CD8+ T lymphocytes with a transgenic ease process involves destruction of the insulin-produc- self-antigen (the influenza virus hemagglutinin) expressed ing beta cells in the pancreas by CD8+ T lymphocytes. by the insulin-producing beta cells in the pancreatic In humans and mice, genetic regions have been iden- islets in 3 different types of mice: normal mice, diabetes- tified in which allelic polymorphism predisposes individ- prone nonobese diabetic (NOD) mice, and mice in which the beta cells express an oncogene that promotes spon- uals to type 1 diabetes. We are studying the effects of taneous transformation and production of tumors. such allelic polymorphism, designated insulin-dependent + In all 3 types of mice, the interaction between anti- diabetes (Idd) loci, on the establishment of CD8 gen and naive CD8+ T lymphocytes specific for hemag- T-cell tolerance. glutinin first occurs in the pancreatic lymph nodes. There Congenic mice that express protective alleles at antigen is recognized on dendritic cells that obtain it in Idd3/5 have normal abortive activation of islet antigen- + from beta cells in the islets and cross-present it to naive specific CD8 T cells in the pancreas, suggesting that T cells in the lymph nodes. In normal mice, this inter- tolerance is restored at the earliest time when naive + action results in an abortive activation of the T cells and CD8 T cells first encounter antigen. In contrast, in + subsequent deletion of the potentially autoreactive T cells NOD mice, such CD8 T cells accumulate in the pan- specific for hemagglutinin. creatic lymph nodes and then enter the islets. This + TOLERANCE OF TISSUE-RESIDENT MEMORY CD8+ difference in the accumulation of CD8 T cells in the T CELLS pancreatic lymph nodes occurs in the absence of all Memory CD8+ T cells are compartmentalized on CD4+ T cells. We are testing the hypothesis that this the basis of their ability to circulate through either difference may be intrinsic to the interaction between 140 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

T cells and cross-presenting dendritic cells in NOD and tive of the cells’ cytokine requirements, the dimeric Idd3/5 mice. receptor for IL-7 (CD127-CD132) is expressed at high ROLE OF CD4+ HELPER T CELLS IN PROMOTING levels on naive and memory T cells, whereas the dimeric TUMOR CELL DESTRUCTION BY CD8+ T CELLS receptor for IL-15 (CD122-CD132) is expressed at neg- CD4+ helper T cells can enhance the performance ligible levels on naive T cells, low levels on memory of CD8+ T cells in different ways, including enhanced CD4+ T cells, and high levels on memory CD8+ T cells. clonal expansion during activation of CD8+ T cells, Neither IL-7 nor IL-15 is produced by T cells; both are enhanced tissue infiltration by the activated effector produced by epithelial, stromal, and antigen-present- CD8+ T cells, and enhanced survival of the effector ing cells. CD8+ T cells. We are assessing the effects of CD4+ IL-2, which is produced by T cells for autocrine pur- helper T cells at various times after activation of CD8+ poses, is closely related to IL-15. Recognition of IL-2 by T cells to evaluate the ability of the helper cells to pro- T cells is mediated by the high-affinity trimeric receptor mote destruction of tumor cells by CD8+ T cells. CD25-CD122-CD132; the dimeric form of the recepter (CD122-CD132) recognizes IL-15. Indeed the IL-15 PUBLICATIONS Cohen, C.J., Zheng, Z., Bray, R., Zhao, Y., Sherman, L.A., Rosenberg, S.A., Mor- receptor can also recognize IL-2 at a lower affinity. gan, R.A. Recognition of fresh human tumor by human peripheral blood lympho- Interestingly, injecting a monoclonal antibody to IL-2, cytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR [published correction appears in J. Immunol. 177:5746, 2006]. J. Immunol. a technique that is thought to deplete the cytokine, 175:5799, 2005. increases the background turnover rate of memory + Martinez, X., Kreuwel, H.T.C., Redmond, W.L., Trenney, R., Hunter, K., Rosen, CD8 T cells. This finding was interpreted to indicate H., Sarvetnick, N., Wicker, L.S., Sherman, L.A. CD8+ T cell tolerance in that IL-2, unlike IL-15, dampens the homeostasis of nonobese diabetic mice is restored by insulin-dependent diabetes resistence alleles. + J. Immunol. 175:1677, 2005. memory CD8 T cells. Recent work, however, indicates that this interpretation is untrue. Wei, C.-H., Trenney, R., Sanchez-Alavez, M., Marquardt, K., Woodland, D.L., Henriksen, S.J., Sherman, L.A. Tissue resident memory CD8+ T cells can be Instead of depleting IL-2, the monoclonal antibody deleted by soluble, but not cross-presented antigen. J. Immunol. 175:6615, 2005. actually boosts the biological activity of the interleukin. Hence, the ability of the monoclonal antibody to IL-2 to elevate turnover of memory CD8+ T cells does not Regulation of Homeostasis of occur in the absence of IL-2, and, more important, injecting the monoclonal antibody complexed with IL-2 Mature T Cells dramatically induces rapid proliferation of memory CD8+ T cells. Indeed, administration of the antibody–IL-2 C.D. Surh, C. Ramsey, J. Purton, E.M.M. van Leeuwen, complex induced 100- to 200-fold greater expansion J.Y. Lee, D. Kim, O. Boyman,* C. Ahn,** J. Sprent*** of memory CD8+ T cells than did administration of IL-2 * Universitaire Vaudois, Lausanne, Switzerland alone. The complex stimulated CD8+ T cells through ** Seoul National University Hospital, Seoul, Korea the dimeric IL-15 receptor rather than through the *** Garvan Institute of Medical Research, Darlinghurst, Australia trimeric IL-2 receptor, because stimulatory activity of he homeostasis of mature T cells is largely gov- the complex was also evident on CD25−CD8+ T cells. erned by 2 related cytokines, IL-7 and IL-15, Exactly how the bound monoclonal antibody T which bind to the receptors belonging to the com- enhances the activity of IL-2 is unknown. Nonetheless, mon γ-chain (CD132) receptor family. Other members it is clear that monoclonal antibodies augment the cyto- of the CD132 family include receptors for IL-2, IL-4, kine activity in vivo but not under in vitro conditions, and IL-9, and IL-21. In conjunction with signals from con- that the Fc part of the antibody is required for its inten- tact with self-peptide–MHC ligands, IL-7 controls sur- sifying role. These findings suggest that the monoclonal vival of naive T cells. Memory T cells, which are at a antibody boosts the cytokine activity by concentrating higher state of activation than naive T cells, depend the cytokine onto the cell surface of antigen-present- on both IL-7 and IL-15 for survival and for intermittent ing cells and/or by prolonging the half-life of the cyto- cell division. Memory CD4+ T cells are generally more kine. The specificity of the monoclonal antibody also dependent on IL-7 than on IL-15 for their homeostasis, determines its ability to enhance the activity of IL-2 to whereas memory CD8+ T cells rely primarily on IL-7 IL-2 receptors. Thus, one particular monoclonal anti- for survival and on IL-15 for periodic cell division. Indica- body to IL-2 could stimulate T cells expressing the IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 141 trimeric IL-2 receptor, such as regulatory T cells, but on artificial bilayers with recombinant forms of TCRαβ, not memory CD8+ T cells that express the dimeric CD3δε, CD3γε, and CD8αβ is in progress. We use a IL-15/IL-2 receptor. combination of single-molecule, multicolor imaging by The ability of the monoclonal antibody to boost the total internal reflection fluorescence microscopy, in col- activity of the bound cytokine appears to be generally laboration with K. Fish, University of Pittsburgh, Pitts- applicable because the biological activity of other cyto- burgh, Pennsylvania, and electron microscopy to examine kines, such as IL-4 and IL-7, can also be enhanced by the dynamics and membrane relationships of each sub- binding to specific monoclonal antibodies. The ability unit within the complex. Similar observations are carried to induce expansion and activation of specific subsets out in the presence of MHC ligands displayed in solu- of T cells by administering complexes composed of tion or at the surface of polystyrene beads and liposomes. cytokine plus monoclonal antibody may offer a new Interactions of MHC and TCR molecules with their way to modulate the immune response for therapeutic respective membrane could provide simple switches purposes. The immune response can be boosted against essential to T-cell activation. This hypothesis is sup- tumors and infectious agents by activating naive and ported by our determination, in collaboration with A.K. memory T cells; alternatively, allergens and tissue grafts Mitra, University of Auckland, Auckland, New Zealand, can be suppressed by inducing expansion of regulatory of the structure of an MHC molecule attached to a phos- T cells and the immune responses against self. pholipid bilayer that shows parallel orientation of the long axis of the molecule with the lipid leaflet. In col- PUBLICATIONS Boyman, O., Kovar, M., Rubinstein, M.P., Surh, C.D., Sprent, J. Selective stimula- laboration with I.A. Wilson, Department of Molecular tion of T cell subsets with antibody-cytokine immune complexes. Science Biology, we are determining 3-dimensional structures 311:1924, 2006. of CD3, TCR complexes, and CD8αβ. Davey, G.M., Starr, R., Cornish, A.L., Burghardt, T., Alexander, W.S., Carbone, AUTOIMMUNE DIABETES F.R., Surh, C.D., Heath, W.R. SOCS-1 regulates IL-15-driven homeostatic proliferation of antigen-naive CD8 T cells, limiting their autoimmune potential. J. Exp. We are using MHC multimers to detect antigen-spe- Med. 202:1099, 2005. cific T-cell populations in diabetes-prone nonobese Gattinoni, L., Finkelstein, S.E., Klebanoff, C.A, Antony, P.A., Palmer, D.C., diabetic mice. Pathogenic T cells are characterized by Spiess, P.J., Hwang, L.N., Yu, Z., Wrzesinski, C., Heimann, D.M., Surh, C.D., Rosenberg, S.A., Restifo, N.P. Removal of homeostatic cytokine links by lymhode- analyzing secretion of cytokines and use of TCRs by pletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J. Exp. Med. 202:907, 2005. single cells. We are also trying to treat insulin-dependent diabetes by depleting antigen-specific T cells in Lee, S.K., Surh, C.D. Role of interleukin-7 in bone and T-cell homeostasis. Immunol. Rev. 208:169, 2005. vivo during the preclinical phase of the disease. For this therapy, we are using MHC molecules to deliver Ramsey, C., Hässler, S., Marits, P., Kämpe, O., Surh, C.D., Peltonen, L., Win- qvist, O. Increased antigen presenting cell-mediated T cell activation in mice and doxorubicin liposomes to autoreactive T cells. The patients without the autoimmune regulator. Eur. J. Immunol. 36:305, 2006. specificity of the intervention will limit side effects Surh, C.D., Boyman, O., Purton, J.F., Sprent, J. Homeostasis of memory T cells. and complications of general immunosuppression. Immunol. Rev. 211:154, 2006. LINKS BETWEEN INNATE AND ADAPTIVE IMMUNITY Surh, C.D., Sprent, J. On the TRAIL of homeostatic memory T cells [published We are studying lipid binding to CD1 to determine correction appears in Nat. Immunol. 7:672, 2006]. Nat. Immunol. 7:439, 2006. the factors that govern the presentation of the lipids to Tan, J.T., Surh, C.D. T cell memory. Curr. Top. Microbiol. Immunol., in press. T cells. A family of lipid transfer proteins known as saposins, which are involved in the catabolism of lipids, are critical for the loading of natural glycolipids onto Structure-Function Studies of CD1 and the selection of natural killer T cells. Other Innate and Adaptive Immunity lipid transfer proteins most likely account for the loading of other endogenous and exogenous ligands. In L. Teyton, B. Atteberry, K. Bennett, C. Cantu, S.Y. Chang, collaboration with A. Bendelac, University of Chicago, L. Develioglu, S. Freigang, H. Issafras, M. Holt, E. Landais, and P.B. Savage, Brigham Young University, Provo, M. Ota, N. Schrantz, J. Sim, R. Stefanko, Utah, we are using RNA interference, genetic techniques, N. Von Allmen-Zurcher, C. Wang, K. Yoshida and recombinant biochemistry to study CD1 within ACTIVATION OF T-CELL RECEPTORS the context of lipid metabolism. At a structural level, ur goal is to understand the molecular switches we are examining recognition of dissimilar ceramides that lead to activation of T cells. Assembly of such as α-galactosyl ceramide (Fig. 1) and isoglobotri- O functional complexes of T-cell receptors (TCRs) hexosyl ceramide (β-linked) by a TCR bearing a unique 142 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE Genetics of Systemic Autoimmunity and T-Cell Homeostasis in Autoimmunity, Aging, and Cancer

A.N. Theofilopoulos, D.H. Kono, R. Baccala, R. Gonzalez-Quintial, M.K. Haraldsson, D. Aït-Azzouzene, J. Schettini, R.M. Chintalapati, C.A. Louis-Dit-Sully

e have continued our research on predispos- ing and effector genes in murine models of W systemic lupus erythematosus (SLE), homeostatic T-cell disturbances in systemic autoimmunity and aging, and the potential use of homeostatic T-cell proliferation for inducing efficient antitumor responses. Fig. 1. Side view of α-galactosyl ceramide bound to murine GENETIC BASIS OF SYSTEMIC AUTOIMMUNITY CD1d. The galactose is accessible for TCR recognition. Susceptibility to SLE is inherited as a multifactor- α-chain (Vα14 in mice, Vα24 in humans) and a lim- ial trait, and genetic predisposition is a major if not an ited set of Vβ partners. essential factor in the disease. We are defining the role INNATE IMMUNE RECEPTORS and identity of susceptibility genes that promote SLE Recognition of unique features of the prokaryotic in both spontaneous and induced mouse models of the world is embedded in a series of receptors of the innate disease. Previously, we identified loci that predispose immune system called pattern recognition molecules. mice to SLE in NZB, NZW, BXSB, and MRL strains, Each of these receptors can sense the presence of a which are SLE prone, and in C57BL/6 mice, which are family of unique prokaryotic compounds such as glyco- not predisposed to autoimmune disease. We also iden- lipids, proteoglycans, DNA, or RNA and allow activa- tified a disease-resistance locus in DBA/2 mice. We tion of macrophages, dendritic cells, and neutrophils. subsequently analyzed the contribution of several of We are collaborating with R. Ulevitch and P. Tobias, these loci to autoimmunity in interval congenic lines. The Department of Immunology, to decipher the structural NZB x NZW F -related Lbw2, Lbw5, and Lbw7 (chromo- basis of this mode of recognition. We expressed recom- 2 somes 4, 7, and 1, respectively); the MRL x B6-Faslpr binant forms of receptor family members from Drosophila, F -related Lmb1–Lmb4 (chromosomes 4, 5, 7, and 10), mice, and humans to compare the biophysical and 2 and the NZB x DBA/2 and SJL x DBA/2 F -related structural characteristics of the receptors and to delin- 2 Hmr1 (chromosome 1) were among those analyzed. eate new activation pathways. On the basis of these studies. we concluded that

PUBLICATIONS lupus-prone strains have substantial genetic hetero- Benlagha, K., Wei, D.G., Veiga, J., Teyton, L., Bendelac, A. Characterization of geneity, strains not predisposed to autoimmune disease the early stages of thymic NKT cell development. J. Exp. Med. 202:485, 2005. can harbor susceptibility genes, a single locus often Shore, D.A., Teyton, L., Dwek, R.A., Rudd, P.M., Wilson, I.A. Crystal structure of promotes multiple traits, and specific combinations of the TCR coreceptor CD8αα in complex with monoclonal antibody YTS 105.18 Fab fragment at 2.88 Å resolution. J. Mol. Biol. 358:347, 2006. loci determine clinical manifestations.* Moreover, phe-

Wei, D.G., Lee, H., Park, S.H., Beaudoin, L., Teyton, L., Lehuen, A., Bendelac, notypes do not always correlate with the initial mapping A. Expansion and long-range differentiation of the NKT cell lineage in mice express- results, and the genetic background plays a major role ing CD1d exclusively on cortical thymocytes. J. Exp. Med. 202:239, 2005. in shaping locus-induced phenotypes. Further fine-map- Zajonc, D.M., Cantu, C. III, Mattner, J., Zhou, D., Savage, P.B., Bendelac, A., ping studies indicated that identifying the underlying Wilson, I.A., Teyton, L. Structure and function of a potent agonist for the semi- invariant natural killer T cell receptor. Nat. Immunol. 8:810, 2005. genes by screening for alterations in gene expression still requires the evaluation of subcongenics. Substantial progress was also made in defining Lmb3 as a nonsense mutation of the gene that encodes IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 143 coronin-1A, and actin-binding protein that plays a role autoantibodies from the sera of patients with SLE, in the formation of filaments. Surprisingly, this mutation induce plasmacytoid dendritic cells to produce large was present in the C57BL/6-Faslpr/Scr substrain, which quantities of IFNα/β. The requirement of forming a com- is not predisposed to autoimmunity, and not in the MRL plex with autoantibodies is consistent with the idea that strain, indicating that Lmb3 most likely is a disease- mammalian nucleic acids themselves are compartmental- modifying or resistance allele. Currently, we are deter- ized away from the endosomes where IFN-α/β–induc- mining the role of coronin-1 in normal immune responses ing TLR3, TLR7, TLR8, and TLR9 reside. Although and autoimmunity. We are also more precisely mapping this mechanism is important for disease perpetuation, and identifying the genes for other loci. primary inducers not predicated on preexisting autoan- TYPE I INTERFERONS IN SLE tibodies need to be identified. A likely candidate might Type I interferons (IFN-α/β) have important effects be apoptotic material that under certain conditions in the innate and adaptive immune systems and may could constitute a “danger signal.” Recent studies, in play a central role in the pathogenesis of autoimmune collaboration with K. Hoebe and B. Beutler, Department diseases, including SLE. We used several approaches of Immunology, indicated that early apoptotic cells can to define the mechanisms of these interferons and to indeed act as efficient inducers of type I interferons and curtail their adverse effects. that the responding cells are not plasmacytoid dendritic We previously found that deletion of the common cells, but precursors of B220−CD8+ lymphoid-type receptor for type I interferons resulted in significant dis- dendritic cells. Importantly, induction was mediated ease reduction in lupus-predisposed NZB mice. Although by a TLR-independent pathway. this finding provides strong support for the involvement On the basis of these findings, we propose that of IFN-α/β in SLE, it has yet to be shown that blockade induction of IFN-α/β in SLE encompasses 2 types of of IFN-α/β activity can inhibit active disease. We pre- apoptosis-generated stimuli that act sequentially in the viously showed the efficacy of intramuscular injections disease process. The early-phase stimulus does not of nonviral vectors encoding the fusion protein consist- require autoantibodies or TLR engagement and is gen- ing of the IFN-γ receptor and the Fc fragment of IgG1 erated by stressed cells with propensity to apoptosis; in ameliorating SLE in MRL-Faslpr lupus-prone mice. A this material is taken up by lymphoid-type dendritic similar strategy to block type I interferons is being used cells and initiates production of IFN-α/β, leading to to treat the prototypic NZB/W strain of lupus-prone activation of antigen-presenting cells, priming of previ- mice. Early assessments indicate that blockade of ously quiescent nontolerant T and B cells, and produc- IFN-α/β can indeed cause regression of active disease. tion of autoantibodies. IFN-α/β may also promote In collaborative studies with R. Schreiber and his maturation and survival of T and B cells directly as colleagues, Washington University of Medicine, St. Louis, well as by inducing production of B-cell trophic fac- Missouri, we will ascertain the efficacy of a recently tors from activated dendritic cells. developed mouse monoclonal antibody to mouse IFN-α The late-phase stimulus consists of apoptosis/necro- receptor 1. Overall, our previous and current studies sis materials and associated nucleic acids complexed have provided the impetus for the potential use of with autoantibodies. These complexes are directed into interferon blockers as therapies in human SLE and endosomal compartments of plasmacytoid or conven- other autoimmune diseases. tional dendritic cells and B cells, through receptors for A major issue in the role of type I interferons in SLE IgG or autoreactive B-cell receptors, where they engage is the stimuli that induce and sustain the production TLRs and amplify production of interferons and responses of these cytokines. Although exogenous stimuli, such as of autoimmune T and B cells.* We are conducting experi- infectious agents, may induce IFN-α/β through engage- ments to define the responsiveness of plasmacytoid ment of Toll-like receptors (TLRs) and precipitate disease, and conventional dendritic cells to apoptotic materials the primary involvement of the endogenous stimuli in SLE and to assess the 2-step hypothesis in disease remains less defined. Therefore, a major focus has been pathogenesis. on identify the endogenous (self) stimuli acting under CYCLIN KINASE INHIBITORS IN SYSTEMIC “sterile” conditions. AUTOIMMUNITY Studies have shown that products of apoptotic/ We previously found that the cell-cycle inhibitor necrotic cells or nucleic acids, when combined with p21 is a nonredundant effector molecule for SLE in 144 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE autoimmune-prone BXSB mice; lack of p21 resulted in We found that these T cells had significantly enhanced apoptosis of T and B lymphocytes, decreased reduced expression of CD127 (the receptor for IL-7) numbers of activated/memory CD4+ T cells, and dis- and CD122 (the receptor for IL-15). In accordance ease reduction. These findings support our earlier with our hypothesis, nonlymphopenic MRL-Faslpr hypothesis that increased numbers of activated/mem- recipients supported proliferation of transferred isogenic ory CD4+ T cells in SLE could be caused by repeated CD8+ T cells, whereas nonlymphopenic normal hosts stimulation by self-antigens leading to accumulation of did not. Moreover, although single-positive CD4+ and cell cyclin kinase inhibitors, such as p21 and p27, and single-positive CD8+ as well as double-negative T cells a replicative senescence-like state. Upon stimulation, proliferated efficiently in a lymphopenic MRL-Faslpr these senescence-like T cells would be resistant to apo- recipient, survival and repopulation occurred with sin- ptosis and proliferation because of their inability to cycle gle-positive, but not double-negative, T cells. but could produce autoimmunity-promoting proinflam- These studies support the concept that excess of matory factors. IL-7 and IL-15 created by downregulation of the corre- A more recent T-cell transfer study confirmed that sponding receptors in expanded autoreactive T cells lack of p21 in T cells is sufficient to reduce clinical creates an environment that mimics the lymphopenic manifestations of autoimmunity. Moreover, BXSB mice condition of cytokine excess, triggering proliferation of lacking the gene for p27, another cell-cycle inhibitor, newly emerging autoreactive T cells. Verification of also have reduced mortality compared with littermate these results may indicate the usefulness of blocking BXSB mice that have the gene, further supporting our T-cell trophic cytokines and/or the receptors of these hypothesis. We are also examining the role of p21 in cytokines to interfere with autoimmune responses. We organ-specific autoimmunity, and we have generated have started experiments to address this possibility. type 1 diabetes–prone nonobese diabetic mice that lack T-CELL HOMEOSTASIS AND AGING the gene for p21. We have backcrossed (>10 genera- Aging has been associated with several T-cell defects, tions) the p21 knockout gene onto the type 1 diabetes- but whether these defects are intrinsic or are imposed –prone nonobese diabetic strain of mice. Studies will by changes in the microenvironment is unclear. To continue to define in more detail the role of cyclin address this issue, we adoptively transferred labeled kinase inhibitors in autoimmunity and to determine T cells into young and old lymphopenic mice and exam- the potential for modulation of the cell cycle in ther- ined degrees of “acute homeostatic expansion.” The apy for SLE. results indicated that aging is associated with impaired T -CELL HOMEOSTASIS AND SLE homeostatic T-cell proliferation. The proliferation was not Systemic autoimmunity is essentially a disease that due to a primary T-cell defect but rather to changes in can be defined on the basis of disturbances in homeo- the microenvironment. Adoptively transferred T cells from stasis of lymphoid cells. We previously speculated that aged donors indeed proliferated normally in lymphopenic frank lymphopenia and associated excess of T-cell trophic young recipients, whereas T cells from young donors had cytokines (IL-7 and IL-15) leading to ”acute homeo- reduced proliferation in aged lymphopenic hosts. static T-cell proliferation” might be an inducing mech- One possibility for this aging-associated defect in the anism for SLE autoimmune processes. We found that microenvironment is reduced levels of IL-7 and IL-15, acute homeostatic T-cell proliferation of adoptively which are necessary for homeostatic expansion. There- transferred T cells indeed recapitulated systemic auto- fore, we treated old, sublethally irradiated recipients immunity in lymphopenic recipient mice predisposed to with recombinant mouse IL-7. We found that the defect SLE. Currently, we are investigating whether a similar was largely corrected. The results suggest that T-cell phenomenon might be mediated by downregulation of trophic cytokine reconstitution may be an effective receptors for IL-7 and/or IL-15 in accumulating chroni- means to correct immunologic senescence. cally activated autoreactive T cells, a process that might T-CELL HOMEOSTATIC PROLIFERATION TO BREAK create an excess of these cytokines and promote prolif- TOLERANCE TO TUMOR ANTIGENS eration of newly generated autoreactive T cells. To assess We previously proposed that lymphopenia-induced this possibility, we studied MRL-Faslpr mice in which a homeostatic proliferation mediated by excess of trophic Fas mutation leads to massive expansion of double-neg- cytokines and recognition of self-peptide–MHC may be ative (CD4−CD8−) T cells. a way to activate low-affinity T cells that recognize tumor IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 145 antigens. Our earlier studies with a melanoma model Initiation of Inflammation by the indicated the validity of this approach when coupled with tumor cell immunization. We are now assessing Innate Immune System the efficacy of this approach in established and metastasizing tumors, specifically in mouse models of P.S. Tobias, H.-K. Lee, L.K. Curtiss,* P. Dawson,** breast and prostate carcinomas. T. Kirkland,*** D. Liebler**** We found that although the size of subcutaneous * Department of Immunology, Scripps Research tumors was again reduced, metastasis was marginally ** Department of Cell Biology, Scripps Research affected, probably because of the time required for *** University of California, San Diego, California **** Vanderbilt University, Nashville, Tennessee regeneration of lymphocytes and acquisition of a diverse repertoire. e focus on understanding the mechanisms Currently, we are assessing the potential usefulness by which cells use the innate immune system of protocols in which homeostatic proliferation as a W to initiate defensive inflammatory responses. priming event is combined with administration of com- First, we seek to understand the structural features of plexes consisting of IL-7 and nonneutralizing antibod- the Toll-like receptors (TLRs) and their allied proteins ies to IL-7. This approach is based on the recent novel lipopolysaccharide-binding protein, CD14, MD-2, and finding of our collaborators O. Boyman, C.D. Surh, and CD36, which enable the receptors to bind their ligands. J. Sprent, Department of Immunology, that such com- Second, we seek to understand the structural changes plexes induce massive and accelerated expansion of by which binding of a microbial ligand to the extracellu- CD8+ (and CD4+) T cells. Preliminary results indicate lar domain of the receptor leads to signal transduction that this modified protocol significantly reduces both the across the cell membrane and initiation of intracellular size of the primary tumor and the degree of metasta- signaling cascades. Third, we seek to understand the sis. We think that this and other contemplated proto- involvement of endogenous and exogenous inflamma- cols to promote priming of T cells may be promising tory stimuli in atherosclerosis. approaches to tumor immunotherapy. Ten TLRs are known. For most of these, ligands derived from microorganisms are known; binding to PUBLICATIONS the ligands initiates signaling, leading to expression of Homann, D., Dummer, W., Wolfe, T., Rodrigo, E., Theofilopoulos, A.N., Oldstone, M.B., von Herrath, M.G. Lack of intrinsic CTLA-4 expression has minimal effect on inflammatory mediators and other defensive responses. regulation of antiviral T-cell immunity. J. Virol. 80:270, 2006. In addition, some TLRs that may be involved in sterile Janssen, E., Tabeta, K., Barnes, M.J., Rutschmann, S., McBride, S., Bahjat, K.S., inflammatory conditions such as arthritis or atheroscle- Schoenberger, S.P., Theofilopoulos, A.N., Beutler, B., Hoebe, K. Efficient T cell activation via a Toll-interleukin 1 receptor-independent pathway. Immunity 24:787, 2006. rosis may have endogenous ligands. However, these ligands are not yet clearly identified. Kono, D.H., Theofilopoulos, A.N. Genetics of autoantibody production in mouse models of lupus. In: Autoantibodies and Autoimmunity: Molecular Mechanisms in To understand the structural features of ligand-recep- Health and Disease. Pollard, K.M. (Ed.). Wiley-VCH, New York, in press. tor binding, we use using 2 approaches. In the tradi- Kono, D.H., Theofilopoulos, A.N. Genetics of murine models of autoimmunity. In: tional mutation approach, amino acid residues in the Dubois’ Systemic Lupus Erythematosus, 7th ed. Wallace, D.J., Hahn, B.H. (Eds.). proteins are mutated, and the proteins are then studied Williams & Wilkins, Baltimore, in press. for functional changes. In the second approach, we use Kono, D.H., Theofilopoulos, A.N. Genetics of SLE in mice. Springer Semin. Immunopathol. 28:83, 2006. cross-linking agents to create covalent attachments of the ligands to the proteins. The proteins are then Sfikakis, P.P., Gourgoulis, G.M., Moulopoulos, L.A., Kouvatseas, G., Theofilopou- los, A.N., Dimopoulos, M.A. Age-related thymic activity in adults following degraded chemically to determine the site of attach- chemotherapy-induced lymphopenia. Eur. J. Clin. Invest. 35:380, 2005. ment. Currently, we are using these approaches to

Watson, L.C., Moffatt-Blue, C.S., McDonald, R.Z., Kompfner, E., Aït-Azzouzene, study binding of endotoxin, bacterial lipopeptides, and D., Nemazee, D., Theofilopoulos, A.N., Kono, D.H., Feeny A.J. Paucity of V-D-D-J polyinosinic-polycytidylic acid to CD14. rearrangements and VH replacement events in lupus prone and nonautoimmune TdT–/– and TdT+/+ mice. J. Immunol. 177:1120, 2006. Binding of ligands to TLRs starts an intracellular signaling cascade that results in activation of a number of cellular responses. Prominent hypothesized mechanisms by which ligand binding to TLRs incurs transmembrane signaling are (1) the ligand induces dimerization of receptors and (2) binding of the ligand induces con- 146 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE formational changes in the receptor. Our studies indicate of bad news. . . . There is nothing intrinsically poiso- that pairs of TLRs are associated even in the absence nous about endotoxin, but it must look awful, or feel of ligand and that the pairs undergo a conformational awful, when sensed by cells. Cells believe that it sig- change upon ligand binding. We are using a variety of nifies the presence of gram-negative bacteria, and they approaches to understand the structural basis for asso- will stop at nothing to avoid this threat.” In other words, ciations among the TLRs and their associated intracel- the innate immune response to infection has caused a lular signaling partners. serious disease in humans. Atherosclerosis is an inflammatory disease of the Clearly, much human suffering could be eased if large arteries. Evidence suggests that inflammatory such overzealous host responses could be tempered. components derived from microbes can induce progres- However, such responses, when not overzealous, are a sion of atherosclerosis. However, most of the develop- normal part of the host’s homeostatic mechanisms, ment of atherosclerotic lesions is due to endogenous designed to respond to the threat of infection by gram- inflammatory factors. Because the TLR system is so negative bacteria. Accordingly, we are attempting to intimately involved with inflammation, we are determin- (1) define the mechanisms of innate immunity and (2) ing whether the TLRs are involved in atherosclerosis. learn how to control these responses without compro- Our initial data clearly indicate that TLR2, whether mising host defenses against pathogens. Recently, we activated by endogenous ligands or by exogenous ligands, contributed to the understanding of innate immunity promotes progression of atherosclerosis. Unexpectedly, through studies of Toll-like receptors (TLRs) and of effec- we found that TLR2 expressed on non–bone marrow– tor mechanisms that mediate host responses to infection. derived cells detects endogenous TLR2 ligands that It is now well appreciated that the innate immune promote atherosclerosis, whereas bone marrow–derived system is positioned at the intersection of multiple host cells detect exogenous TLR2 ligands that promote pathways, including those for microbial and viral recog- atherosclerosis. For these experiments, we are using nition, enhancement of adaptive immune responses, and, mouse models of the disease and mice deficient in possibly, cancer immunosurveillance. Each pathway individual TLRs. depends on ligand recognition by specific cellular receptors that are either membrane bound (plasma membrane PUBLICATIONS Lee, H.K., Dunzendorfer, S., Soldau, K., Tobias, P.S. Double-stranded RNA-medi- as well as endosomal compartments) or cytosolic. The ated TLR3 activation is enhanced by CD14. Immunity 24:153, 2006. most important class of membrane-bound receptors are

Mullick, A.E., Tobias, P.S., Curtiss, L.K. Modulation of atherosclerosis in mice by the TLRs. Among cytosolic receptors, an important Toll-like receptor 2. J. Clin. Invest. 115:3149, 2005. family known as the NLR/ Nod/Caterpillar family has

Viriyakosol, S., Tobias, P.S., Kirkland, T.N. Mutational analysis of membrane and been identified. Within this family, 2 proteins, Nod1 and soluble forms of human MD-2. J. Biol. Chem. 281:11955, 2006. Nod2, are involved in recognition of bacterial ligands distinct from the ligands for TLRs. Activation of TLR and Nod signaling pathways leads to production of Molecular Mechanisms of multiple cytokines with proinflammatory and anti-inflammatory activities. Such responses are central to host Host-Pathogen Interactions responses to infection. However, when a breakdown occurs in the normal regulatory mechanisms that con- R.J. Ulevitch, V.V. Kravchenko, C. Fearns, T.-H. Chuang, trol these pathways, disease may result. J.C. Mathison, Q. Pan, J. da Silva Correia, K. Iwata, Perhaps the most well-understood link between innate K.D. Janda, G. Kaufmann, M. Meijler immunity and human disease is in the host response nfection by microbial pathogens often sets in motion to infection. When dysregulation of innate immune chains of events that cause severe injury to the host, responses occurs, clinical syndromes such as septic I and nowhere is this phenomenon illustrated more shock and acute respiratory distress syndrome ensue. dramatically than in the response by humans to infec- Dysregulation of innate immune responses may also tion by gram-negative bacteria. In his book Lives of a play a role in human diseases in which chronic inflam- Cell, Lewis Thomas characterized the host response to mation is responsible for disease progression, includ- the endotoxin, or lipopolysaccharide, of gram-negative ing autoimmune and autoinflammatory diseases. Genetic bacteria as being “read by our tissues as the very worst studies in humans have revealed strong associations IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 147 among various members of the Nod family of proteins activated protein kinase p38; double-stranded viral and human diseases. RNA additionally induces the phosphorylation of eukary- During the past year, we made considerable prog- otic translation initiation factor 2α. Now we have shown ress in several different areas. We have identified sev- that p38 and phosphorylation of eukaryotic translation eral unique pathways of innate immunity. These findings initiation factor 2α are 2 biochemical markers of the are briefly summarized here. effects induced by N-(3-oxo-acyl)homoserine lactones, CASPASE 12 AS A NEGATIVE REGULATOR OF the secreted products of a number of gram-negative INNATE IMMUNITY bacteria, including Pseudomonas aeruginosa, an Caspases function in both apoptosis and inflamma- opportunistic pathogen in humans. Furthermore, tory cytokine processing and thereby have a role in N-(3-oxo-dodecanoyl)homoserine lactone induces dis- resistance to sepsis. During the past year we described tension of mitochondria and the endoplasmic reticulum a novel role for a caspase in dampening responses to and transcription of the gene for c-jun. These effects bacterial infection. We showed that in mice, gene-tar- occur in a wide variety of cell types, including alveolar geted deletion of caspase-12 makes animals resistant macrophages and bronchial epithelial cells, and require to peritonitis and septic shock. The resulting survival the structural integrity of the lactone ring motif and its advantage was conferred by the ability of the caspase- natural stereochemistry. 12–deficient mice to clear bacterial infection more These findings suggest that N-(3-oxo-acyl)homo- efficiently than did wild-type littermates. Caspase-12 serine lactones might be recognized by receptors of the dampened the production of the proinflammatory cyto- innate immune system. However, we found that sig- kines IL-1β, IL-18 (IFN-γ–inducing factor), and IFN-γ, naling mediated by N-(3-oxo-dodecanoyl)homoserine but not TNF-α and IL-6, in response to various bacter- lactone does not require the presence of the canonical ial components that stimulate TLR and Nod pathways. innate immune system receptors, TLRs or Nod1 and The IFN-γ pathway was crucial in mediating survival Nod2. These data offer a new understanding of the of caspase-12–deficient mice that had sepsis; admin- effects of N-(3-oxo-dodecanoyl)homoserine lactone on istration of neutralizing antibodies to IFN-γ receptors host cells and its role in persistent airway infections ablated the survival advantage that otherwise occurred caused by P aeruginosa. Our results should be useful in these animals. in the development of new therapeutic interventions Mechanistically, caspase-12 associated with cas- for devastating diseases such as cystic fibrosis in which pase-1 and inhibited its activity. Notably, the protease P aeruginosa or other homoserine lactone–producing function of caspase-12 was not necessary for this effect, organisms are important in disease progression. because the catalytically inactive caspase-12 mutant ROLE OF NOD1 IN THE GROWTH OF ESTROGEN- Cys299Ala also inhibited caspase-1 and IL-1β produc- SENSITIVE BREAST TUMOR CELL LINES tion to the same extent as did wild-type caspase-12. Nod1, a cytosolic protein that senses ligands con- In this regard, caspase-12 seems to be the counter- taining meso-diaminopimelic acid derived from peptido- part of cFLIP, an antiapoptotic protein, for regulating glycan, plays a role in host responses to invasive the inflammatory branch of the caspase cascade. In bacteria. We have identified a novel function for Nod1: mice, caspase-12 deficiency confers resistance to control of tumor formation. We used cell lines derived sepsis, and its presence exerts a dominant-negative from the human breast cancer epithelial cell line MCF-7 suppressive effect on caspase-1, resulting in enhanced in a xenograft model of mice with severe combined vulnerability to bacterial infection and septic mortality. immunodeficiency to characterize a pathway linking These findings have broad implications for new therapies Nod1 to the growth of estrogen-sensitive tumors. for sepsis and related problems. In MCF-7 cells, the absence of Nod1 correlates HOMOSERINE LACTONES AND HOST IMMUNITY with tumor growth, an increased sensitivity to estro- Receptors in the innate immune system function as gen-induced cell proliferation, and a failure to undergo sensors of infection and trigger the immune responses Nod1-dependent apoptosis. Conversely, overexpression through ligand-specific signaling pathways. The ligands of Nod1 in MCF-7 cells results in inhibition of estro- are pathogen-associated products, such as components gen-dependent tumor growth and reduction of estro- of bacterial walls and viral nuclear acids. A common gen-induced proliferative responses in vitro. We are response to such ligands is the activation of mitogen- using a combination of genetics and protein pathway 148 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE mapping to investigate the molecular details of this in archived tissue samples suggests that at least 3000 Nod1 pathway. persons in the United Kingdom are currently incubating vCJD asymptomatically. PUBLICATIONS da Silva Correia, J., Miranda, Y., Austin-Brown, N., Hsu, J., Mathison, J., Xiang, R., Uniquely, the infectious agent in transmissible Zhou, H., Li, Q., Han, J., Ulevitch, R.J. Nod1-dependent control of tumor growth. spongiform encephalopathies, the prion, is though to Proc. Natl. Acad. Sci. U. S. A. 103:1840, 2006. be composed largely of PrPSc, an abnormally shaped Kravchenko, V.V., Kaufmann, G.F., Mathison, J.C., Scott, D.A., Katz, A.Z., Wood, version of the cellular prion protein PrPC, a molecule M.R., Brogan, A.P., Lehmann, M., Mee, J.M., Iwata, K., Pan, Q., Fearns, C., Knaus, U.G., Meijler, M.M., Janda, K.D., Ulevitch, R.J. N-(3-Oxy-acyl)homoserine of unknown function found in all healthy individuals. lactones signal cell activation through a mechanism distinct from the canonical Once established within an infected host, prions repli- pathogen-associated molecular pattern recognition receptor pathways. J. Biol. C Chem. 281:28822, 2006. cate by converting the normal PrP form of the protein into additional molecules of the disease-associated Pan, Q., Kravchenko, V., Katz, A., Huang, S., Ii, M., Mathison, J.C., Kobayashi, Sc K., Flavell, R.A., Schreiber, R.D., Goeddel, D., Ulevitch, R.J. NF-κB-inducing form. Over time, PrP accumulates in the CNS, and kinase regulates selected gene expression in the Nod2 signaling pathway. Infect. Immun. 74:2121, 2006. its appearance is closely associated with profound neuropathologic changes. Raetz, C.R., Garrett, T.A., Reynolds, C.M., Shaw, W.A., Moore, J.D., Smith, D.C., Sc Jr., Ribeiro, A.A., Murphy, R.C., Ulevitch, R.J., Fearns, C., Reichart, D., Glass, In the favored model of prion propagation, PrP C.K., Benner, C., Subramaniam, S., Harkewicz, R., Bowers-Gentry, R.C., Buczyn- acts as a template, sequestering endogenous PrPC and ski, M.W., Cooper, J.A., Deems, R.A., Dennis, E.A. Kdo2-lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4. J. Lipid Res. triggering its conformational rearrangement into nascent 47:1097, 2006. PrPSc and prion infectivity. Although almost none of

Saleh, M., Mathison, J.C., Wolinski, M.K., Bensinger, S.J., Fitzgerald, P., Droin, the molecular details of this pivotal process are under- N., Ulevitch, R.J., Green, D.R., Nicholson, D.W. Enhanced bacterial clearance stood, the persistence of individual prion strains (each and sepsis resistance in caspase-12-deficient mice. Nature 440:1064, 2006. of which is associated with a distinct disease pheno- Wirz, S.A., Tobias, P.S., Ulevitch, R.J., Aribibe, L., Bartfai, T. TLR2 is required for the altered transcription of p75NGF receptors in gram positive infection. Neu- type) suggests that assembly of the prion replicative rochem. Res. 31:297, 2006. complex is a tightly choreographed process. Implicit in this view of prion propagation is a direct and specific binding interaction between PrPC and Prion Diseases: Insights Into the PrPSc. To systematically map defined regions of PrP sequence that bind tightly to PrPSc, we generated a Biology of an Infectious Protein large and comprehensive panel of motif-grafted recombinant antibodies containing successive and overlapping L. Solforosi, A. Bellon, Z. Cheng, P. Sidiropoulos, G. Abalos, polypeptide grafts that collectively span PrP residues J. Cruite, R.A. Williamson 19–231. In PrPSc-binding studies conducted under he prion diseases, or transmissible spongiform stringent conditions with these hybrid-antibody reagents, encephalopathies, are diseases of protein confor- we identified 3 distinct and independent high-affinity T mation that cause profound neurodegeneration PrPSc recognition motifs. and death. They include bovine spongiform encephalop- The first of these binding motifs lies at the N-termi- athy, also known as mad cow disease; scrapie in sheep; nal region of the mature PrP molecule, within residues and chronic wasting disease, which is spreading rapidly PrP 23–33; the second motif, within PrP residues in deer and elk within the United States. The epidemic 98–110; and the third, within PrP residues 136–158. of bovine spongiform encephalopathy in the United Mutational analyses of these PrPSc-binding regions Kingdom predated the emergence of a variant form of revealed that reactivity of the 23–33 and 98–110 PrP Creutzfeldt-Jacob disease (vCJD) in humans, the inci- peptide segments depends largely on the presence of dence of which can be most readily explained by the multiple positively charged amino acid residues. Intrigu- consumption of foods contaminated with the prion that ingly, in an acidic environment akin to that found causes bovine spongiform encephalopathy. The trans- intracellularly within the endocytic pathway in which mission of vCJD prions via blood products obtained PrPSc is found, PrP grafts corresponding to the N-ter- from apparently healthy donors in whom vCJD later minal region of PrP, between residues 29 and 100, developed has reignited concern about the widespread also acquire the ability to strongly recognize misfolded dissemination of prions in humans. Alarmingly, retro- PrP conformers. These studies yield new insight into spective immunocytochemical detection of prion disease critical peptidic components composing one face of the IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 149 prion replicative interface and suggest the potential for Expression of endoglin after oral vaccination was a 2-phase PrPC-PrPSc binding interaction that is pH verified in Peyer’s patches by using confocal micros- dependent. Importantly, elucidating how these differ- copy, indicating that CD11c+ dendritic cells express ent PrP conformers interact now enhances the prospect endoglin intracellularly. Moreover, the endoglin vaccine of efficiently inhibiting their association and thereby markedly suppressed D2F2 breast tumor metastases to halting prion replication and disease. the lung, resulting in a 60% prolongation in life span. In vivo, suppression of pulmonary metastases was abrogated by depletion of CD8+ T cells but not by depletion Endoglin (CD105) as a Target of CD4+ T cells. Antitumor activity of the vaccine correlated with T-cell activation as indicated by upregulation for a DNA Vaccine Against of CD28 and with activation of dendritic cells as indicated by increased expression of CD80 and CD86 on Breast Cancer CD11c+ dendritic cells. Immunization with the DNA vaccine evoked the generation of endoglin-specific cyto- S.H. Lee, N. Mizutani, M. Mizutani, Y. Luo, H. Zhou, toxic T lymphocytes that lysed endoglin-positive murine C.D. Kaplan, R. Xiang endothelial cells. Importantly, tumor angiogenesis was ntiangiogenic therapy has become an attractive markedly suppressed in Matrigel assays, indicating a concept for tumor therapy because the growth significant decrease in neovascularization only in mice A of new capillary blood vessels from preexisting immunized with the endoglin vaccine. vasculature is an essential feature of tumor growth and Taken together, our data suggest that a CD8+ metastasis. The goal of this approach has been to deliver T cell–mediated immune response effectively suppressed antiangiogenic agents to appropriate targets in the tumor dissemination of pulmonary metastases of D2F2 breast vasculature to eliminate or suppress the blood supply carcinoma cells by eliminating proliferating endothelial to tumors, ablating or suppressing growth of the tumors cells, causing suppression of angiogenesis in the tumor without seriously disturbing blood flow to normal tissues. vasculature. We anticipate that vaccine strategies such Endoglin (CD105) is a suitable target for such an as this one will contribute to future therapies for antiangiogenic strategy because it is a coreceptor in breast cancer. the transforming growth factor β (TGF-β) receptor com- PUBLICATIONS plex that is overexpressed on proliferating endothelial Lee, S.H., Mizutani, N., Mizutani, M., Luo, Y., Zhou, H., Kaplan, C.D., Kim. cells in the neovasculature of breast tumors. CD105 S.W., Xiang, R., Reisfeld, R.A. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol. Immunother. 55:1565, 2006. and its ligand TGF-β are important modulators of angiogenesis, and expression of endoglin on proliferating Zhou, H., Luo, Y., Mizutani, M., Mizutani, N., Reisfeld, R.A., Xiang R. T cell- mediated suppression of angiogenesis results in tumor protective immunity. Blood endothelial cells is upregulated by TGF-β and hypoxic 106:2026, 2005. conditions. In solid tumors, such as breast cancer, endoglin is almost exclusively expressed on endothelial cells of both peritumoral and intratumoral blood vessels and on tumor stromal components. We tested the hypothesis that antiangiogenic or antitumor effects can be achieved in a prophylactic setting by using an oral DNA vaccine encoding murine endoglin carried by double-attenuated Salmonella typhimurium to a secondary lymphoid organ, that is, Peyer’s patches in the small intestine. We found that this DNA-based vaccine elicited the activation of antigen-presenting dendritic cells and induced immune responses mediated by CD8+ T cells against endoglin- positive murine endothelial cells. For these studies, we used a syngeneic model of D2F2 murine breast carcinoma cells, which do not express endoglin. 150 IMMUNOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE The Membrane-Proximal We are particularly interested now in performing a similarly detailed structure-function analysis of the External Region of gp41 and monoclonal antibody Z13, because its epitope on the MPER overlaps those of 2F5 and 4E10 and therefore HIV Type 1 Vaccine Design is important for understanding all 3 sites on gp41. By clarifying the effects that the membrane and constraints J.D. Nelson, R. Jensen, I.A. Wilson, P.E. Dawson, imposed by the envelope trimer have on the MPER, our D.R. Burton, M.B. Zwick findings should enable the design of HIV-1 vaccine IV type 1 (HIV-1) is a major world health pro- candidates with stable, homogeneous, and nativelike blem for which the preferred solution is an presentations of the MPER of gp41. effective vaccine. However, typical candidates H PUBLICATIONS for HIV-1 vaccines have not come close to eliciting the Brunel, F.M., Zwick, M.B., Cardoso, R.M.F., Nelson, J.D., Wilson, I.A., Burton, concentration of neutralizing antibodies associated with D.R., Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, protection. HIV-1 neutralizing antibodies target the enve- 2006. lope glycoproteins gp120 and gp41, which assemble as Moore, P.L., Crooks, E.T., Porter, L., Zhu, P., Cayanan, C.S., Grise, H., Corcoran, P., Zwick, M.B., Franti, M., Morris, L., Roux, K.H., Burton, D.R., Binley, J.M. a trimer, (gp120-gp41)3, on the surface of the virion. Nature of nonfunctional envelope proteins on the surface of human immunodefi- We have shown that of the roughly 6 reported broadly ciency virus type 1. J. Virol. 80:2515, 2006. neutralizing monoclonal antibodies against HIV-1, 3 of van Houten, N.E., Zwick, M.B., Menendez, A., Scott, J.K. Filamentous phage as them, 2F5, 4E10, and Z13, bind to the membrane-proxi- an immunogenic carrier to elicit focused antibody responses against a synthetic mal external region (MPER) of gp41. These antibodies, peptide. Vaccine 24:4188, 2006. particularly 4E10, can neutralize primary isolates of Yuste, E., Sanford, H.B., Carmody, J., Bixby, J., Little, S., Zwick, M.B., Gree- nough, T., Burton, D.R., Richman, D.D., Desrosiers, R.C., Johnson, W.E. Simian HIV-1 from around the globe with remarkable breadth immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV- and potency. 1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma. J. Virol. 80:3030, 2006. Clearly, it would be desirable to exploit the MPER, and gp41 in general, for vaccine development, but Zwick, M.B. The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring. AIDS 19:1725, 2005. eliciting neutralizing antibodies against gp41 has not been straightforward. One problem is that the structure of gp41 in the native trimer is unknown. Another difficulty is that the envelope trimer is labile and produces nonfunctional forms of gp41, including gp120-gp41 monomers and “stumps” of gp41 from which gp120 has been shed. Thus, the poor concentrations of neutralizing antibodies to gp41 elicited by vaccination and during natural infection apparently are due at least in part to a poor presentation to the immune system of native gp41 relative to nonfunctional forms of gp41. In collaboration with I.A. Wilson, Department of Molecular Biology, and P.E. Dawson, Department of Cell Biology, we identified a minimal peptide epitope that binds with high affinity to 4E10 and adopts a largely helical conformation in the 4E10-bound complex. To our knowledge, no structure-function analysis has been done on an antibody whose natural epitope is closer to the membrane than that of 4E10. Thus, we have an excellent opportunity not only to better understand the MPER of gp41 and its potential in HIV-1 vaccine design but also to gain insight into the role of lipid in antibody recognition near a membrane. Molecular Biology

Depiction of the P22 bacteriophage virion as solved by electron cryomicroscopy. This structure reveals the major components of the infection machinery, as well as the manner in which DNA is spooled coaxially within the icosahedral capsid. The contour level is set so that the cross section of individual dsDNA strands can be seen with only the outer, most ordered shell of DNA visible. A dodecameric structure at the center of the infection machinery functions as a pressure sensor, which during the packaging of DNA during virus maturation, sends a termination signal to the packaging machinery once the capsid has been fully packaged. Reconstruction and graphics by graduate student Gabriel Lander. Work done in the laboratory of John E. Johnson, Ph.D., in collaboration with Bridget Carragher and Clint Potter, Department of Cell Biology, National Research Resource for Automated Microscopy. Kurt Wüthrich, Ph.D. Cecil H. and Ida M. Green Professor of Structural Biology MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 153

DEPARTMENT OF H. Jane Dyson, Ph.D. Scott Lesley, Ph.D. Vaughn V. Smider, Ph.D. MOLECULAR BIOLOGY Professor Assistant Professor of Assistant Professor of Biochemistry Molecular Biology John H. Elder, Ph.D.*** STAFF Professor Tianwei Lin, Ph.D. Robyn L. Stanfield, Ph.D. Associate Professor Assistant Professor Martha J. Fedor, Ph.D.* Peter E. Wright, Ph.D.* Associate Professor Clare McGowan, Ph.D.† James Steven, Ph.D. Professor and Chairman Associate Professor Assistant Professor Cecil H. and Ida M. Green James Arthur Fee, Ph.D. Investigator in Medical Professor of Research Duncan E. McRee, Ph.D. Raymond C. Stevens, Research Adjunct Associate Professor Ph.D.††† Elizabeth D. Getzoff, ActiveSight Professor Ruben Abagyan, Ph.D. Ph.D.**** San Diego, California Professor Professor Charles D. Stout, Ph.D. David P. Millar, Ph.D. Associate Professor Carlos F. Barbas III, David B. Goodin, Ph.D. Associate Professor Ph.D.***** Associate Professor Peiqing Sun, Ph.D. Professor Louis Noodleman, Ph.D. Associate Professor David S. Goodsell Jr., Ph.D. Janet and W. Keith Kellogg II Associate Professor Chair Associate Professor J. Gregor Sutcliffe, Ph.D. Arthur J. Olson, Ph.D. Professor Rajesh Belani, Ph.D. Joel M. Gottesfeld, Ph.D. Professor Professor Adjunct Assistant Professor John A. Tainer, Ph.D.* †† Professor Jennifer Harris, Ph.D. James C. Paulson, Ph.D. Ola Blixt, Ph.D. Assistant Professor of Professor Assistant Professor of Fujie Tanaka, Ph.D. Biochemistry Molecular Biology Vijay Reddy, Ph.D. Assistant Professor Christian A. Hassig, Ph.D. Assistant Professor Michael N. Boddy, Ph.D. Elizabeth Anne Thomas, Adjunct Assistant Professor Assistant Professor Steven I. Reed, Ph.D.† Ph.D. Kalypsis, Inc. Professor Assistant Professor Charles L. Brooks III, Ph.D. San Diego, California James R. Williamson, Professor Paul Russell, Ph.D. Peter B. Hedlund, M.D., Ph.D. Ph.D.***** Professor David A. Case, Ph.D. Assistant Professor of Professor Professor Molecular Biology Michel Sanner, Ph.D. Associate Dean, Kellogg Associate Professor School of Science and Mirko Hennig, Ph.D.** Geoffrey Chang, Ph.D.* Technology Associate Professor Assistant Professor Harold Scheraga, Ph.D. Medical University of South Adjunct Professor Ian A. Wilson, D.Phil.* Jerold Chun, M.D., Ph.D.*** Carolina George W. and Grace L. Todd Professor Professor Charleston, South Carolina Professor of Chemistry, Curt Wittenberg, Ph.D.† Emeritus Luis De Lecea, Ph.D.** John E. Johnson, Ph.D. Professor Associate Professor Professor Cornell University Stanford University Ithaca, New York Kurt Wüthrich, Ph.D. Palo Alto, California Gerald F. Joyce, M.D., Cecil H. and Ida M. Green Paul R. Schimmel, Ph.D.***** Professor of Structural Ph.D.***** Aymeric Pierre De Parseval, Professor Biology Ph.D. Dean, Faculty Ernest and Jean Hahn Assistant Professor of Professor of Molecular Xiang-Lei Yang, Ph.D. Molecular Biology Ehud Keinan, Ph.D. Biology and Chemistry Assistant Professor of Adjunct Professor Molecular Biology Lluis Ribas De Pouplana, Anette Schneemann, Ph.D. Ph.D. Richard A. Lerner, M.D., Associate Professor Todd O. Yeates, Ph.D. Adjunct Assistant Professor Ph.D.***** Adjunct Professor Barcelona Science Park President, Scripps Research Subhash C. Sinha, Ph.D.* University of California Barcelona, Spain Lita Annenberg Hazen Professor Associate Professor Los Angeles, California of Immunochemistry Ashok Deniz, Ph.D. Cecil H. and Ida M. Green Gary Siuzdak, Ph.D. Qinghai Zhang, Ph.D. Assistant Professor Chair in Chemistry Adjunct Associate Professor Assistant Professor 154 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

SERVICE FACILITIES Ryan Burnett, Ph.D. Beatriz Gonzalez Alonso, Ronald M. Brudler, Ph.D.†††† Ph.D. John Chung, Ph.D. Zhongguo Chen, Ph.D. Lintao Bu, Ph.D. Manager, Nuclear Magnetic David Alvarez-Carbonell, Resonance Facilities Brian Collins, Ph.D.** Ph.D.** Tommy Bui, Ph.D. Momenta Pharmaceuticals Memorial Sloan-Kettering Rosa Maria Cardoso, Ph.D. Gerard Kroon Boston, Massachusetts Cancer Center Assistant Manager, Nuclear New York, New York Justin E. Carlson, Ph.D. Magnetic Resonance Adrienne Elizabeth Dubin, Ph.D. Facilities Juliano Alves, Ph.D. Andrew Barry Carmel, Ph.D. Maria Alejandra Gamez- Michael E. Pique Yu An, Ph.D. Qing Chai, M.D., Ph.D. Director, Computer Graphics Abascal, Ph.D. Development Brigitte Anliker, Ph.D.** Eli Chapman, Ph.D. Reto Horst, Ph.D. Institut für Biochemie und Nahid Razi, Ph.D. Molekularbiologie II Amarnath Chatterjee, Ph.D. Assistant Core Manager, Julio Kovacs, Ph.D. Heinrich-Heine-Universitaet Consortium for Functional Düsseldorf, Germany Anju Chatterji, Ph.D.** Ying Chuan Lin, Ph.D. Bayer Healthcare, Glycomics Andrew James Annalora, Diagnostics Division Mikhail Popkov, Ph.D.** Peter Sobieszcsuk, Ph.D. Ph.D. Berkeley, California Amunix, Inc. Core Manager, Consortium San Jose, California Susana Chaves, Ph.D. for Functional Glycomics Roger Armen, Ph.D. Richard R. Rivera, Ph.D. Joseph W. Arndt, Ph.D. Anton Vladislavovich Cheltsov, Ph.D.** SENIOR STAFF SCIENTIST Kenji Sugase, Ph.D. Karunesh Arora, Ph.D. Burnham Institute Wayne A. Fenton, Ph.D. La Jolla, California Koji Tamura, Ph.D.** Mabelle Ashe, Ph.D. Tokyo University of Science Jianhan Chen, Ph.D. Wojciech Augustyniak, Ph.D. STAFF SCIENTISTS Chiba, Japan Yen-Ju Chen, Ph.D. Jamie Mitchell Bacher, Ph.D. Svitlana Berezhna, Ph.D. Xiaoqin Ye, M.D., Ph.D. Zhiyong Chen, Ph.D. Sung-Hun Bae, Ph.D. Brian M. Lee, Ph.D.** Dirk M. Zajonc, Ph.D.** La Jolla Institute for Allergy Srinivas Chittaboina, Ph.D. Southern Illinois University Manidipa Banerjee, Ph.D. Carbondale, Illinois & Immunology Jungwoo Choe, Ph.D. San Diego, California Christopher Baskerville, Maria Martinez-Yamout, Ph.D. Ph.D.** Chung Jen Chou, Ph.D. RESEARCH ASSOCIATES Garrett M. Morris, Ph.D. Veterans Administration Medical Center Li-Chiou Chuang, Ph.D. Sunny Abraham, Ph.D.** Chiaki Nishimura, Ph.D. San Diego, California Ambit Biosciences Jean-Pierre Clamme, Ph.D.** Jeffrey Speir, Ph.D. San Diego, California Lipika Basummalick, Ph.D.** Université Louis Pasteur current employer unknown Strasbourg Melanie Ann Adams, Ph.D. Manal Swairjo, Ph.D.** relocated to Bay Area Strasbourg, France Western University of Health Fabio Agnelli, Ph.D. Sciences Konstantinos Beis, Ph.D. Linda Maria Columbus, Ph.D. Pomona, California Moballigh Ahmad, Ph.D.** Per Bengston, Ph.D.** Stephen Connelly, Ph.D. University of Illinois Mutsuo Yamaguchi, Ph.D. Lund University Urbana-Chapaign, Illinois Adam Corper, Ph.D. Lund, Sweden Xueyong Zhu, Ph.D. Alexander Ivanov Alexandrov, Qizhi Cui, Ph.D.†††† Christine, Beuck, Ph.D. Ph.D. la P. Da Costa, Ph.D. SENIOR RESEARCH William Henry Bisson, Stephen G. Aller, Ph.D. ASSOCIATES Ph.D.** Sanjib Das, Ph.D.** Marcius Da Silva Almeida, Burnham Institute Takeda David Barondeau, Ph.D.** La Jolla, California San Diego, California Texas A&M Unversity Ph.D.** College Station,Texas Universidade Federal do Rio David Boehr, Ph.D. Surya Kanta De, Ph.D. de Janeiro Kirk Beebe, Ph.D. Rio De Janeiro, Brazil David Bostick, Ph.D. Robert De Bruin, Ph.D.

Sohela De Rozieres, Ph.D.** Bong Kwan Han, Ph.D.** Dae Hee Kim, Ph.D. Derrick Meinhold, Ph.D. Biomatrica University of California San Diego, California San Diego, Calfornia Eda Koculi, Ph.D. Elena Menichelli, Ph.D.

Qingdong Deng, Ph.D.** Byung Woo Han, Ph.D. Bethany Koehntop, Ph.D. Jonathan Mikolosko, Ph.D. Klypsy, Inc. Milka Kostic, Ph.D. Peter J. Mikulecky, Ph.D. San Diego, California Shoufa Han, Ph.D. Irina Kufareva, Ph.D. Mauro Mileni, Ph.D. Paula Desplats, Ph.D. Wenge Han, Ph.D. Shantanu Kumar, Ph.D. Susumu Mitsumori, Ph.D.** Claire Louise Dovey, Ph.D. Jason W. Harger, Ph.D.** Illumina, Inc. Shionogi Research Sharon Kwan, Ph.D. Laboratories Zhanna Druzina, Ph.D. San Diego, California Osaka, Japan Bianca Lam, Ph.D. Li-Lin Du, Ph.D. Rodney Harris, Ph.D. Marissa Mock, Ph.D. Emma Langley, Ph.D. Michelle Duquette-Huber, David M. Herman, Ph.D. Seongho Moon, Ph.D.** Ph.D. Jason Lanman, Ph.D. Deron Herr, Ph.D. Samsung Electronic Scott Eberhardy, Ph.D.** Chang-Wook Lee, Ph.D. Seoul, South Korea Schering-Plough Joreg Hinnerwisch, Ph.D. Chul Won Lee, Ph.D. Samrat Mukhopadhyay, Union, New Jersey Kenichi Hitomi, Ph.D. Ph.D. Jinhyuk Lee, Ph.D.** Stephen Edgcomb, Ph.D. Wen-Xu Hong, Ph.D. University of Kansas Tetsuji Mutoh, Ph.D. Susanna V. Ekholm-Reed, Lawrence, Kansas Sujatha Narayan, Ph.D. Ph.D. Yunfeng Hu, Ph.D. June Hyung Lee, Ph.D. Hung Nguyen, Ph.D. Li Fan, Ph.D. Kwan Hoon Hyun, Ph.D. Kelly Lee, Ph.D. George Nicola, Ph.D. Daniel Felitsky, Ph.D. Wonpil Im, Ph.D.** University of Kansas Edward Lemke, Ph.D. Tadateru Nishikawa, Ph.D. Allan Chris Merrera Ferreon, Lawrence, Kansas Ph.D. Chenglong Li, Ph.D.** Kyoko Noguchi, Ph.D. Masanori Imai, Ph.D. Ohio State University Josephine Chu Ferreon, Ph.D. Columbus, Ohio Wataru Nomura, Ph.D. Veli-Pekka Jaakola, Ph.D. Pierre Henri Gaillard, Ph.D.** Liao Liang, Ph.D. Brian V. Norledge, Ph.D. Centre National de la Kai Jenssen, Ph.D. Severn School Recherche Scientifique Vasco Liberal, Ph.D. Severna Park, Maryland Marseille, France Glenn C. Johns, Ph.D. William M. Lindstrom, Ph.D. Ionian Technologies Wendy Fernandez Ochoa, Yann Gambin, Ph.D. Upland, California Hui-Yue Christine Lo, Ph.D.** Ph.D.†††† University of California Hui Gao, Ph.D.** Eric C. Johnson, Ph.D.** San Diego, California Arena Pharmaceuticals Bruker BioSpin Corporation Kunheng Luo, Ph.D. San Diego, California Fremont, California Amy Odegard, Ph.D. Ann MacLaren, Ph.D. Elsa D. Garcin, Ph.D. Hope Johnson, Ph.D. Lisa Renee Olano, Ph.D. Darly Joseph Manayani, Ph.D. Shannon E. Gardell, Ph.D. Margaret Alice Johnson, Ph.D. Brian L. Olson, Ph.D. Jeff Mandell, Ph.D. Joshua Gill, Ph.D. Susanna Juraja, Ph.D. Maria Victoria Martin- Mary O’Reilly, Ph.D. Christian Kannemeier, Ph.D. Edith Caroline Glazer, Ph.D. Sanchez, Ph.D.** Brian Paegel, Ph.D. University of Geneva Mili Kapoor, Ph.D. Bettina Groschel, Ph.D. Geneva, Switzerland Sandeep Patel, Ph.D.** Andrey Aleksandrovich University of Delaware Fang Guo, Ph.D.** Santiago Cavero Martinez, Karyakin, Ph.D. Newark, Delaware Department of Immonology Ph.D. Scripps Research Yang Khandogin, Ph.D. Stephanie Pebernard, Ph.D. Hanna-Stina Martinsson Min Guo, Ph.D. Ilja V. Khavrutskii, Ph.D. Ahlzén, Ph.D. Bill Francesco Pedrini, Ph.D.

Mahender Gurram, Ph.D. Reza Khayat, Ph.D. Tsutomu Matsui, Ph.D. Robert Pejchal, Ph.D. 156 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Vladimir Pelmenschikov, Daniela Andrea Slavin, Ph.D. Shun-ichi Wada, Ph.D.†††† Wei Zhang, Ph.D.** Ph.D. Southwest Medical Center Elisabetta Soragni, Ph.D. Ross Walker, Ph.D. Houston, Texas Jefferson Perry, Ph.D. San Diego Supercomputer Holly Heaslet Soutter, Ph.D.** La Jolla, California Yong Zhao, Ph.D. Suzanne Peterson, Ph.D. Pfizer Global Research & Development Jessica Williams, Ph.D. Peizhi Zhu, Ph.D.** Jessica Petrillo, Ph.D. Ann Arbor, Michigan University of Michigan Robert Scott Williams, oran Pljevaljcic, Ph.D. Arbor, Michigan Greg Springsteen, Ph.D. Ph.D. Stephanie Pond, Ph.D. Furman University Greenville, South Carolina Eric L. Wise, Ph.D. SCIENTIFIC ASSOCIATES Owen Pornillos, Ph.D.** Celgene Corporation S.V. Ramasastry Sripada, Jonathan Wojciak, Ph.D. Enrique Abola, Ph.D. San Diego, California Ph.D. Vance Wong, Ph.D. Andrew S. Arvai, M.S. Daniel Joseph Price, Ph.D.** Thomas Steinbrecher, Ph.D. Timothy I. Wood, Ph.D.** Ognian V. Bohorov, Ph.D. GlaxoSmithKline Gudrun Stengel, Ph.D.** Walter Reed Army Medical Research Triangle Park, Dennis Carlton, B.S. University of Lund Center North Carolina Washington, D.C Lund, Sweden Vadim Cherezov, Ph.D. John Prudden, Ph.D. Shih-Che Su, Ph.D. Eugene Wu, Ph.D.** Ellen Yu-Lin Tsai Chien, Ph.D. Duke University Medical Grazia Daniela Raffa, Ph.D.** Magnus Sundstrom, Ph.D. Università di Roma La Center Xiaoping Dai, Ph.D. Sapienza Blair R. Szymczyna, Ph.D. Durham, North Carolina Rome, Italy Marc Deller, D.Phil Florence Muriel Tama, Wei Xie, Ph.D. Christopher L. Reyes, Ph.D. Ph.D.** Gye Won Han, Ph.D. Lan Xu, Ph.D. Biogen Idec Research University of Arizona Michael Allen Hanson, Ph.D. San Diego, California Tucson, Arizona Yoshiki Yamada, Ph.D. Marcy A. Kingsbury, Ph.D. Alim Seit-Nebi, Ph.D. Nardos Tassew, Ph.D.** Atsushi Yamagata, Ph.D. University of Toronto Diane Marie Kubitz, B.A. Riturparna Sinha Roy, Ph.D. Toronto, Canada Qi Yan, Ph.D.** Miramar College Padmaja Natarajan, Ph.D. Stanislav Rudyak, Ph.D.†††† Hiroaki Tateno, Ph.D. San Diego, California Marianne Patch, Ph.D.** Sean Ryder, Ph.D.** Rebecca E. Taurog, Ph.D. Yong Yao, Ph.D.** Qualcomm University of Massachusetts Burnham Institute San Diego, California Medical School Ewan Richardson Taylor, Ph.D. La Jolla, California Worcester, Massachusetts Gabriela Perez-Alvarado, Manami R. Saha, Ph.D.†††† Hua Tian, Ph.D. Yongjun Ye, Ph.D. Ph.D.** Southern Illinois University Mauricio Carrillo Tripp, Ph.D. Sanjay Adrian Saldanha, Kye Sook Yi, Ph.D. Carbondale, Illinois Ph.D. Ulrich Ignaz Tschulena, Ph.D. Yong Yin, Ph.D. Nicholas Preece, Ph.D.†††† Andre Schiefner, Ph.D. Julie L. Tubbs, Ph.D. Kenji Yoshimoto, Ph.D. Lin Wang, Ph.D. Lauren J. Schwimmer, Naoto Utsumi, Ph.D. Ph.D. Naoto Yoshizuka, Ph.D. Frank van Drogen, Ph.D.** VISITING INVESTIGATORS Jennifer S. Scorah, Ph.D. Institute für Biochemie Veronica Yu, Ph.D.†††† Stephen J. Benkovic, Ph.D. Pedro Serrano-Navarro, Ph.D. Zürich, Switzerland Yuan Yuan, Ph.D. Pennsylvania State Craig McLean Shepherd, Ajay Vashisht, Ph.D. University Markus Zeeb, Ph.D. Ph.D. University Park, Philip Arno Venter, Ph.D. Ying Zeng, Ph.D. Pennsylvania David S. Shin, Ph.D. Petra Verdino, Ph.D. Astrid Graslund, Ph.D. Develeena Shivakumar, Ph.D. Haile Zhang, Ph.D. William Frederick Waas, Stockholm University David A. Shore, Ph.D. Ph.D. Qing Zhang, Ph.D. Stockholm, Sweden

Arne Holmgren, M.D., Ph.D. * Joint appointment in The Skaggs Karolinska Institutet Institute for Chemical Biology Stockholm, Sweden ** Appointment completed; new location shown Barry Honig, Ph.D. *** Joint appointment in the Columbia University Molecular and Integrative New York, New York Neurosciences Department

Arthur Horwich, M.D. **** Joint appointments in the Department of Immunology and Yale University The Skaggs Institute for New Haven, Connecticut Chemical Biology Shie-Liang Hsieh, Ph.D. ***** Joint appointments in the National Yang-Ming Department of Chemistry and University The Skaggs Institute for Chemical Biology Taipei, Taiwan † Joint appointment in the Tai-Huang Huang, Ph.D. Department of Cell Biology Academica Sinica †† Joint appointment in the Taipei, Taiwan Department of Molecular and Experimental Medicine Sunghoon Kim, Ph.D. ††† Joint appointment in the Seoul National University Department of Chemistry Seoul, Korea †††† Appointment completed Ayori Mitsutake, Ph.D. Keio University Yokohama, Japan

Joseph David Ng, Ph.D. University of Alabama, Huntsville Huntsville, Alabama

Victoria A. Roberts, Ph.D. University of California San Diego, California

Robert D. Rosenstein, Ph.D. Lawrence Berkeley National Laboratory Berkeley, California

Lincoln Scott, Ph.D. Cassia, LLC San Diego, California

Deborah Tahmassebi, Ph.D. University of San Diego San Diego, California 158 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

only a few highlights are mentioned below. The Depart- ment of Molecular Biology is also home to two major National Institutes of Health initiatives, the Joint Center for Structural Genomics and the Consortium for Func- tional Glycomics. One of the outstanding achievements of the past year was the determination of the structure of the intact and infectious P22 virion by electron cryomicroscopy. Research led by Jack Johnson has provided a remarkably detailed view of the virion structure at an unprecedented 17-Å resolution. The structure revealed the DNA tightly spooled around the portal in the interior of the capsid and suggested that the virus uses a pressure-sensing mechanism to control DNA packaging. The structure also provides insights into the mechanisms of virion assembly and injection of DNA into target cells. Structural biology continues to be a major focus in the department, and many new x-ray and nuclear magnetic resonance structures of major biomedical signifi- Peter E. Wright, Ph.D. cance were completed during the past year. Geoffrey Chang and colleagues reported new structural studies Chairman’s Overview of the Escherichia coli multidrug transporter EmrD, obtaining new insights into the mechanisms by which a esearch in the Department of Molecular Biology diverse range of drugs are transported through the cell encompasses a broad range of disciplines, extend- membrane. Such understanding is of major importance, R ing from structural and computational biology at given the rapidly growing problem of drug resistance in one extreme to molecular genetics at the other. During bacteria. John Tainer and his coworkers used a combina- the past year, our scientists have continued to make rapid tion of electron cryomicroscopy and x-ray crystallogra- progress toward understanding the fundamental molecu- phy to determine the structure of the Type IV pilus lar events underlying the processes of life. Major advances filament of Neisseria gonorrhoeae. These studies pro- have been made in elucidating the structural biology of vide new insights into assembly and disassembly mecha- signal transduction and viral assembly, in understand- nisms and are of importance because of the role played ing mechanisms of viral infectivity, in determining the by Type IV pili in allowing antibiotic resistant strains to structures of membrane proteins and multidrug trans- escape the immune system and cause persistent infec- porters, in understanding the molecular basis of nucleic tions. Dr.Tainer and colleagues have also determined acid recognition and DNA repair, and in determining the new structures of DNA repair enzymes; these include mechanisms of protein folding and ribosome assembly. the xeroderma pigmentosum group B helicase, an Progress has been made in elucidating the molecular enzyme that plays an essential role in nucleotide-exci- events involved in regulation of the cell cycle, in tumor sion repair by removing DNA lesions caused through development, in induction of sleep, in the molecular exposure to ultraviolet light, and the exonuclease origins of neuronal development and of CNS disorders, domain of WRN, a protein that protects against prema- in the regulation of transcription, and in the decoding ture aging and cancer. Defects in the gene for WRN of genetic information in translation.Finally, new advances result in Werner’s syndrome, an inherited disease that have been made in the design of novel low molecular causes premature aging. weight compounds that can specifically regulate genes, Research in the laboratories of Jane Dyson and Peter and in the area of biomolecular engineering, building Wright has provided new insights into the role of protein novel functions into viruses, antibodies, and zinc finger conformational fluctuations in enzyme catalysis. Protein proteins, RNA, and DNA. Progress in these and other dynamics have long been thought to play an important areas is described in detail on the following pages, and role in catalysis. This new work shows how the dynamic MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 159 energy landscape of the enzyme dihydrofolate reductase Molecular biology remains a field of enormous oppor- channels the protein through the reaction cycle. Confor- tunity and excitement. The scientists in this department mational transitions between the various conformational are taking full advantage of powerful new technologies substates of the enzyme occur at a rate that is directly to advance our understanding of fundamental biological relevant to catalysis. processes at the molecular level. Their discoveries will Several research groups are working in areas directly ultimately be translated into new advances in biotechnol- related to drug discovery and protein therapeutics. Joel ogy and in medicine. Gottesfeld and his colleagues have developed small- molecule histone deacetylase inhibitors that reactivate frataxin, the gene responsible for the neurodegenerative disease Friedreich’s ataxia, a disease that is associated with the expansion of triplet repeats in DNA. These compounds hold great promise as potential therapeutics for Freidreich’s ataxia. Subhash Sinha, Carlos Barbas, and Richard Lerner have developed a unique self-assembly strategy to direct antibodies against specific cellular targets. Their novel approach has led to new compounds targeted against metastatic breast cancer. Many of the research groups in this department are applying the tools of molecular and structural biology to understand the molecular basis of human disease. In research led by James Paulson and Ian Wilson, glycan microarray technology is being used to identify mutations that could allow avian influenza viruses to adapt to the human population. The glycan array is a powerful surveillance tool for mapping the pathways by which new human pathogenic viruses can emerge. This research has revealed a potential mutational pathway that could switch the specificity of the highly pathogenic H5N1 avian influenza virus and allow it to adapt to humans. Strikingly, the 3-dimensional structure of the H5N1 hemagglutinin, the protein responsible for binding the virus to host cell receptors, bears a closer resemblance to the hemagglutinin from the virus that caused the 1918 influenza pandemic than to that associated with more recent influenza outbreaks. Finally, research during the past year has greatly advanced our understanding of the complex mechanisms of cell-cycle regulation. Curt Wittenberg and his colleagues have identified a yeast protein that plays a central role in repressing transcription during the cell cycle. The protein functions in a parallel manner to the important metazoan transcriptional regulator E2F. Work in Steven Reed’s laboratory has provided new insights into the mechanisms of multiubiquitinylation and degradation of cyclin E, a process that is essential for the normal regulation of the cell cycle. Misregulation of either of these processes, transcriptional repression or cyclin turnover, is associated with cancer. 160 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

INVESTIGATORS’REPORTS Structural Biology of Immune Recognition, Molecular Assemblies, and Anticancer Targets

I.A. Wilson, R.L. Stanfield, J. Stevens, X. Zhu, M.A. Adams, Y. An, K. Beis, D.A. Calarese, R.M.F. Cardoso, J.E. Carlson, P.J. Carney, J.-W. Choe, S. Connelly, A.L. Corper, T.H. Cross, Fig. 1. A, Structure of the H5 A/Vietnam/1203/2004 (Viet04) X. Dai, E.W. Debler, W.L. Densley, M.-A. Elsliger, S. Ferguson, hemagglutinin trimer, represented as a ribbon diagram. The recep- B.W. Han, G.W. Han, M.J. Jimenez-Dalmaroni, J.G. Luz, tor binding domain, cleavage, and basic patch sites are highlighted J.R. Mikolosko, A. Schiefner, D.A. Shore, R.S. Stefanko, on one monomer. Only 2 of the 9 glycosylation sites per monomer (positions 34 and 169 in the HA1 chain) had interpretable carbo- J.A. Vanhnasy, P. Verdino, E. Wise, L. Xu, X. Xu, D.M. Zajonc hydrates in the electron density maps. B, Glycan microarray analy- e are working toward a better understanding ses of wild-type human Viet04 hemagglutinin and mutations at of the structure and function of a variety of positions 226 and 228, known to be important for adaptation of H3 viruses from avian 2-3 specificity to human 2-6 receptor immune-related receptors and of other med- α α W specificity. Binding to the different avian and human α2-3 and α2- ically relevant proteins. We use x-ray crystallography 6 sialosides on the array are highlighted. to determine structures for these molecules in complex with their ligands and coreceptors. This research is instru- to change from its avian receptor binding (α2-3-linked mental for the design of future drugs and vaccines to sialic acids) to adapt to human receptors (α2-6-linked target these proteins. sialic acids; Fig. 1B) and have elucidated a possible INFLUENZA VIRUS route by which H5 viruses could gain a foothold in the Influenza virus is a highly contagious and deadly human population. agent that causes acute respiratory illness. The current IL-2 RECEPTOR H5N1 avian influenza virus has reached epizootic lev- IL-2 is a cytokine that functions as a T-cell growth els in domestic and wild birds, with worldwide debate factor and a central immune system regulator. Its impor- whether the next influenza pandemic could arise from tance is underlined by its broad use as a therapeutic one of these avian strains. Hemagglutinin is the princi- agent against cancers of the immune system, and IL-2 pal viral surface antigen and is responsible for binding to antagonists are used to prevent rejection of transplanted host receptors through interaction with sialylated glycans. organs. We have determined the structure of the het-

The structure of the hemagglutinin from a highly path- erotrimeric IL-2 receptor ectodomains (IL-2Rαβγc) in ogenic H5N1 influenza virus (A/Vietnam/1203/2004; complex with IL-2 at 3.0-Å resolution (Fig. 2). Surpris-

Fig. 1A) is more closely related to the human 1918 H1 ingly, IL-2Rα makes no contacts with IL-2Rβ or IL-2Rγc, hemagglutinin than to the other human, avian, and swine and only minor changes occur in IL-2 in response to hemagglutinins. We are also examining crystal structures receptor binding. Thus, our findings support the notion of (1) various influenza neuraminidases to determine the that IL-2Rα delivers IL-2 to the signaling complex and specificity of the enzymes and their involvement in interacts as a regulator of signal transduction. This research action/escape of the virus from current drugs and (2) was performed in collaboration with K.A. Smith, Cornell influenza viral proteins that interact with components of University Weill Medical College, New York, New York. the apoptosis signaling pathway. THE INNATE IMMUNE SYSTEM In collaboration with O. Blixt and J. Paulson of the Toll-like receptors (TLRs) play key roles in activat- Consortium for Functional Glycomics, La Jolla, Califor- ing immune responses during infection. The 2.1-Å nia, we used their recently described glycan microar- structure of the human TLR3 ectodomain revealed a ray technology to assess the propensity of the avian large horseshoe-shaped solenoid structure assembled receptor H5N1 A/Vietnam/1203/2004 hemagglutinin from 23 leucine-rich repeats. Seven conserved hydro- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 161

Department of Immunology, we are studying the membrane-bound part of the NADPH complex to correlate how mutations in NADPH oxidase can cause chronic granulomatous disease. The nucleotide oligomerization binding domain 2 is an important intracellular receptor that recognizes bacterial peptidoglycans. Mutations in this receptor are associated with the inflammatory Crohn’s disease. Structural studies are under way on the domains and on full-length protein, in collaboration with R. Ulevitch, Department of Immunology. CATALYTIC ANTIBODIES Abuse of cocaine is a major public health problem; however, no treatments approved by the Food and Drug Fig. 2. Architecture of the trimeric human IL-2 receptor (desig- Administration are available for cocaine abuse, addiction, nated IL2R in the figure) signaling complex. View of the quaternary or overdose. Development of effective treatments for IL-2 signaling assembly composed of α, β, and γc chains of the IL- 2R and IL-2, with the C terminus of the β and γ chains close to the cocaine abuse has been frustrated by the complex neu- membrane. IL-2 binds to the elbow regions of IL-2Rβ and IL-2Rγc, rochemistry of cocaine addiction. Nevertheless, within as in other cytokine receptors such as human growth hormone the past decade, immunotherapy for cocaine abuse has receptor and erythropoietin receptor. The novel IL-2Rα chain docks been evaluated in preclinical and clinical trials. In colon top of this assembly but does not form any contacts with the laboration with K.D. Janda, Department of Chemistry, other 2 receptor subunits. Six N-linked carbohydrates (S1–S6) are displayed as ball-and-stick models. S1 is wedged between D1 and we determined high-resolution structures for the cocaine D2 of IL-2Rβ and thus contributes to the stabilization of a specific catalytic antibody 7A1 for all major steps along the D1/D2 interdomain angle. IL-2Rβ and IL-2Rγ form a 3-way junc- catalytic reaction pathway, through cocrystallization tion with IL-2 at the heart of the quaternary high-affinity IL-2 sig- with substrate, products, and transition-state analogs naling complex and provide a structural basis for the cooperativity (Fig. 3). On the basis of this comprehensive series of in assembly of the complete IL-2 signaling complex. phobic residues in the leucine-rich repeat motif form a tight hydrophobic core, and conserved asparagines contribute extensive hydrogen-bonding networks for solenoid stabilization. TLR3 is largely masked by carbohydrate, but the only glycosylation-free face may provide potential ligand-binding sites and an oligomerization interface. We are doing biochemical analysis of the interaction between the TLR3 ectodomain and various double- stranded RNA oligomers and structural investigations of TLR1, TLR2, TLR6, and the TLR2 coreceptor CD36. These projects are a collaboration with B. Beutler and R. Ulevitch, Department of Immunology. Neutrophils and other phagocytes play an important role in innate immunity by serving as a first line of defense against invading pathogens. Generation of superoxide by the phagocyte NADPH oxidase complex initi- Fig. 3. Crystal structure of the antibody 7A1 Fab′ fragment in complex with cocaine. The secondary structure of the Fab′ and the ates this process by catalyzing the transfer of metabolic substrate cocaine are shown. Cocaine is trapped in the active site electrons across the plasma membrane for reduction of and is hydrolyzed to nontoxic metabolites. molecular oxygen. Individuals deficient in this enzymatic activity have chronic granulomatous disease, crystal structures, a catalytic mechanism has been pro- characterized by recurrent, life-threatening bacterial posed, as well as possible mutations to improve cata- and fungal infections. In collaboration with G. Bokoch, lytic proficiency. 162 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

COFACTOR-CONTAINING ANTIBODIES biochemical studies with our collaborators, R.A. Lerner, Although antibodies are generally thought to function K.D. Janda, P.G. Schultz, and F.E. Romesberg, Depart- without use of cofactors, they are major carrier proteins ment of Chemistry. in human circulation for the biologically important EVOLUTION OF LIGAND RECOGNITION AND cofactor riboflavin. A riboflavin-containing bright-yellow SPECIFICITY antibody, IgG GAR, was purified from a patient with To enhance our understanding of how recognition multiple myeloma 30 years ago and is the only avail- and specificity for different ligands can be accomplished able material for studies of the structure and function by different antibodies that have high levels of sequence of natural cofactor-containing antibodies. Our recent homology, we are studying the evolution of ligand-bind- 3.0-Å crystal structure of GAR reveals the location in ing properties by site-directed mutagenesis. The most the antibody-combining site for the riboflavin poten- active catalytic Diels-Alder antibody known to date, 1E9, tial cofactor (Fig. 4). This research was carried out and the steroid-binding antibody DB3 are derived from the same germ line and have 85% sequence identity. Through sequential amino acid exchanges, the specificity of 1E9 was changed to that of DB3. Thus, only a few binding site residues are responsible for achieving either efficient catalysis of the Diels-Alder reaction or, when mutated, a strong steroid binder. In collaboration with D. Hilvert, ETH, Zürich, Switzerland, we are structurally characterizing these 1E9 mutants to show how relatively minor changes can be rationally used to modify antibody specificity and function. HIV TYPE 1 NEUTRALIZING ANTIBODIES The search for an effective HIV type 1 vaccine has prompted the study of the few known broadly neutralizing antibodies to HIV type 1 in complex with their antigens, in order to structurally characterize important viral epitopes. The potent and broadly neutralizing antibodies include 4E10 and Z13, which bind to conserved and overlapping epitopes on the membrane-proximal region of gp41, and 2G12, which binds to a carbohy- Fig. 4. The antigen-binding site of the original yellow antibody drate cluster rich in mannose on gp120. These crystal IgG GAR. The riboflavin cofactor is inserted into the combining site structures are then used as the basis for rational design with its isoalloxazine ring stacked between aromatic residues TyrH33, PheH58, and TyrH100A. Together with hydrogen bonds of immunogens for a candidate vaccine against HIV between the N5 atom of the ring to AsnH50 and the ribityl side type 1. This research is done in collaboration with chain to ArgH52 and GluH56, these interactions reveal the struc- D. Burton, Department of Immunology; P. Dawson, tural basis for high-affinity riboflavin binding. Department of Cell Biology; C.-H. Wong, Department of Chemistry; S. Danishefsky, Sloan-Kettering Institute, in collaboration with R.A. Lerner and P. Wentworth, New York, New York; J.K. Scott, Simon Fraser University, Jr., Department of Chemistry. Burnaby, British Columbia; J. Moore, Cornell University, BLUE AND PURPLE FLUORESCENT ANTIBODIES Ithaca, New York; H. Katinger, R. Kunert, and G. Stiegler, Catalytic antibodies are designed to accelerate chem- University für Bodenkultur, Vienna, Austria; R. Wyatt and ical reactions by acting on the electronic ground state. P. Kwong, Vaccine Research Center, National Institutes However, antibodies have been generated that can inter- of Health, Bethesda, Maryland; and the Neutralizing act with and direct the photochemical behavior of the Antibody Consortium of the International AIDS Vaccine electronically excited state of stilbene, a model com- Initiative, New York, New York. pound for studies in photochemistry and photophysics. CLASSICAL AND NONCLASSICAL MHC AND T-CELL We are exploring the structural basis of the diverse RECEPTOR SIGNALING fluorescent properties of these complexes by using x-ray An inflammatory joint disease with many similarities crystallography in combination with biophysical and to human rheumatoid arthritis develops spontaneously in MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 163

KRN T-cell receptor (TCR) transgenic mice (F1 K/B x N mice). Class II MHC I-Ag7 presentation to KRN of self- peptide derived from glucose-6-phosphate isomerase is a critical step in the initiation of the disease. In collaboration with L. Teyton, Department of Immunology, we determined the crystal structures of I-Ag7–glucose-6- phosphate isomerase peptide and of the TCR KRN. We are attempting to crystallize the KRN–I-Ag7 complex to enhance our understanding of how this autoimmune disease is mediated at the molecular level. The CD3 TCR coreceptor comprises several distinct cell-surface glycoproteins that associate with TCR to enable intracellular signal transduction upon the formation of complexes consisting of TCR and MHC-peptides. Structural investigation into the interaction between the TCR and CD3 subunits can aid in elucidation of the events that lead to T-cell activation. The CD8 glycoprotein is essential for the class I MHC-restricted T-cell response to peptide antigen, analogous to the CD4 core- Fig. 5. Structure of mouse CD1d with inositol-dimannoside. ceptor of class II–restricted T cells. CD8 is expressed at Close-up view of the binding site shows the hydrogen-bonding net- the cell surface as CD8αα and CD8αβ. We have deter- work between the glycolipid and CD1d. Both alkyl chains of the mined structures for both CD8αα and CD8αβ in com- ligand are deeply buried inside the binding groove (not shown), whereas the complex inositol-dimannoside headgroup is optimally plex with antibody Fab fragments. Comparison of both positioned above the binding groove to directly interact with the TCR. forms of the CD8 coreceptor have provided insight into how the α and β forms contribute to the functionality of endocytic pathways of eukaryotic cells. In collaboration CD8. These studies are a collaboration with S. Davis, with W. Balch, Department of Cell Biology, we have University of Oxford, Oxford, England, and L. Teyton, determined the 2.0-Å structure of the Rab1 GTPase- Department of Immunology. regulated N-terminal domain of the p115 tether involved The CD1 family is structurally related to MHC mole- in transport and structural organization of the Golgi com- cules, but members of the family present lipid antigens plex. The structure reveals a dimeric handshakelike rather than peptides to CD1-restricted TCRs. We have assembly consisting of 2 α-solenoid chains, each with determined several structures of mouse CD1d in com- 12 novel armadillo-like, tetherin trihelical repeat elements plex with α-galacturonosyl ceramide, cis-tetracosenoyl that form a superhelical elliptical cylinder. This structure sulfatide, or mycobacterial phosphatidylinositol diman- supports a model for binding of Rab1 on opposing mem- noside. For each CD1d-ligand, the lipid tails are embed- branes to promote membrane tether assembly for mem- ded in the CD1 hydrophobic binding groove, and a brane docking and fusion and for understanding the large restricted set of CD1d residues orient and stabilize the family of molecular tethers. various different antigenic headgroups for TCR recognition (Fig. 5). Collaborators in research on CD1 and JOINT CENTER FOR STRUCTURAL GENOMICS TCRs include D.B. Moody and M.B. Brenner, Harvard The Joint Center for Structural Genomics is a large Medical School, Boston, Massachusetts; C.-H. Wong, consortium of scientists from Scripps Research; the Department of Chemistry; L. Teyton, Department of Stanford Synchrotron Radiation Laboratory; the Univer- Immunology; M. Kronenberg, La Jolla Institute for Allergy sity of California, San Diego; the Burnham Institute; and Immunology, San Diego, California; V. Kumar, Torrey and the Genomics Institute of the Novartis Research Pines Institute for Molecular Studies, San Diego, Cali- Foundation. The center is funded by the Protein Structure fornia; and W. Severn and G. Painter, Industrial Research Initiative of the National Institute of General Medical Ltd., Upper Hut, New Zealand. Sciences. Its purpose is the high-throughput structure PROTEIN TRAFFICKING determination of large protein families with no struc- Molecular tethers play a critical role in the organi- tural representatives, a biologically important group of zation of the membrane architecture of the exocytic and targets that are conserved in the central machinery of 164 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE life; the complete proteome from Thermotoga maritima; Johnson, M.A., Peti, W., Herrmann, T., Wilson, I.A., Wüthrich, K. Solution structure of Asl1650, an acyl carrier protein from Anabaena sp PCC 7120 with a vari- and targets suggested by the community. To date, mem- ant phosphopantetheinylation-site sequence. Protein Sci. 15:1030, 2006. bers of the consortium have pioneered many novel high- Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved throughput methods, constructed a high-throughput hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a pipeline, and determined more than 270 nonredun- new fold. Proteins 61:1132, 2005. dant structures. Luz, J.G., Yu, M., Su, Y., Wu, Z., Zhou, Z., Sun, R., Wilson, I.A. Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi’s sarcoma-associated herpesvirus at 1.7 Å. J. Mol. Biol. 352:1019, 2005. PUBLICATIONS Almeida, M.S., Herrmann, T., Peti, W., Wilson, I.A., Wüthrich, K. NMR structure Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of of the conserved hypothetical protein TM0487 from Thermotoga maritima: impli- phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga mar- cations for 216 homologous DUF59 proteins. Protein Sci. 14:2880, 2005. itima at 2.15 Å resolution. Proteins 63:1106, 2006.

Brunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R., Moody, D.B., Zajonc, D.M., Wilson, I.A. Anatomy of CD1-lipid antigen complexes. Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neu- Nat. Rev. Immunol. 5:387, 2005. tralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006. Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C., Burton, D.R., Stanfield, R.L., Wilson, I.A. Antibody vs HIV in a clash of evolution- Wüthrich, K. Towards miniaturization of a structural genomics pipeline using ary titans. Proc. Natl. Acad. Sci. U. S. A. 102:14943, 2005. micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005.

Calarese, D.A., Lee, H.K., Huang, C.Y., Best, M.D., Astronomo, R.D., Stanfield, Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of the global R.L., Katinger, H., Burton, D.R., Wong, C.-H., Wilson, I.A. Dissection of the car- regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a bohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc. new fold. Proteins 61:449, 2005. Natl. Acad. Sci. U. S. A. 102:13372, 2005. Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a putative modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution Cheng, H., Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, reveals a new fold. Proteins 61:444, 2005. S.J., Boger, D.L. Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as Shore, D.A., Teyton, L., Dwek, R.A., Rudd, P.M., Wilson, I.A. Crystal structure of potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway. the TCR co-receptor CD8αα in complex with monoclonal antibody YTS 105.18 Bioorg. Med. Chem. 13:3577, 2005. Fab fragment at 2.88 Å resolution. J. Mol. Biol. 358:347, 2006.

Cheng, H., Hwang, I., Chong, Y., Tavassoli, A., Webb, M.E., Zhang, Y., Wilson, Stanfield, R.L., Gorny, M.K., Zolla-Pazner, S., Wilson, I.A. Crystal structures of I.A., Benkovic, S.J., Boger, D.L. Synthesis and biological evaluation of N-[4-[5- human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 2219 in com- (2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]ben- plex with three different V3 peptides reveal a new binding mode for HIV-1 cross- zoyl]-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine reactivity. J. Virol. 80:6093, 2006. biosynthetic pathway. Bioorg. Med. Chem. 13:3593, 2005. Stanfield, R.L., Zemla, A., Wilson, I.A., Rupp, B. Antibody elbow angles are influ- Choe, J., Kelker, M.S., Wilson, I.A. Crystal structure of human Toll-like receptor 3 enced by their light chain class. J. Mol. Biol. 357:1566, 2006. (TLR3) ectodomain. Science 309:581, 2005. Stauber, D.J., Debler, E.W., Horton, P.A., Smith, K.A., Wilson, I.A. Crystal struc- Chong, Y., Hwang, I., Tavassoli, A., Zhang, Y., Wilson, I.A., Benkovic, S.J., Boger, ture of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor. D.L. Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10- Proc. Natl. Acad. Sci. U. S. A. 10:2788 2006. CF3CO-DDACTHF. Bioorg. Med. Chem. 13:3587, 2005. Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C., DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a Wilson, I.A. Glycan microarray analysis of the hemagglutinins from modern and single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60 pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. Å resolution.Proteins 63:256, 2006. 355:1143, 2006.

Giabbai, B., Sidobre, S., Crispin, M.D., Sanchez-Ruiz, Y., Bachi, A., Kronenberg, Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson, M., Wilson, I.A., Degano, M. Crystal structure of mouse CD1d bound to the self I.A. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza ligand phosphatidylcholine: a molecular basis for NKT cell activation. J. Immunol. virus. Science 312:404, 2006. 175:977, 2005. Van Rhijn, I., Zajonc, D.M., Wilson, I.A., Moody, D.B. T-cell activation by lipopeptide antigens. Curr. Opin. Immunol. 17:222, 2005. Glaser, L., Stevens, J., Zamarin, D., Wilson, I.A., Garcia-Sastre, A., Tumpey, T.M., Basler, C.F., Taubenberger, J.K., Palese, P. A single amino acid substitution Wilson, I.A., Stanfield, R.L. MHC restriction: slip-sliding away. Nat. Immunol. in 1918 influenza virus hemagglutinin changes receptor binding specificity. J. Virol. 6:434, 2005. 79:11533, 2005. Wiseman, R.L., Johnson, S.M., Kelker, M.S., Foss, T., Wilson, I.A., Kelly, J.W. Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo Kinetic stabilization of an oligomeric protein by a single ligand binding event. J. mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins Am. Chem. Soc. 127:5540, 2005. 60:797, 2005. Wu, D., Zajonc, D.M., Fujio, M., Sullivan, B.A., Kinjo, Y., Kronenberg, M., Wil- Huang, C.C., Tang, M., Zhang, M.Y., Majeed, S., Montabana, E., Stanfield, R.L., son, I.A., Wong, C.-H. Design of natural killer T cell activators: structure and func- Dimitrov, D.S., Korber, B., Sodroski, J., Wilson, I.A., Wyatt, R., Kwong, P.D. tion of a microbial glycosphingolipid bound to mouse CD1d. Proc. Natl. Acad. Sci. Structure of a V3-containing HIV-1 gp120 core. Science 310:1025, 2005. U. S. A. 103:3972, 2006.

Jaroszewski, L., Schwarzenbacher,R., McMullan, D., et al. Crystal structure of Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution.Pro- factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold. teins 61:669, 2005. Proteins 62:292, 2006.

Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367 Zajonc, D.M., Cantu, C. III, Mattner, J., Zhou, D., Savage, P.B., Bendelac, A., from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the Wilson, I.A., Teyton, L. Structure and function of a potent agonist for the semi- cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006. invariant natural killer T cell receptor. Nat. Immunol. 6:810, 2005. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 165

Zajonc, D.M., Maricic, I., Wu, D., Halder, R., Roy, K., Wong, C.-H., Kumar, V., Wilson, I.A. Structural basis for CD1d presentation of a sulfatide derived from myelin and its implications for autoimmunity. J. Exp. Med. 202:1517, 2005.

Zhang, Y., Wang, L., Schultz, P.G., Wilson, I.A. Crystal structures of apo wild-type M jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine. Protein Sci. 14:1340, 2005.

Zhu, X., Dickerson, T.J., Rogers, C.J., Kaufmann, G.F., Mee, J.M., McKenzie, K.M., Janda, K.D., Wilson, I.A. Complete reaction cycle of a cocaine catalytic antibody at atomic resolution. Structure 14:205, 2006.

Zhu, X., Wentworth, P., Jr., Kyle, R.A., Lerner, R.A., Wilson, I.A. Cofactor-containing antibodies: crystal structure of the original yellow antibody. Proc. Natl. Acad. Sci. U. S. A. 103:3581, 2006.

Structure and Function of Proteins as Molecular Machines

E.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau, Fig. 1. The NADPH-binding site in the crystallographic structure of R.M. Brudler, T.H. Cross, E.D. Garcin-Hosfield, C. Hitomi, the neuronal NOS reductase module. A triad of amino acid residues K. Hitomi, C.J. Kassmann, M.E. Pique, M.E. Stroupe, conserved in NOS reductases and homologous flavoproteins (Tyr1322, Ser1313, and Arg1314) stabilize the 2′ phosphate group (2′P) that J.L. Tubbs, T.I. Wood distinguishes NADPH from NADH. In contrast, Arg1400 is specific to ur goals are to understand how proteins func- the calcium-regulated neuronal and endothelial NOS enzymes, in which tion as molecular machines. We use structural, it performs a isozyme-specific regulatory function. In the absence of calcium-bound calmodulin, Arg1400 helps stabilize the regulato- molecular, and computational biology to study O ry C-terminal tail, inhibiting nonproductive electron transfer. proteins of biological and biomedical interest, especially proteins that work synergistically with coupled chromo- integrated biochemical data with our structures of NOS phores, metal ions, or other cofactors. oxygenase, NOS reductase, and calmodulin in complex NITRIC OXIDE SYNTHASES with peptides derived from NOS to propose a model To synthesize nitric oxide, a cellular signal and for the assembled holoenzyme that provides a moving- defensive cytotoxin, nitric oxide synthases (NOSs) domain mechanism for electron flow from NOS reductase require calmodulin-orchestrated interactions between to the NOS oxygenase heme. Preliminary small-angle their catalytic, heme-containing oxygenase module and x-ray scattering measurements in solution provide molec- their electron-supplying reductase module. Crystallo- ular envelopes for NOS proteins that support our model. graphic structures of wild-type and mutant NOS oxy- Our model also explains how regulatory site-specific genase dimers with substrate, intermediate, inhibitors, phosphorylation and dephosphorylation activate and cofactors, and cofactor analogs, determined in collabo- inactivate nitric oxide synthesis in vivo. ration with J. Tainer, Department of Molecular Biology, PHOTOACTIVE PROTEINS AND CIRCADIAN CLOCKS and D. Stuehr, the Cleveland Clinic, Cleveland, Ohio, To understand in atomic detail how proteins trans- provided insights into the catalytic mechanism and late sunlight into defined conformational changes for dimer stability. biological functions, we are exploring the reaction Our structure-based drug design projects are aimed mechanisms of the blue-light receptors photoactive at selectively inhibiting inducible NOS, to prevent inflam- yellow protein (PYP), photolyase, and cryptochrome. matory disorders, or neuronal NOS, to prevent migraines, PYP is the prototype for the Per-Arnt-Sim domain pro- while maintaining blood pressure regulation by endothe- teins of circadian clocks, whereas proteins of the pho- lial NOS. Our structure of the neuronal NOS reductase tolyase and cryptochrome family catalyze DNA repair has provided news insights into the complex regulatory or act in circadian clocks. To understand the protein mechanisms of this enzyme family. photocycle, we combined ultra-high-resolution and We have designed and assayed site-directed mutant time-resolved crystallographic structures of the dark enzymes that support our mechanistic hypotheses for state and 2 photocycle intermediates of PYP with site- isozyme-specific inhibition and regulation (Fig. 1). We directed mutagenesis; ultraviolet-visible spectroscopy; 166 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE time-resolved Fourier transform infrared spectroscopy; targeting to specific cellular locations. By completing deuterium-hydrogen exchange mass spectrometry, in the metalloprotein design cycle from prediction to highly collaboration with V. Woods, University of California, accurate structures, we can rigorously evaluate and San Diego; and quantum mechanical and electrostatic improve algorithms for the design of metal sites. computational methods, in collaboration with L. Noodle- In related research, we discovered that the architec- man, Department of Molecular Biology. ture of GFP and RFP promotes a remarkable range of Cryptochrome flavoproteins are homologs of light- posttranslational modification chemistry. High-resolu- dependent DNA repair photolyases that function as blue- tion crystallographic structures of GFP and RFP inter- light receptors in plants and as components of circadian mediates in fluorophore cyclization and oxidation lead clocks in animals. We determined the first crystallo- to a novel mechanism for the spontaneous synthesis of graphic structure of a cryptochrome, which revealed this tripeptide fluorophore within the protein scaffold. commonalities with photolyases in DNA binding and Remarkably, the same protein architectural features redox-dependent function but showed differences in that favor peptide cyclization can drive peptide hydrol- active-site and interaction-surface features. Recently, we ysis (Fig. 2) and red shift the spectral properties of the showed that this cryptochrome binds the same antenna cofactor found in a photolyase homolog but uses different residues for the cofactor-binding site. New structures of photolyases from 2 other branches of the photolyase/cryptochrome family that repair cyclobutane pyrimidine dimers and photoproducts help us decipher the cryptic structure, function, and evolutionary relationships of these fascinating redox-active proteins. We are also studying clock proteins with PYP-like and Per-Arnt-Sim domains that bind to mammalian cryptochromes. Our goal is to determine the detailed chemistry and atomic structure of these proteins, define their mechanisms of action and interaction, and use our results to understand their biological function and regulation. PROTEIN DESIGN AND POSTRANSLATIONAL MODIFICATION CHEMISTRY An ultimate goal for protein engineers is to design and construct new protein variants with desirable catalytic or physical properties. As members of the Scripps Research Metalloprotein Structure and Design Group, we are testing our understanding of affinity, selectivity, and activity of metal ions by transplanting metal sites from structurally characterized metalloproteins into new protein scaffolds. To aid our design efforts, we have organized quantitative information and interactive viewing of protein metal sites at the Metalloprotein Database and Browser (available at http://metallo.scripps.edu). Fig. 2. Spontaneous peptide hydrolysis and decarboxylation For green fluorescent protein (GFP) and the homolo- reactions promoted by the protein architecture of GFP. A, Crystallo- gous red fluorescent protein (RFP), we designed, con- graphic structure of a designed GFP variant reveals peptide-bond structed, and characterized metal-ion biosensors, in cleavage and decarboxylation chemistry at the site of GFP fluoro- which binding of metal ions is signaled by changes in phore synthesis. S65G and Y66S mutations converted the fluorophore tripeptide SYG sequence to GSG. The simulated annealing spectroscopic properties of the naturally occurring flu- omit electron density map (mesh) clearly shows the resultant break orophores. Use of GFP allows optimization with random in the polypeptide chain at this site. B, Corresponding reaction and mutagenesis, noninvasive expression in living cells, and posttranslational products for this self-cleaving GFP variant. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 167 chromophore. Decarboxylation reactions in designed vari- potential for structure-based design of inhibitors relevant ants of GFP (Fig. 2) support a role for the GFP environ- to the development of novel therapeutic agents and ment in facilitating formation of radicals and 1-electron chemical tools, and structural implications by biochem- chemistry. Together, our results provide the ground- istry and mutagenesis. For DNA repair, we collaborate work for the design of proteins with novel catalytic or with P. Russell and N. Boddy, Department of Molecular reporter properties. Biology, to couple our structures with genetics and phenotypes. For protein design, we collaborate with E. Get- PUBLICATIONS Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP zoff, Department of Molecular Biology, to understand and posttranslational chemistry: structures of designed variants that achieve backbone control the formation of self-synthesizing chromophores fragmentation, hydrolysis, and decarboxylation. J. Am. Chem. Soc. 128:4685, 2006. in green fluorescent protein and its homologs.

Barondeau, D.P., Tainer, J.A., Getzoff, E.D. Structural evidence for an enolate inter- PROTEIN MODIFICATIONS AND FUNCTION mediate in GFP fluorophore biosynthesis. J. Am. Chem. Soc. 128:3166, 2006. The finding that the number of protein-coding genes Brudler, R., Gessner, C.R., Li, S., Tyndall, S., Getzoff, E.D., Woods, V.L., Jr. PAS in the human genome is more than 10-fold lower than domain allostery and light-induced conformational changes in photoactive yellow protein upon I2 intermediate formation, probed with enhanced hydrogen/deuterium the number of proteins found in human cells by the exchange mass spectrometry. J. Mol. Biol. 363:148, 2006. Human Genome Project is surprising. This huge increase Panda, K., Haque, M.M., Garcin-Hosfield, E.D., Durra, D., Getzoff, E.D., Stuehr, in protein diversity must primarily be due to alternative D.J. Surface charge interactions of the FMN module governs catalysis by nitric- splicing and posttranslational modification of proteins. oxide synthase. J. Biol. Chem., in press. A particularly important and intriguing posttranslational Stroupe, M.E., Getzoff, E.D. The role of siroheme in sulfite and nitrite reductases. In: Tetrapyrroles. Warren, M.J., Smith, A. (Eds.). Landes Bioscience, Georgetown, modification is the spontaneous peptide backbone cycliza- TX, in press. tion and oxidation chemistry required to convert 3 amino

Tiso, M., Konas, D.W., Panda, K., Garcin, E.D., Sharma, M., Getzoff, E.D., acids into a fluorophore for the family of green fluores- Stuehr, D.J. C-terminal tail residue Arg1400 enables NADPH to regulate electron cent proteins. transfer in neuronal nitric-oxide synthase. J. Biol. Chem. 280:39208, 2005. REACTIVE OXYGEN AND XENOBIOTIC CONTROL Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff, ENZYMES E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry 44:16211, 2005. Superoxide dismutases and nitric oxide synthases are master regulators for reactive oxygen species involved in injury, pathogenesis, aging, and degenerative diseases. We are characterizing the hydrogen-bonding networks Structural Biology of Molecular that underlie the activity of mitochondrial manganese Interactions and Design superoxide dismutases. For human copper, zinc superoxide dismutase, we are probing how single-site muta- J.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras, tions cause the neurodegeneration in Lou Gehrig disease B.R. Chapados, L. Craig, T.H. Cross, L. Fan, C. Hitomi, or familial amyotrophic lateral sclerosis. For nitric oxide K. Hitomi, J.L. Huffman, C.J. Kassmann, I. Li, G. Moncalian, synthases, we are examining the structure and chem- M.E. Pique, D.S. Shin, O. Sundheim, R.S. Williams, istry that control levels of nitric oxide, which acts as T.I. Wood, A. Yamagata an important signal and cytotoxin with implications for inflammatory and neurodegenerative diseases. ur studies reveal overall themes and common DNA REPAIR AND GENETIC EVOLUTION relationships for fundamental principles and All the information for heredity is encoded in DNA O processes of protein regulators and effectors of molecules that are constantly under attack from sun- DNA damage responses, reactive oxygen species, and light, ionizing radiation, and other environmental car- pathogenesis. We combine x-ray crystallography and cinogens. Surprisingly, however, most DNA damage is solution small-angle x-ray scattering methods, often at due to chemical reactions and free radicals that arise our advanced synchrotron facility SIBLYS, to gain a from normal cellular metabolism that is necessary for clear view of the structural chemistry that drives biology. life. Thus, paradoxically, life is impossible even in the Further fusing these techniques with electron micros- absence of environmental toxins unless coupled to DNA copy, we bridge the size gaps between high-resolution repair. Mutations that cause defects in DNA repair macromolecular structures and lower resolution multi- systems may cause cancer and degenerative diseases protein machine complexes. We then investigate the associated with aging, but fortunately the mutations associated dynamic reversible interactions within cells, can also be exploited for cancer therapy. 168 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

AGING AND THE WRN STRUCTURE Mutation of the DNA repair protein WRN can give rise to Werner syndrome, which is characterized by rapid aging and cancer disorders. We have characterized the structure of the WRN nuclease component (Fig. 1).

Fig. 2. Conserved XPB helicase core and DNA-induced open-to- closed conformational changes. XPB contains 4 conserved functional domains: the damage recognition domain (DRD), 2 helicase domains (HD1 and HD2), and a thumb insert (ThM). The interaction of the helicase with DNA may induce a rotation of about 170° of domain HD2 and ThM to form the closed conformation as observed in the crystal structure of hepatitis C virus (HCV) NS3 helicase bound to a single-stranded DNA.

and Neisseria meningitidis.Pili play key roles in sur- Fig. 1. Hexameric ring model for the WRN nuclease (WRN exo) face motility, adhesion, formation of microcolonies and component. A, WRN x-ray crystal structures aligned as a ring by biofilms, natural transformation, and signaling. We are homology comparisons. B, DNA processing is altered in the WRN characterizing structures of type IV pilin subunits: the Trp145A mutant. C, Electron density map (3σ, 5σ) of dGMP bound assembled pilus fiber, the pilus membrane protein part- to WRN exo. D, The similar internal and external dimensions of Ku70/80 (left) and the WRN exo hexamer model (right). ners, and the assembly ATPase. Pili induce a calcium influx in host cells that plays a role in pathogenesis by This component is an editing nuclease resembling those altering endocytic trafficking and lysosome homeosta- found in DNA polymerases. Furthermore, the editing sis in infected cells. Because calcium is a central sec- of DNA ends by the WRN exonuclease is stimulated ond messenger that regulates several signal cascades, for broken DNA end joining by the Ku DNA end-binding pilus-induced calcium bursts most likely influence complex. Our findings suggest how the editing of DNA bacterial infectivity in key ways. For infections caused ends during DNA damage responses can critically affect by N meningitidis, these calcium bursts are expected aging and carcinogenesis. to activate neuronal nitric oxide synthases, resulting in NUCLEOTIDE EXCISION REPAIR toxic levels of nitric oxide that may in part explain the Nucleotide excision repair, a critical defense mecha- fatal effects of N meningitidis infections of the brain. nism that removes DNA lesions caused by the mutational effects of sunlight (ultraviolet radiation) and toxic PUBLICATIONS chemicals, is also central to the success of anticancer Ayala, I., Perry, J.P., Szczepanski, J., Tainer, J.A., Vala, M.T., Nick, H.S., Silver- man, D.N. Hydrogen bonding in human manganese superoxide dismutase contain- drugs such as cisplatin. We have focused on understanding 3-fluorotyrosine. Biophys. J. 89:4171, 2005. ing the mechanisms of nuclear excision repair for poten- Ayala, P., Wilbur, J.S., Wetzler, L.M., Tainer, J.A., Snyder, A., So, M. The pilus tial improvements in cancer treatment. We determined and porin of Neisseria gonorrhoeae cooperatively induce Ca2+ transients in infected epithelial cells. Cell. Microbiol. 7:1736, 2005. the crystal structure of an enzyme called xeroderma pigmentosum group B (XPB) helicase (Fig. 2). We found Barondeau, D.P., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone several unexpected functions of XPB helicase in nuclear fragmentation, hydrolysis, and decarboxylation. J. Am. Chem. Soc. 128:4685, 2006. excision repair. These findings helped us address impor- Barondeau, D.P., Tainer, J.A., Getzoff, E.D. Structural evidence for an enolate tant questions about the enzyme’s role in DNA tran- intermediate in GFP fluorophore biosynthesis. J. Am. Chem. Soc. 128:3166, scription and repair. XPB helicase recognizes DNA 2006. damage that causes blockages in reading the DNA code Craig, L., Volkmann, N., Arvai, A.S., Pique, M.E., Yeager, M., Egelman, E.H., and aids initiation of efficient repair. Tainer, J.A. Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. Mol Cell. 23:651, 2006. BACTERIAL PILI AND INFECTIOUS DISEASES Type IV pili are essential virulence factors for many Doi, Y., Katafuchi, A., Fujiwara, Y., Hitomi, K., Tainer, J.A., Ide, H., Iwai, S. Synthe- sis and characterization of oligonucleotides containing 2′-fluorinated thymidine glycol gram-negative bacteria, such as Neisseria gonorrhoeae as inhibitors of the endonuclease III reaction. Nucleic Acids Res. 34:1540, 2006. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 169

Fan, L., Arvai, A., Cooper, P.K., Iwai, S., Hanaoka, F., Tainer, J.A. Conserved XPB overproducing integral membrane proteins suitable for core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair. Mol. Cell 22:27, 2006. biophysical study. We use several experimental methods, including detergent/lipid protein biochemistry, 3-dimen- Fan, L., Kim, S., Farr, C.L., Schaefer, K.T., Randolph, K.M., Tainer, J.A., Kaguni, L.S. A novel processive mechanism for DNA synthesis revealed by structure, mod- sional crystallization of integral membrane proteins, eling and mutagenesis of the accessory subunit of human mitochondrial DNA polyprotein x-ray crystallography, and functional analysis merase. J. Mol. Biol. 358:1229, 2006. of transporters. Fan, L., Perry, J.J.P., Tainer, J.A. Reactive oxygen control and DNA repair struc- We are addressing the molecular basis of MDR in tural biology: implications for aging and neuropathology. Neuroscience, in press. the treatment of infectious disease and cancer. A major Hitomi, K., Iwaia, S., Tainer, J.A. The intricate structural chemistry of base exci- cause of MDR is drug efflux pumps imbedded in the cell sion repair machinery: implications for DNA damage recognition, removal, and repair. DNA Repair (Amst.), in press. membrane. Through our structural studies on MDR

Ivanov, I., Chapados, B.R., McCammon, J.A., Tainer, J.A. Proliferating cell nuclear transporters, we are gaining insights into the molec- antigen loaded onto double-stranded DNA: dynamics, minor groove interactions ular mechanics of translocating amphipathic substrates and functional implications. Nucleic Acids Res. in press. across the cell membrane and the rational design of Pascal, J.M., Tsodikov, O.V., Hura, G.L., Song, W., Cotner, E.A., Classen, S., powerful inhibitors. Tomkinson, A.E., Tainer, J.A., Ellenberger, T. A flexible interface between DNA ligase and PCNA supports conformational switching and efficient ligation of DNA. We are combining chemistry and biology with struc- Mol. Cell. 24:279-91, 2006. ture for the discovery and design of potent MDR reversal

Perry, J.J.P., Yannone, S.M., Holden, L.G., Hitomi, C., Asaithamby, A., Han, S., agents for cancer chemotherapy in collaboration with Cooper, P.K., Chen, D.J., Tainer, J.A. WRN exonuclease structure and molecular M.G. Finn, Department of Chemistry; I. Urbatsch, Texas mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 13:414, 2006. Tech University Health Sciences Center, Lubbock, Texas; and S. Reutz, Novartis International AG, Basel, Switzer- Putnam, C.D., Hura, G.L., Tainer, J.A. Combining x-ray solution and crystal diffraction and scanning force microscopies to characterize reversible macromolecular land. In collaboration with M. Saier, University of Califor- interactions and conformational states. Q. Rev. Biophys., in press. nia, San Diego, and Q. Zhang, Department of Molecular Putnam, C.D., Tainer, J.A. Protein mimicry of DNA and pathway regulation. DNA Biology, we are probing the structures and function of Repair (Amst.) 4:1410, 2005. bacterial MDR transporters. In a collaboration with Sundheim, O., Vågbø, C.B., Bjørås, M., de Sousa, M.M.L., Talstad, V., Aas, P.A., R.A. Milligan, Department of Cell Biology, we are using Drabløs, F., Krokan, H.E., Tainer, J.A., Slupphaug, G. Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage. EMBO J. electron cryomicroscopy to visualize the low-resolution 25:3389, 2006. structures of our transporters.

Tsutakawa, S., Tainer, J.A. Combined methods of SAXS and crystallography to char- Recently, we determined the x-ray structure of an acterize dynamic protein conformations at atomic resolution. J. Struct. Biol., in press. MDR transporter called EmrD. EmrD is from the Major

Wood, T.I., Barondeau, D.P., Hitomi, C., Kassmann, C.J., Tainer, J.A., Getzoff, Facilitator Superfamily, and it expels amphipathic com- E.D. Defining the role of arginine 96 in green fluorescent protein fluorophore pounds across the inner membrane of E coli. The struc- biosynthesis. Biochemistry 44:16211, 2005. ture reveals an interior that is composed mostly of hydrophobic residues, a finding consistent with the role of EmrD in transporting amphipathic molecules. Two Structural Biology of Integral long loops extend into the inner leaflet side of the cell Membrane Proteins membrane. This region can recognize and bind substrate directly from the lipid bilayer. We propose that multi- substrate specificity, binding, and transport are facili- G. Chang, S. Aller, A. Chen, Y. Chen, X. He, A. Karyakin, tated by these loop regions and the internal cavity. C.R. Reyes, P. Szewczyk, A. Ward, S. Wada, J. Yu, Y. Yin

he structural biology of integral membrane proteins PUBLICATIONS Yin, Y., He, X., Szewczyk, P., Nguyen, T., Chang, G. Structure of the multidrug is an exciting frontier.We are interested in 5 areas: transporter EmrD from Escherichia coli. Science 312:741, 2006. T (1) the molecular structural basis for lipid and drug transport across the cell membrane by multidrug- resistance (MDR) transporters, (2) the high-resolution structure of yeast and mammalian MDR transporters, (3) signal transduction by receptors, (4) the discovery and design of potent MDR reversal agents, and (5) the development of an in vitro cell-free system capable of 170 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE Structure and Function of In collaboration with J.A. Fee, Department of Molec- ular Biology, we are studying the mechanism of action

Membrane-Bound Enzymes of cytochrome ba3 oxidase, the terminal enzyme of respiration. The high-resolution structure of the enzyme C.D. Stout, H. Heaslet, M. Yamaguchi, V.M.M. Luna, from Thermus thermophilus, crystallized in the pres- A. Annalora, J. Chartron, V. Sundaresan ence of a detergent, has been determined. Crystallo- graphic experiments, in concert with mutagenesis and e focus on the structure and function of mem- spectroscopy, can be used to visualize intermediates in brane-bound enzymes and the development of the reduction of oxygen to water and to define the paths W methods for crystallizing membrane proteins. of oxygen molecules and protons into the active site. We study the mechanism of transhydrogenase, a mito- In collaboration with E.F. Johnson, Department of chondrial respiratory enzyme complex that couples proton Molecular Biology; J.R. Halpert, University of Texas translocation with hydride transfer. We use x-ray crys- Medical Branch, Galveston, Texas; and others, we are tallography, biochemical and spectroscopic methods, characterizing structures of mammalian cytochrome electron microscopy studies in collaboration with M. P450s. These membrane-associated enzymes are involved Yeager, Department of Cell Biology, and nuclear mag- in the biosynthesis of lipophilic hormones and specifi- netic resonance studies in collaboration with J. Dyson, cally metabolize a remarkable diversity of exogenous Department of Molecular Biology. Crystal structures of compounds and drugs. More than 60 genes for P450 transhydrogenase soluble domains, alone and in com- occur in the human genome. High-resolution structures, plex, have been determined (Fig. 1). Currently, our pri- including substrate and inhibitor complexes, have been determined for the P450s 1A2, 2C5, 2C8, 2C9, 2A6, 2A13, 3A4, and 2B4. For 2B4, 3 structures of the enzyme in markedly different conformations provide insight to substrate binding and membrane insertion. A major effort to determine the basis of HIV resistance to antiviral drugs is ongoing in collaboration with A.J. Olson and J.H. Elder, Department of Molecular Biology; B.E. Torbett, Department of Molecular and Experimental Medicine; and D.E. McRee, ActiveSight, San Diego, California. One aspect of this project entails determining the crystal structure of HIV protease-resistant mutants in complex with a wide range of inhibitors. Fig. 1. Superposition of 3 heterotrimers of transhydrogenase solu- Additional research projects involve crystallographic col- ble domains observed in cocrystals. The presence of additional copies laborations on iron-sulfur enzymes, with K.S. Carroll, of the smaller soluble domain (dIII, lower right) in the crystal lattice provides a possible model for the intact enzyme in the membrane. University of Michigan, Ann Arbor, Michigan; electron transfer proteins, with J.A. Fee, Department of Molec- mary effort is to determine the structure of the intact ular Biology; and synthetic self-assembling peptides, 200-kD enzyme in its membrane-bound configuration. with M.R. Ghadiri, Department of Chemistry. We are developing applications of nanodiscs for biophysical studies of integral membrane proteins in collab- PUBLICATIONS Chartron, J., Carroll, K.S., Shiau, C., Gao, H., Leary, J.A., Bertozzi, C.R., Stout, oration with P. Dawson, Department of Cell Biology, and C.D. Substrate recognition, protein dynamics, and novel iron-sulfur cluster in S.G. Sligar, University of Illinois, Urbana-Champaign, Pseudomonas aeruginosa adenosine 5′-phosphosulfate reductase. J. Mol. Biol. 364:152, 2006. Illinois. Nanodiscs are composed of phospholipid-binding peptides that self-assemble into discrete, water-soluble, Heaslet, H., Kutilek, V., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout, C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease bilayer-containing particles. Integral membrane proteins NL4-3. J. Mol. Biol. 356:967, 2006. incorporated into these particles retain their enzymatic Hillier, B.J., Sundaresan, V., Stout, C.D., Vacquier, V.D. Expression, purification, activity, are amenable to biochemical assays, and may crystallization and preliminary x-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin. Acta Crystallogr. Sect. F Struct. Biol. have superior properties for crystallization in the absence Cryst. Commun. 62(Pt. 1):16, 2006. of detergents. Both transhydrogenase and cytochrome Johnson, E.F., Stout, C.D. Structural diversity of human xenobiotic-metabolizing cyto- ba3 oxidase have been incorporated into nanodiscs. chrome P450 monooxygenases. Biochem. Biophys. Res. Commun. 338:331, 2005. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 171

Yadav, M.K., Leman, L.J., Price, D.J., Brooks, C.L. III, Stout, C.D., Ghadiri, M.R. detailed mechanisms whereby the oxygen molecule is Coiled coils at the edge of configurational heterogeneity: structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a sin- reduced to 2 water molecules. The outstanding questions gle solvent-exposed amino acid substitution. Biochemistry 45:4463, 2006. pertain to the flow of protons into the enzyme from its

Zhao, Y., White, M.A., Muralidhara, B.K., Sun, L., Halpert, J.R., Stout, C.D. in side, across the hydrophobic core of the membrane, Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug to exit on its out side. The mystery lies in how all this bifonazole: insight into P450 conformational plasticity and membrane interaction. J. Biol. Chem. 281:5973, 2006. scalar chemistry comes together to “pump” 4 protons across the membrane. Although the enzyme has been examined by using virtually every available spectroscopic Cytochrome ba3 From technique, no one has addressed directly the pathways of those protons becoming either water or part of the Thermus thermophilus: New proton gradient. Windows on the Mechanisms Much of the past work was done with enzymes in a single clade typified by the mitochondrial enzyme (derived of Energy Transduction by from an ancient bacterium) and with enzymes isolated from common bacteria, notably Rhodobacter sphaeroides, Cytochrome c Oxidases Paraccocus denitrificans, and Escherichia coli (the quinol oxidase). The enzymes from these sources are J.A. Fee, Y. Chen highly similar in amino acid sequence, 3-dimensional relatively small integral-membrane protein con- structure, electron-transfer paths, mechanism of oxy- taining 2 iron and 3 copper atoms distributed in gen reduction, and, most likely, mechanisms of proton A 3 redox active sites generates approximately one pumping. Our research is based on a 1988 description third of a human’s metabolic energy. That enzyme is of a highly sequence-divergent form of the enzyme from cytochrome c oxidase, and its mechanism of action Thermus thermophilus, cytochrome ba3, that represents remains a mystery. The enzyme was first recognized by a distinct clade of enzymes widely distributed among Charles MacMunn as “histohaematin” in the 1880s and archaebacteria. Respectively, these clades represent was studied intensely by Otto Warburg as “atmungs- A- and B-type oxidases. ferment” during the 1920s and 1930s and by David We recently developed a homologous expression Keilin as “cytochrome” into the late 1950s. Today, system for cytochrome ba3. This system allows easy cytochrome c oxidase is still the subject of an inter- purification of the enzyme in amounts of 2–3 mg/L of national effort. culture medium by using an N-terminal heptahistidine Cytochrome c oxidase catalyzes the following decep- tag on subunit I. The recombinant enzyme is equally tively simple reaction: active with native, wild-type protein, and the results of a 2.3-Å x-ray structure determination, done in col- 2+ + laboration with C.D. Stout, Department of Molecular 4 cytochrome c + O2 + 8 H in → 4 cytochrome 3+ + Biology, revealed the expected details at full occupancy c + 2 H2O + 4 H out, and at least one notable surprise. where the subscripts in and out refer, respectively, All the A-type oxidases have a glutamic acid residue to matrix and the intermembrane space of the mitochon- within the hydrophobic interior of the enzyme that is drion or, in bacteria, to the cytoplasm and the periplas- thought to be at the “end” of the D pathway of proton mic space. The free energy of dioxygen reduction is thus transport and close to the Fea3-CuB site of dioxygen captured as a proton gradient; the out side is positive reduction (Fig. 1A). Mutation of this residue to, for exam- and the in side is negative. ple, glutamine blocks all but a small percentage of the During the past 5 decades, enormous progress has enzyme’s electron-transfer activity, and infrared studies been realized in understanding the chemical properties indicate that this residue “senses” changes in the chemi- of the 3 redox centers, and the enzyme from several cal structure of the dioxygen reduction site. Indeed, sci- different sources has been crystalized and its structure entists think that glutamate 286 actually donates the determined at resolutions ranging from about 3 to 1.8 Å. pumped proton to the exit part of the molecule, becom- Moreover, much has been learned about the pathways ing deprotonated with a pKa of about 9.4. However, of electron transfer within the enzyme and the no direct evidence exists for this notion, and because 172 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Hunsicker-Wang, L.M., Pacoma, R.L., Chen, Y., Fee, J.A., Stout, C.D. A novel cryoprotection scheme for enhancing the diffraction of crystals of recombinant cytochrome ba3 oxidase from Thermus thermophilus. Acta Crystallogr. D Biol. Crystal- logr. 61(Pt. 3):340, 2005.

Developing Reagents for Stabilization of Membrane Proteins

Q. Zhang, M.G. Finn,* X. Ma, R.S. Roy * Department of Chemistry, Scripps Research

ntegral membrane proteins are extremely unstable outside the hydrophobic membrane bilayer, a situa- I tion that makes their in vitro biophysical and structural characterization difficult. An artificial environment is therefore needed to stabilize the proteins in their Fig. 1. Cross-eyed stereo view of the Fea3-CuB binuclear active- native state. We focus on developing new membrane- site structures (leftmost Fe and Cu) of the cytochrome aa3 from R simulating reagents for the stabilization of membrane sphaeroides (A) and the cytochrome ba from T thermophilus (B), 3 proteins for structural and functional studies. emphasizing the overlapping position of the glutamate 286 (Glu- 238) in R sphaeroides and the isoleucine 235 (Ile-235) in T ther- Detergents, structurally similar to cell lipids, self- mophilus. Under the influence of oxygen reduction, protons most assemble into micellar structures and are indispensable likely enter the protein from the upper left of the figure and exit at in dissolving integral membrane proteins into single the lower right. particles to facilitate protein crystallization. We intend to incorporate more hydrophobicity in the interior of glutamate 286 resides in a highly hydrophobic region of detergent micelles to improve their stability and con- the structure, its pKa most likely is much higher. sequently their ability to stabilize integral membrane The surprise in the structure of cytochrome ba3 is proteins. This change is accomplished by appending that an isoleucine residue is isopositional with gluta- branches along the alkyl chains of detergents and, most mate 286 as isoleucine 235, as shown in Fig. 1B. We interestingly, by adding a short branch at the interface mutated this residue to both a glutamine and a gluta- between the hydrophobic tail and the hydrophilic head. mate residue in the cytochrome with no apparent loss These branches may behave in 2 distinct ways like small of electron-transfer activity. To determine if the substi- amphiphile additives successfully used in crystallization tuted glutamate residue can be used to monitor changes of integral membrane proteins, thereby decreasing the in the Fea3-CuB pair, as it does in the A-type oxidases, micellar radius and extruding water from the hydro- we have initiated an infrared study of these mutant pro- phobic core of the micelles. teins in collaboration with R. Gennis, University of Illi- We are also working on a unique class of molecules nois, Urbana-Champaign, Illinois, and J. Heberle, Jülich with facial amphiphilicity. The facial amphiphiles are Research Center, Jülich, Germany. How these studies structurally distinct from the classical detergents that will advance our understanding of proton-pumping have end polarity. Although not clear, the binding mode mechanisms remains unclear. What is clear is that with integral membrane proteins by the facial amphi- cytochrome ba3 provides new openings to explore the philes should differ from that of classical detergents. mechanism of the cytochrome oxidases. A smaller protein–facial amphiphile complex may be formed because of the amphiphile’s small aggregation PUBLICATIONS Chen, Y., Hunsicker-Wang, L.M., Pacoma, R.L., Luna, E., Fee, J.A. A homologous number, which is expected to be beneficial for obtain- expression system for obtaining engineered cytochrome ba3 from Thermus ther- ing well-ordered protein crystals. We have shown that mophilus HB8. Protein Expr. Purif. 40:299, 2005. our newly designed facial amphiphiles can maintain Farver, O., Chem, Y., Fee, J.A., Pecht, I. Electron transfer among the CuA-, heme the full catalytic function of an ATP-binding cassette b- and a3-centers of Thermus thermophilus cytochrome ba3. FEBS Lett. 580:3417, 2006. transporter protein. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 173

The structural determination of integral membrane second center is the Accelerated Technologies Center proteins with our synthesized amphiphiles is being inves- for Gene to 3D Structure (http://www.atcg3d.org). Here tigated in collaboration with members of the Center for we are doing collaborative work with Dr. Kuhn and with Innovative Membrane Protein Technologies of the Joint researchers from deCODE biostructures, Bainbridge Center for Structural Genomics at Scripps Research. Island, Washington; Lyncean Technologies, Palo Alto, California; and the University of Chicago, Chicago, Illi- PUBLICATIONS Bosco, D.A., Fowler, D.M., Zhang, Q., Nieva, J., Powers, E.T., Wentworth, P., Jr., nois. In 2005, scientists at the centers showed that Lerner, R.A., Kelly, J.W. Elevated levels of oxidized cholesterol metabolites in Lewy high-resolution electron density maps and refined models body disease brains accelerate α-synuclein fibrillization [published correction appears in Nat. Chem. Biol. 2:346, 2006]. Nat. Chem. Biol. 2:249, 2006. can be obtained from in situ diffraction of crystals grown in microcapillaries. In 2007, the first laboratory-sized synchrotron will be installed at Scripps Research. The Structural Neurobiology and synchrotron has performance characteristics comparable to those of a synchrotron beam line in terms of Development of Protein intensity and tunability and will enable us to use direct diffraction analysis of ongoing in situ crystallization Therapeutic Agents experiments to accelerate the determination of macromolecular structures. R.C. Stevens, E.E. Abola, A.I. Alexandrov, H.M. Archer, STRUCTURAL NEUROBIOLOGY J.W. Arndt, G.A. Asmar-Rovira, R.R. Benoit, M.H. Bracey, Although we have developed high-throughput meth- A. Brooun, Q. Chai, V.G. Cherezov, E. Chien, A. Gámez, ods to accelerate the determination of protein structures, M.T. Griffith, C. Grittini, M.A. Hanson, V.-P. Jaakola, J. Joseph, our primary interest is using these tools to study the K. Masuda, M. Mileni, K. Moy, M. Nelson, C. Roth, chemistry and biology of neurotransmission and of K. Saikatendu, V. Subramanian, J. Velasquez, L. Wang, diseases that affect neurons, particularly childhood M.K. Yadav neurologic disorders. Our goals are to understand how HIGH-THROUGHPUT STRUCTURAL BIOLOGY neuronal cells function on a molecular level and, on ut of frustration with the rate at which informa- the basis of that understanding, create new molecules tion on structural biology became known in the and materials that mimic neuronal signal transduction O past, we focused on developing new tools to and recognition. change the field of structural biology by accelerating the BIOSYNTHESIS OF NEUROTRANSMITTERS rate of determination of protein structures. This endeavor For neuronal signal transduction, the presynaptic cell included pioneering microliter expression/purification synthesizes neurotransmitters that then traverse the for structural studies, nanovolume crystallization, auto- synaptic cleft. We are using the high-throughput meth- mated collection of images, and synchrotron beam line ods to determine the inclusive structures of complete automation. These technologies were initially tested biochemical pathways. Specifically, we are interested by staff at the Joint Center for Structural Genomics in determining the structures of all the enzymes in the (htpp://www.jcsg.org), where the power of the new biosynthesis pathways of neurotransmitters in order to tools was demonstrated. Although the Joint Center for understand the mechanistic details of each individual Structural Genomics 2 has continued as a successful enzymatic reaction at the atomic level. This approach second-phase structural genomics production center, also allows us to determine the best path of drug dis- in collaboration with P. Kuhn, Department of Cell Biol- covery for the biosynthesis of neurotransmitters. ogy, we have created 2 new technology-focused centers THERAPEUTIC AGENTS FOR TREATMENT OF funded by the National Institutes of Health. PHENYLKETONURIA The first center is the Joint Center for Innovative Mem- In addition to the basic hydroxylase enzymology brane Protein Technologies (http://jcimpt.scripps.edu). questions under investigation, recent clinical studies Here, in collaboration with K. Wüthrich, Q. Zhang, and suggest that some patients with the metabolic disease G. Chang, Department of Molecular Biology; M.G. Finn, phenylketonuria are responsive to (6R)-L-erythro-5,6,7,8- Department of Chemistry; and P. Kuhn and M. Yeager, tetrahydrobiopterin, the natural cofactor of phenylala- Department of Cell Biology, we do research exclusively nine hydroxylase. We are doing studies in collaboration on eukaryotic and prokaryotic membrane proteins. The with scientists at BioMarin Pharmaceutical Inc., Novato, 174 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

California, to correlate how structure can be used to predict which patients with phenylketonuria most likely will respond to treatment with this cofactor. Phase 3 clinical trials for the treatment of mild phenylketonuria with the proprietary form of the cofactor, Phenoptin, have been completed. For classical phenylketonuria, we are developing an enzyme replacement therapeutic agent that is being tested in animal models. The therapy is based on administration of a modified form of phenylalanine ammonia lyase discovered in our structural studies (Fig. 1). Last,

Fig. 1. A, Crystal structure of phenylalanine ammonia lyase (PAL) determined at 1.6-Å resolution. This protein structure was engineered and chemically modified as a once-a-week injectable Fig. 2. Serotype structures of botulinum neurotoxin (BoNT), its therapeutic agent for treatment of phenylketonuria. B, ENU2 mice light chain (LC), the closely related tetanus neurotoxin (TeNT), and are used as a model for phenylketonuria in preclinical studies. C the crystal structure of 150-kD botulinum neurotoxin A bound to a and D, A reduction in phenylalanine and immune response levels fragment of a neutralizing monoclonal antibody. occurs in ENU2 mice after the injection of PAL that has been chemically modified (pegylated). These PEG-PAL formulations show stand and redesign the toxin’s mechanism of action promise as therapeutic agents for treatment of phenylketonuria. and to determine additional therapeutic applications of the toxin. we are determining the structural basis of diseases CANNABINOID SIGNALING caused by several other enzymes involved in the biosyn- In collaboration with B.F. Cravatt, Department of thesis of neurotransmitters. Cell Biology, we solved the structure of fatty acid amide NEUROTOXINS hydrolase, a degradative integral membrane enzyme The clostridial neurotoxins include tetanus toxin and responsible for setting intracellular levels of endocannabi- the 7 serotypes of botulinum toxin. We are determining noids, to 2.8 Å. Fatty acid amide hydrolase is intimately the molecular events involved in the binding, pore for- associated with CNS signaling processes such as retro- mation, translocation, and catalysis of botulinum neu- grade synaptic transmission, a process that is also rotoxin. Although botulinum toxin is most known for modulated by the illicit substance δ-9-tetrahydrocannabi- its deadly effects, it is now being used therapeutically nol. With our knowledge of the 3-dimensional struc- to treat involuntary muscle disorders such as cerebral ture, we are trying to understand how the enzyme works palsy and neuromuscular dystonias. Previously, we at a basic level and how it might be the basis for poten- determined the structure of the 150-kD holotoxin form tial drug discovery. of the toxin, the holotoxin bound to antibodies, the catalytic domains of several serotypes (A, B, D, F, G), PUBLICATIONS and the catalytic domain bound to substrates and inhibi- Arndt, J.W., Chai, Q., Christian, T., Stevens, R.C. Structure of botulinum neurotoxin type D light chain at 1.65 Å resolution: repercussions for VAMP-2 substrate tors (Fig. 2). These structures are being used to under- specificity. Biochemistry 45:3255, 2006. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 175

Arndt, J.W., Jacobson, M.J., Abola, E.E., Tepp, W.H., Johnson, E.A., Stevens, R.C. Scriver, C.R., Hurtubise, M., Prevost, L., Phommarinh, M., Konecki, D., Erlandsen, A structural perspective of the sequence variability within botulinum neurotoxin H., Stevens, R.C., Waters, P.J., Ryan, S., McDonald, D., Sarkissan, C. A PAH subtypes A1-A4. J. Mol. Biol. 362:733, 2006. gene knowledge base: content, informatics, utilization. In: PKU and BH4: Advances in Phenylketonuria and Tetrahydrobiopterin Research. Blau, N. (Ed.). SPS Publica- Blau, N., Koch, R., Matalon, R., Stevens, R.C. Five years of synergistic scientific tions, Heilbrun, Germany, 2006, p. 434. effort on phenylketonuria therapeutic development and molecular understanding. Mol. Genet. Metab. 86(Suppl. 1):S1, 2005. Swaminathan, S., Stevens, R.C. Three-dimensional protein structures of botulinum neurotoxin light chains serotypes A, B, and E. In: Treatments from Toxins: The Collins, B., Stevens, R.C., Page, R. Crystallization optimum solubility screening: Therapeutic Potential of Clostridial Neurotoxins. Foster, K.A., Hambleton, P., Shone, using crystallization results to identify the optimal buffer for protein crystal formation. C.C. (Eds.). CRC Press: Boca Raton, FL, in press. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61(Pt. 12):1035, 2005. Xu, Q., Schwarzenbacher, R., Krishna, S.S., et al. Crystal structure of acireductone dioxygenase (ARD) from Mus musculus at 2.06 Å resolution. Proteins 64:808, 2006. DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60 Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence Å resolution. Proteins 63:256, 2006. factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold. Proteins 62:292, 2006. Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W.W., Roach, L.S., Ismagilov, R.F., 60:797, 2005. Kuhn, P., Stevens, R.C. In situ data collection and structure refinement from microcapillary protein crystallization. J. Appl. Crystallogr. 38:900, 2005. Han, G.W., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of the ApbE protein (TM1553) from Thermotoga maritima at 1.58 Å resolution. Proteins 64:1083, 2006.

Jaroszewski, L., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of High-Throughput Approaches to Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution. Proteins 61:669, 2005. Protein Structure and Function Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367 from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the S.A. Lesley, M. Deller, D. Carlton, H. Johnson, Y. Elias, cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006. T. Clayton Joseph, J.S., Saikatendu, K.S., Subramanian, V., Neuman, B.W., Brooun, A., Griffith, M., Moy, K., Yadav, M.K., Velazquez, J., Buchmeier, M.J., Stevens, R.C., enomic information from the large number of Kuhn, P. Crystal structure of non-structural protein-10 (nsp10) from the SARS sequenced species has provided as many ques- coronavirus reveals a novel fold with two zinc-binding motifs. J. Virol. 80:7894, 2006. G tions as it has answered. Evaluating protein structure and function is of primary importance for under- Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a standing the basic biology of the cell and is a challenge new fold. Proteins 61:1132, 2005. in the context of the genome. To address this challenge, Matalon, R., Michals-Matalon, K., Koch, R., Grady, J., Tyring, S., Stevens, R.C. we have established high-throughput approaches for Response of patients with phenylketonuria in the US to tetrahydrobiopterin. Mol. evaluating structural and functional diversity of proteins. Genet. Metab. 86(Suppl. 1):S17, 2005. We have developed the capacity to clone, express, purify, Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of and crystallize large numbers of proteins in parallel as phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at 2.15 Å resolution. Proteins 63:1106, 2006. part of our structural genomics effort with the Joint Cen-

Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of ter for Structural Genomics, and we hope to apply these phosphoribosylformyl-glycinamidine synthase II, PurS subunit (TM1244) from same tools to characterize the molecular basis of the Thermotoga maritima at 1.90 Å resolution. Proteins 65:249, 2006. specificity of enzyme substrates. Pérez, B., Desviat, L.R., Gómez-Puertas, P., Martínez, A., Stevens, R.C., Ugarte, M. The goals of the Joint Center for Structural Genomics Kinetic and stability analysis of PKU mutations identified in BH4-responsive patients. Mol. Genet. Metab. 86(Suppl. 1):S11, 2005. are to develop a high-throughput and cost-effective structure pipeline and to use the pipeline to determine novel Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C., Wüthrich, K. Towards miniaturization of a structural genomics pipeline using protein folds and explore protein structure-function rela- micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005. tionships. We have used this approach in the extensive

Ratia, K., Saikatendu, K.S., Santarsiero, B.D., Barretto, N., Baker, S.C., Stevens, study of the thermophilic bacterium Thermotoga mar- R.C., Mesecar, A.D. Severe acute respiratory syndrome coronavirus papain-like pro- itima and for targets from mouse and other bacterial tease: structure of a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S. A. 103:5717, 2006. genomes. Our technologies have enabled us to perform comprehensive structural studies of these proteomes. Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K., Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structural To date, these efforts have resulted in more than 300 basis of severe acute respiratory syndrome coronavirus ADP-ribose-1′′-phosphate novel protein structures from the center. dephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005. Functional studies of selected targets have been Schwarzenbacher,R., McMullan, D., Krishna, S.S., et al. Crystal structure of a performed. For example, in collaboration with A. Kohen, glycerate kinase (TM1585) from Thermotoga maritima at 2.70 Å resolution reveals a new fold. Proteins 65:243, 2006. University of Iowa, Iowa City, we explored the mecha- 176 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE nism of thymidylate synthase from T maritima. This Jin, K.K., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of TM1367 from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the protein has a novel fold and a unique flavin-dependent cyclophilin (peptidylprolyl isomerase) fold. Proteins 63:1112, 2006 biochemical mechanism. The gene for thymidylate syn- Klock, H.E., Schwarzenbacher, R., Xu, Q., et al. Crystal structure of a conserved thase is an essential one, and the protein is an important hypothetical protein (gi: 13879369) from mouse at 1.90 Å resolution reveals a potential antibacterial target because of its structural new fold. Proteins 61:1132, 2005. dissimilarity with the human protein. Klock, H.E., White, A., Koesema, E., Lesley, S.A. Methods and results for semi- We have also developed the method of deuterium automated cloning using integrated robotics. J. Struct. Funct. Genomics 6:89, 2005. exchange by mass spectrometry in collaboration with Kreusch, A., Han, S., Brinker, A., Zhou, V., Choi, H., He, Y., Lesley, S.A., Cald- well, J., Gu, X. Crystal structures of a new class of HSP90 inhibitors, dihydrox- V. Woods, University of California, San Diego, to char- yphenylpyrazoles. Bioorg. Med. Chem. Lett. 15:1475, 2005. acterize protein regions with highly flexible regions Kreusch, A., Han, S., Brinker, A., Zhou, V., Choi, H.S., He, Y., Lesley, S.A., Caldwell, that interfere with crystallization. Subsequent elimina- J., Gu, X.J. Crystal structures of human HSP90α complexed with dihydrox- tion of these regions dramatically improves crystalliza- yphenylpyrazoles. Bioorg. Med. Chem. Lett. 15:1475, 2005. tion and has resulted in structures for several Lesley, S.A., Wilson, I.A. Protein production and crystallization at the Joint Center problematic structures. for Structural Genomics. J. Struct. Funct. Genomics 6:71, 2005. Expression of membrane proteins continues to be Levin, I., Miller, M.D., Schwarzenbacher, R., et al. Crystal structure of an indigoi- dine synthase A (IndA)-like protein (TM1464) from Thermotoga maritima at 1.90 one of the most difficult challenges in studying this Å resolution reveals a new fold. Proteins 59:864, 2005. important protein class. Our structural genomics efforts Mason, A., Agrawal, N., Washington, M.T., Lesley, S.A., Kohen, A. A lag-phase in in collaboration with S. Eshaghi, Karolinska Institutet, the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mecha- Stockholm, Sweden, have led to the structure of the nistic missing link. Chem. Commun. (Camb.) 1781, 2006, Issue 16. integral membrane protein CorA, a magnesium trans- Mathews, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of S-adeno- porter from T maritima. In collaboration with P. Schultz, sylmethionine:tRNA ribosyltransferase-isomerase (QueA) from Thermotoga maritima at 2.0 Å resolution reveals a new fold. Proteins 59:869,2005. Department of Chemistry, we are exploring the use of unnatural amino acids to enhance the purification and Mathews, I.I., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at crystallization of integral membrane proteins. 2.15 Å resolution. Proteins 63:1106, 2006.

McMullan, D., Canaves, J.M., Quijano, K., Abdubek, P., Nigoghossian, E., Hau- PUBLICATIONS gen, J., Klock, H.E., Vincent, J., Hale, J., Paulsen, J., Lesley, S.A. High-through- Arndt, J.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an α/β ser- put protein production for x-ray crystallography and use of size-exclusion ine hydrolase (YDR428C) from Saccharomyces cerevisiae at 1.85 Å resolution. chromatography to validate computational biological unit predictions. J. Struct. Proteins 58:755, 2005. Funct. Genomics 6:135, 2005.

Chamberlain, P.P., Sandberg, M.L., Sauer, K., Cooke, M.P., Lesley, S.A., Spraggon, Page, R., Deacon, A.M., Lesley, S.A., Stevens, R.C. Shotgun crystallization strat- G. Structural insights into enzyme regulation for inositol 1,4,5-trisphosphate 3- egy for structural genomics, II: crystallization conditions that produce high resolu- kinase B. Biochemistry 44:14486, 2005. tion structure for T maritima proteins. J. Struct. Funct. Genomics 6:209, 2005.

Columbus, L., Lipfert, J., Klock, H., Millett, I., Doniach, S., Lesley, S.A. Expres- Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of the global sion, purification, and characterization of Thermotoga maritima membrane proteins regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a for structure determination. Protein Sci. 15:961, 2006. new fold. Proteins 61:449, 2005.

DiDonato, M., Krishna, S.S., Schwarzenbacher, R., et al. Crystal structure of a Rife, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a putative single-stranded DNA-binding protein (TM0604) from Thermotoga maritima at 2.60 modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution Å resolution. Proteins 63:256, 2006. reveals a new fold. Proteins 61:444, 2005.

Eshaghi, S., Niegowski, D., Kohl, A., Martinez Molina, D., Lesley, S.A., Nordlund, P. Wang, Y., Klock, H., Yin, H., Wolff, K., Bieza, K., Niswonger, K., Matzen, J., Crystal structure of a divalent metal ion transporter CorA at 2.9 Å resolution [pub- Gunderson, D., Hale, J., Lesley, S., Kuhen, K., Caldwell, J., Brinker, A. Homoge- lished correction appears in Science 313:1389, 2006]. Science 313:354, 2006. neous high-throughput screening assays for HIV-1 integrase 3β-processing and strand transfer activities. J. Biomol. Screen. 10:456, 2005. Han, G.W., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of an apo mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a form- 60:797, 2005. iminotetrahydrofolate cyclodeaminase (TM1560) from Thermotoga maritima at 2.80 Å resolution reveals a new fold. Proteins 58:976, 2005. Han, G.W., Schwarzenbacher, R., Page, R., et al. Crystal structure of an alanine- glyoxylate aminotransferase from Anabaena sp. at 1.70 Å resolution reveals a non- Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of virulence covalently linked PLP cofactor. Proteins 58:971, 2005. factor CJ0248 from Campylobacter jejuni at 2.25 Å resolution reveals a new fold. Proteins 62:292, 2006. Han, S., Zhou, V., Pan, S., Liu, Y., Hornsby, M., McMullan, D., Klock, H., Lesley, S.A., Gray, N., Caldwell, J., Gu, X.J. Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors. Bioorg. Med. Chem. Lett. 15:5467, 2005.

Jaroszewski, L., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of Hsp33 chaperone (TM1394) from Thermotoga maritima at 2.20 Å resolution. Proteins 61:669, 2005. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 177 Nuclear Magnetic Resonance nonstructural proteins that perform enzymatic functions of the virus within the host cell. These functions include Spectroscopy in Protein RNA-processing steps and other functions that are currently unknown. The genome also encodes viral struc- Structural Biology and Structural tural proteins, which form part of the mature viral particle along with genomic RNA. Genomics Several of the replicase proteins of coronaviruses have little or no apparent relationship to other known M. Almeida, W. Augustyniak, L. Columbus, M. Geralt, proteins, and little is known about how they function R. Horst, M. Johnson, B. Pedrini, W.J. Placzek, P. Serrano, during viral infection. In addition, the reasons for the K. Wüthrich severe signs and symptoms caused by SARS-CoV in ur research program focuses on 2 areas. First, comparison with other human coronaviruses, which in a collaboration with A. Horwich, Yale Univer- usually cause much less severe infections, are cur- O sity, New Haven, Connecticut, who is a visiting rently unknown. We are using NMR spectroscopy for scientist at Scripps Research, we are investigating struc- structural and functional investigations of SARS-CoV tural and mechanistic aspects of the function of GroE- proteins to gain information about the viral life cycle type chaperonin systems in Escherichia coli. This and to identify possible new antiviral strategies. research concerns the process of protein folding in NONSTRUCTURAL PROTEIN 1 healthy and diseased organisms and thus is directly Nonstructural protein 1 (nsp1) is the leader protein related to the currently extensively discussed protein of the SARS-CoV genome and the first to be translated misfolding diseases. Because of the large size of the and cleaved by the viral protease to its mature form. GroE-type supramolecular structures, this project It has little apparent relationship to proteins of other depends on continuous improvement of existing solu- coronaviruses and may perform a function unique to tion nuclear magnetic resonance (NMR) techniques SARS-CoV. We used NMR spectroscopy to investigate and development of new techniques. the solution structure and dynamics of nsp1. The pro- Second, we develop and apply NMR methods for tein adopts a new 3-dimensional fold, with a distorted, use in structural genomics. We participate in the Joint 6-stranded β-barrel covered by an α-helix. This stable, Center for Structural Genomics (JCSG), the JCSG Cen- folded globular domain carries a long, flexibly disor- ter for Innovative Membrane Protein Technologies, and dered polypeptide “tail” at the C terminus. We used the Consortium for Functional and Structural Proteomics bioinformatics techniques to search for local structural of SARS-CoV–Related Proteins (FSPS). On the one hand, features that might provide insight into the functional we explore the use of automated microscale NMR equip- properties of this protein. We detected a possible pro- ment for the screening of recombinant protein prepara- tease active site on one end of the β-barrel, indicating tions for folded globular domains. On the other hand, that nsp1 may be a previously unrecognized viral pro- we use NMR spectroscopy to determine the structures of tease. Follow-up studies indicated that formation of a selected proteins from the proteomes under study. The functional active site may require the presence of the following sections highlight our research on proteins long C-terminal tail, or of other protein cofactors. from the severe acute respiratory syndrome–associated NONSTRUCTURAL PROTEIN 3 coronavirus (SARS-CoV) proteome, which is pursued The viral element nsp3 is a large protein of about under the auspices of the FSPS (http://sars.scripps.edu) 2000 amino acid residues that most likely includes and the JCSG (http://www.jcsg.org). multiple functional domains. We designed smaller con- THE SARS-COV PROBLEM structs of this protein encompassing predicted individual In 2003, a major global outbreak of SARS was domains, and used 1-dimensional 1H NMR screening caused by a newly emerged coronavirus. The coron- to identify those domains that were independently folded. avirus genome is composed of a single plus-strand We then determined the solution structure of the N-ter- RNA of about 30 kb and is the largest genome among minal domain, nsp3a. Unexpectedly, we found that its known RNA viruses. About two thirds of the genome structure is similar to that of the α/β roll fold of ubiq- is devoted to encoding the replicase polyprotein, which uitin. This structural motif is most commonly found in is cleaved by viral proteases to release the mature proteins of eukaryotes that are involved in cellular sig- 178 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE naling pathways. Therefore, the nsp3a domain may be PUBLICATIONS Almeida, M.S., Herrmann, T., Peti, W., Wilson, I.A., Wüthrich, K. NMR structure used by the virus to interact with signaling proteins of of the conserved hypothetical protein TM0487 from Thermotoga maritima: impli- the host cell and to interfere with cellular pathways in cations for 216 homologous DUF59 proteins. Protein Sci. 14:2880, 2005. order to increase virulence. Columbus, L., Peti, W., Etezady-Esfarjani, T., Herrmann, T., Wüthrich, K. NMR We are also investigating a possible second func- structure determination of the conserved hypothetical protein TM1816 from Ther- motoga maritima. Proteins 60:552, 2005. tion of this protein in RNA processing. Using NMR spec- Horst, R., Bertelsen, E.B., Fiaux, J., Wider, G., Horwich, A.L., Wüthrich, K. troscopy and mass spectrometry, we identified RNA Direct NMR observation of a substrate protein bound to the chaperonin GroEL. molecules that bind to nsp3a. We are studying these Proc. Natl. Acad. Sci. U. S. A. 102:12748, 2005. interactions to determine possible enzymatic or scaf- Peti, W., Herrmann, T., Zagnitko, O., Grzechnik, S.K., Wüthrich, K. NMR struc- folding functions. ture of the conserved hypothetical protein TM0979 from Thermotoga maritima. Proteins 59:387, 2005. NONSTRUCTURAL PROTEIN 7 The viral component nsp7 is highly conserved Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson, M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural geno- between the different coronaviruses and probably per- mics of the severe acute respiratory syndrome coronavirus: nuclear magnetic reso- forms an essential core function in this virus family. nance structure of the protein nsp7. J. Virol. 79:12905, 2005. Interestingly, the solution structure of nsp7 also shows Peti, W., Page, R., Moy, K., O’Neil-Johnson, M., Wilson, I.A., Stevens, R.C., Wüthrich, K. Towards miniaturization of a structural genomics pipeline using a new fold that was not previously observed in any micro-expression and microcoil NMR. J. Struct. Funct. Genomics 6:259, 2005. known protein structure. The structure consists of 4 helices, and although many 4-helix bundle proteins are known, nsp7 does not form a bundle. Rather 3 helices are assembled into a flat sheet, with the helices antipar- Nuclear Magnetic Resonance allel, and the fourth helix is stacked across one side of this sheet (Fig. 1). The other surface of the flat sheet of 3-Dimensional Structure and Dynamics of Proteins in Solution

P.E. Wright, H.J. Dyson, R. Burge, J. Ferreon, N. Greenman, T.-H. Huang, B.B. Koehntop, M. Kostic, B. Lee, C.W. Lee, M. Landes, M. Martinez-Yamout, T. Nishikawa, K. Sugase, J. Wojciak, M. Zeeb, E. Manlapaz, L.L. Tennant, J. Chung, D.A. Case, J. Gottesfeld, R. Evans,* M. Montminy* * Salk Institute, La Jolla, California

e use multidimensional nuclear magnetic resonance (NMR) spectroscopy to investi- W gate the structures, dynamics, and interactions of proteins in solution. Such studies are essential for understanding the mechanisms of action of these Fig. 1. Ensemble of 20 conformers representing the polypeptide proteins and for elucidating structure-function relation- backbone in the solution structure of SARS-CoV nsp7. The 3 helices α2, α3, and α4 assemble into a flat sheet, with the α1 ships. The focus of our current research is protein-pro- helix stacked diagonally across one surface of this sheet. Reprinted tein and protein–nucleic acid interactions involved in the with permission from Peti, W., et al. J. Virol. 79:12905, 2005. regulation of gene expression. Copyright 2005 American Society for Microbiology. TRANSCRIPTION FACTOR–NUCLEIC ACID COMPLEXES NMR methods are being used to determine the contains hydrophobic and negatively charged patches, 3-dimensional structures and intramolecular dynamics which most likely are sites for protein-protein inter- of zinc finger motifs from several eukaryotic transcrip- actions. Currently, we are using NMR spectroscopy to tional regulatory proteins, both free and complexed with identify interactions with other SARS-CoV proteins. Such target nucleic acid. Zinc fingers are among the most interactions could be targeted for the design of inhibitory abundant domains in eukaryotic genomes. They play a molecules with antiviral activity. central role in the regulation of gene expression at both MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 179 the transcriptional and the posttranscriptional level, cate that the insertion increases the flexibility of the mediated through their interactions with DNA, RNA, linker between fingers 3 and 4 and abrogates binding of or protein components of the transcriptional machin- the fourth zinc finger to its cognate site in the DNA ery. The C2H2 zinc finger, first identified in transcription major groove, thereby modulating DNA-binding activity. factor IIIA (TFIIIA), is used by numerous transcription The x-ray structure of the DNA complex has been deter- factors to achieve sequence-specific recognition of DNA. mined, providing insights into the mechanism by which

There is growing evidence, however, that some C2H2 zinc disease-causing mutations interfere with DNA binding. finger proteins control gene expression both through NMR studies of the RNA complex are in progress. We their interactions with DNA regulatory elements and, at have also determined the structure of a novel zinc the posttranscriptional level, by binding to RNA. finger protein that binds to double-stranded RNA and

The best-characterized example of a C2H2 zinc fin- have begun experiments to define the mechanism of ger protein that binds specifically to both DNA and to RNA recognition. RNA is TFIIIA, which contains 9 zinc fingers. We showed Several novel zinc binding motifs have recently been previously that different subsets of zinc fingers are identified that mediate gene expression at the posttran- responsible for high-affinity binding of TFIIIA to DNA scriptional level by regulating mRNA processing and (fingers 1–3) and to 5S RNA (fingers 4–6). To obtain metabolism. Regulatory proteins of the TIS11 family insights into the mechanism by which the TFIIIA zinc bind specifically, through a pair of novel CCCH zinc fingers recognize both DNA and RNA, we have used fingers, to the adenosine-uridine–rich element in the NMR methods to determine the structures of the com- 3′ untranslated region of short-lived cytokine, growth fac- plex formed by zf1-3 (a protein containing fingers 1–3) tor, and proto-oncogene mRNAs and control expres- with DNA and by zf4-6 (a protein consisting of fingers sion by promoting rapid degradation of the message. We 4–6) with a fragment of 5S RNA. recently determined the NMR structure of the complex Three-dimensional structures were determined pre- formed between the tandem zinc finger domain of TIS11d viously for the complex of zf1-3 with the cognate 15-bp and its binding site on the adenosine-uridine–rich ele- oligonucleotide duplex. The structures contain several ment. This structure showed sequence-specific recog- novel features and reveal that prevailing models of DNA nition of single-stranded RNA through formation of a recognition, which assume that zinc fingers are inde- network of hydrogen bonds between the polypeptide pendent modules that contact bases through a limited backbone and the Watson-Crick edges of the bases. set of amino acids, are outmoded. PROTEIN-PROTEIN INTERACTIONS IN In addition to its role in binding to and regulating TRANSCRIPTIONAL REGULATION the 5S RNA gene, TFIIIA also forms a complex with Transcriptional regulation in eukaryotes relies on the 5S RNA transcript. NMR structures of the complex protein-protein interactions between DNA-bound factors formed by zinc fingers 4–6 with a truncated form of 5S and coactivators that, in turn, interact with the basal RNA have been completed and give important insights transcription machinery. The transcriptional coactiva- into the structural basis for 5S RNA recognition. Fin- tor CREB-binding protein (CBP) and its homolog p300 ger 4 of the protein recognizes both the structure of play an essential role in cell growth, differentiation, and the RNA backbone and the specific bases in the loop E development. Understanding the molecular mechanisms motif of the RNA, in a classic lock-and-key interaction. by which CBP and p300 recognize their various target Fingers 5 and 6, with a single residue between them, proteins is of fundamental biomedical importance. CBP undergo mutual induced-fit folding with the loop A and p300 have been implicated in diseases such as region of the RNA, which is highly flexible in the absence leukemia, cancer, and mental retardation and are novel of the protein. targets for therapeutic intervention. NMR studies of 2 alternate splice variants of the We previously determined the structure of the kinase- Wilms tumor zinc finger protein are in progress. These inducible activation domain of the transcription factor proteins differ only through insertion of 3 additional CREB bound to its target domain (the KIX domain) in amino acids (the tripeptide lysine-threonine-serine) in CBP. Ongoing work is directed toward mapping the inter- the linker between fingers 3 and 4, yet have marked actions between KIX and the transcriptional activation differences in their DNA-binding properties and subdomains of the proto-oncogene c-Myb and of the mixed- cellular localization. 15N relaxation measurements indi- lineage leukemia protein. The solution structure of the 180 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE ternary complex between KIX, c-Myb and the mixed- Folding of Proteins and lineage leukemia protein has been completed and provides insights into the structural basis for the ability of Protein Fragments the KIX domain to interact simultaneously and allosteri- cally with 2 different effectors. Our work has also pro- P.E. Wright, H.J. Dyson, C. Nishimura, D. Felitsky, Y. Yao, vided new understanding of the thermodynamics of the J. Chung, L.L. Tennant, V. Bychkova,* T. Uzawa,** coupled folding and binding processes involved in inter- S. Takahashi** action of KIX with transcriptional activation domains. * Institute of Protein Research, Puschino, Russia ** Kyoto University, Kyoto, Japan We are using R2 relaxation dispersion experiments to elucidate the mechanism by which folding of the kinase- he molecular mechanism by which proteins fold inducible activation domain of CREB is coupled to bind- into their 3-dimensional structures remains one ing to its KIX target domain. These experiments reveal T of the most important unsolved problems in struc- formation of a transient and largely unfolded encounter tural biology. Nuclear magnetic resonance (NMR) spec- complex, which then folds on the surface of the KIX troscopy is uniquely suited to provide information on domain to form the helical structure observed in the the structure of transient intermediates formed during fully bound state. protein folding. Previously, we used NMR methods to Recently, we determined the structure of the com- show that many peptide fragments of proteins have a plex between the hypoxia-inducible factor Hif-1α and tendency to adopt folded conformations in water solu- the CH1 domain of CBP. The interaction between Hif-1α tion. The presence of transiently populated folded struc- and CBP/p300 is of major therapeutic interest because tures, including reverse turns, helices, nascent helices, of the central role Hif-1α plays in tumor progression and hydrophobic clusters, in water solutions of short and metastasis; disruption of this interaction leads to peptides has important implications for initiation of pro- attenuation of tumor growth. A protein named CITED2 tein folding. Formation of elements of secondary structure functions as a negative feedback regulator of the hypoxic probably plays an important role in the initiation of pro- response by competing with Hif-1α for binding to the tein folding by reducing the number of conformations CH1 domain of CBP.We determined the structure of the that must be explored by the polypeptide chain and by complex formed between CITED2 and the CH1 domain directing subsequent folding pathways. and were able to show that the CH1 domain is folded APOMYOGLOBIN FOLDING PA THWAY into a stable 3-dimensional structure even in the absence A major program in our laboratory is directed of binding partners. The intrinsically unstructured Hif-1α toward a structural and mechanistic description of and CITED2 domains use partly overlapping surfaces the apomyoglobin folding pathway. Previously, we of the CH1 motif to achieve high-affinity binding and used quenched-flow pulse-labeling methods in con- compete effectively with each other for CBP/p300. We junction with 2-dimensional NMR spectroscopy to map are continuing to map the multiplicity of interactions the kinetic folding pathway of the wild-type protein. between CBP/p300 domains and their numerous bio- With these methods, we showed that an intermediate logical targets to understand the complex interplay of in which the A, G, and H helices and part of the B helix interactions that mediate key biological processes in adopt hydrogen-bonded secondary structure is formed health and disease. within 6 milliseconds of the initiation of refolding. Fold- ing then proceeds by stabilization of additional structure PUBLICATIONS De Guzman, R.N., Goto, N.K., Dyson, H.J., Wright, P.E. Structural basis for coopera- in the B helix and in the C and E helices. We are using tive transcription factor binding to the CBP coactivator. J. Mol. Biol. 355:1005, 2006. carefully selected myoglobin mutants and both optical

Kostic, M., Matt, T., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Solution stopped-flow spectroscopy and NMR methods to further structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53. probe the kinetic folding pathway. For some of the J. Mol. Biol. 363:433, 2006. mutants studied, the changes in amino acid sequence Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A., resulted in changes in the folding pathway of the pro- Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of 5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006. tein. These experiments are providing novel insights into both the local and the long-range interactions that stabilize the kinetic folding intermediate. Of particular importance, long-range interactions have been observed MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 181 that indicate nativelike packing of some of the helices dipolar couplings arise from the well-known statistical in the kinetic molten globule intermediate. properties of flexible polypeptide chains. Residual dipo- Apomyoglobin provides a unique opportunity for lar couplings provide valuable insights into the dynamic detailed characterization of the structure and dynamics and conformational propensities of unfolded and partly of a protein-folding intermediate. Conditions were pre- folded states of proteins and hold great promise for chart- viously identified under which the apomyoglobin molten ing the upper reaches of protein-folding landscapes. globule intermediate is sufficiently stable for acquisition To probe long-range interactions in unfolded and of multidimensional heteronuclear NMR spectra. Analysis partially folded states of apomyoglobin, we introduced of 13C and other chemical shifts and measurements of spin-label probes at several sites throughout the poly- polypeptide dynamics provided unprecedented insights peptide chain. These experiments led to the surprising into the structure of this state. discovery that structures with nativelike topology exist The A, G, and H helices and part of the B helix are within the ensemble of conformations formed by the folded and form the core of the molten globule. This acid-denatured state of apomyoglobin. They also indi- core is stabilized by relatively nonspecific hydrophobic cated that the packing of helices in the molten globule interactions that restrict the motions of the polypeptide state is similar to that in the native folded protein. chain. Fluctuating helical structure is formed in regions The view of protein folding that results from our outside the core, although the population of helix is low work on apomyoglobin is one in which collapse of the and the chain retains considerable flexibility. The F helix polypeptide chain to form increasingly compact states acts as a gate for heme binding and only adopts sta- leads to progressive accumulation of secondary struc- ble structure in the fully folded holoprotein. ture and increasing restriction of fluctuations in the The acid-denatured (unfolded) state of apomyoglobin polypeptide backbone. Chain flexibility is greatest at is an excellent model for the fluctuating local interactions the earliest stages of folding, in which transient elements that lead to the transient formation of unstable elements of secondary structure and local hydrophobic clusters of secondary structure and local hydrophobic clusters are formed. As the folding protein becomes increasingly during the earliest stages of folding. NMR data indicated compact, backbone motions become more restricted, substantial formation of helical secondary structure in the hydrophobic core is formed and extended, and the acid-denatured state in regions that form the A and nascent elements of secondary structure are progres- H helices in the folded protein and also revealed non- sively stabilized. The ordered tertiary structure charac- native structure in the D and E helix regions. teristic of the native protein, with well-packed side Because the A and H regions adopt stabilized helical chains and relatively low-amplitude local dynamics, structure in the earliest detectable folding intermediate, appears to form rather late in folding. these results lend strong support to folding models in We recently introduced a variation on the classic which spontaneous formation of local elements of sec- quench-flow technique, which makes use of the capa- ondary structure plays a role in initiating formation of bilities of modern NMR spectrometers and heteronu- the A-[B]-G-H molten globule folding intermediate. In clear NMR experiments, to study the proteins labeled addition to formation of transient helical structure, for- along the folding pathway in an unfolded state in an mation of local hydrophobic clusters has been detected by aprotic organic solvent. This method allows detection using 15N relaxation measurements. Significantly, these of many more amide proton probes than in the classic clusters are formed in regions where the average sur- method, which required formation of the fully folded face area buried upon folding is large. In contrast to protein and the measurement of its NMR spectrum in acid-denatured unfolded apomyoglobin, the urea-dena- water solutions. This method is particularly useful in tured state is largely devoid of structure, although resid- documenting changes in the folding pathway that result ual hydrophobic interactions have been detected by using in the destabilization of parts of the protein in the molten relaxation measurements. globule intermediate. We recently showed that self- We measured residual dipolar couplings for unfolded compensating mutations designed to change the amino states of apomyoglobin by using partial alignment in acid sequence such that the average area buried upon strained polyacrylamide gels. These data provide novel folding is significantly changed while the 3-dimensional insights into the structure and dynamics of the unfolded structure of the final folded state remains the same. polypeptide chain. We have shown that the residual These studies showed that the average area buried 182 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE upon folding is an accurate predictor of those parts of the apomyoglobin molecule that will fold first and participate in the molten globule intermediate. Quench-flow hydrogen exchange experiments performed on a series of hydrophobic core mutants indicated that the overall helix-packing topology of the kinetic folding intermediate is like that of the native protein, despite local nonnative interactions in packing of the G and H helices. Finally, using a rapid mixing device, we have reduced the dead time of the kinetic refolding experiments and have shown that a compact helical intermediate is formed within 400 microseconds after initiation of apomyoglobin refolding. FOLDING-UNFOLDING TRANSITIONS IN CELLULAR METABOLISM Many species of bacteria sense and respond to their Fig. 1. Folding of protein and protein fragments. Ribbon diagram showing the lowest energy structure of the complex between own population density by an intricate autoregulatory HSL and E coli SdiA. mechanism known as quorum sensing; the bacteria release extracellular signal molecules, called autoinduc- basis of their interactions, but unfolded proteins are ers, for cell-cell communication within and between impossible to characterize structurally by x-ray crystal- bacterial species. A number of bacteria appear to use lography, and spectroscopic methods of all kinds are quorum sensing for regulation of gene expression in limited. It is necessary to explore unfolded proteins response to fluctuations in cell population density. under conditions that approximate their physiologic Processes regulated in this way include symbiosis, milieu: in solution, at physiologic pHs and salt concentrations, and in the presence of specific cofactors. virulence, competence, conjugation, production of antibi- Structural insights will be obtained not only from the otics, motility, sporulation, and formation of biofilms. delineation of 3-dimensional structures but also from We determined the 3-dimensional solution structure the description of conformational ensembles and of the of a complex composed of the N-terminal 171 residues motions of polypeptide chains under various conditions. of the quorum-sensing protein SdiA of Escherichia coli To gain new insights into the structural basis for the and an autoinducer molecule, N-octanoyl-1-homoser- ability of unfolded and partly folded proteins to function ine lactone (HSL) (Fig. 1). The SdiA-HSL system shows in living systems, we are studying the interactions of the “folding switch” behavior associated with quorum- “client” proteins and cochaperones with a well-known sensing factors produced by other bacterial species. In eukaryotic chaperone, Hsp90. Some of the protein com- the presence of HSL, the SdiA protein is stable and folded ponents are much larger than have traditionally been and can be produced in good yields from an E coli expres- studied by using solution NMR. However, we have sion system. In the absence of the autoinducer, the pro- designed a set of experiments that will allow us to draw tein is expressed into inclusion bodies. Samples of the valid conclusions about the extent and role of disorder in SdiA-HSL complex can be denatured but cannot be Hsp90 interactions. In particular, we will apply tech- refolded in aqueous buffers. The solution structure of niques recently developed in our laboratory for the the complex provides a likely explanation for this behav- analysis of hydrogen-deuterium exchange from unstable ior. The autoinducer molecule is tightly bound in a deep partially folded proteins by trapping the 2H-labeled pocket in the hydrophobic core and is bounded by spe- species in the aprotic solvent dimethyl sulfoxide. This cific hydrogen bonds to the side chains of conserved powerful new technique will be used to probe the struc- residues. The autoinducer thus forms an integral part ture, stability, and interactions of client proteins and of the hydrophobic core of the folded SdiA. cochaperones with Hsp90. CHAPERONE–COCHAPERONE–CLIENT PROTEIN

INTERACTIONS PUBLICATIONS Understanding the role of unfolded states in cellular Dyson, H.J., Wright, P.E. According to current textbooks, a well-defined three- dimensional structure is a prerequisite for the function of the protein: is this cor- processes will require an understanding of the structural rect? IUBMB Life 58:107, 2006. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 183

Dyson, H.J., Wright, P.E., Scheraga, H.A. The role of hydrophobic interactions in features for a broad range of timescales (picoseconds initiation and propagation of protein folding. Proc. Natl. Acad. Sci. U. S. A. 103:13057, 2006. to milliseconds). A major focus is on the characterization of all inter- Kamikubo, Y., Kroon, G., Curriden, S.A., Dyson, H.J., Loskutoff, D.J. The reduced, denatured somatomedin B domain of vitronectin refolds into a stable, bio- mediates in the dihydrofolate reductase reaction cycle. logically active form. Biochemistry 45:3297, 2006. We have identified functionally important motions in Martinez-Yamout, M.A., Venkitakrishnan, R.P., Preece, N.E., Kroon, G., Wright, loops that control access to the active site of dihydro- P.E., Dyson, H.J. Localization of sites of interaction between p23 and Hsp90 in solution. J. Biol. Chem. 281:14457, 2006. folate reductase on timescales similar to those of the hydride transfer chemistry and the rate-determining Nishimura, C., Dyson, H.J., Wright, P.E. Identification of native and non-native structure in kinetic folding intermediates of apomyoglobin. J. Mol. Biol. 355:139, 2006. step of product release. These motions differ in amplitude and timescale depending on the presence of sub- Papadopoulos, E., Oglecka, K., Mäler, L., Jarvet, J., Wright, P.E., Dyson, H.J., Gräslund, A. NMR solution structure of the peptide fragment 1-30, derived from strate and/or cofactor in the active site, priming the mouse Doppel protein, in DHPC micelles. Biochemistry 45:159, 2006. nicotinamide ring of the cofactor and the pterin ring of Yao, Y., Martinez-Yamout, M.A., Dickerson, T.J., Brogan, A.P., Wright, P.E., Dyson, the substrate for hydride transfer. In addition, measure- H.J. Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones. J. Mol. Biol. 355:262, 2006. ments of the population distribution of aliphatic side- chain rotamers provided evidence for coupled motion Yao, Y., Martinez-Yamout, M.A., Dyson, H.J. Backbone and side chain 1H, 13C and 15N assignments for Escherichia coli SdiA1-171, the autoinducer-binding of active-site side chains that could enhance the cata- domain of a quorum sensing protein [letter]. J. Biomol. NMR 31:373, 2005. lytic process. Most recently, we used relaxation dispersion measurements to obtain direct information on microsecond- Nuclear Magnetic Resonance millisecond timescale motions in dihydrofolate reductase, Studies of the Structure and allowing us to characterize the structures of excited states involved in some of these catalysis-relevant pro- Dynamics of Enzymes cesses. Fluctuations between these states, which involve motions of the nicotinamide ring of the cofactor into and H.J. Dyson, P.E. Wright, S.H. Bae, D. Boehr, G. Kroon, out of the active site, occur on a timescale that is directly M. Martinez-Yamout, N.E. Preece, S.C. Sue, L.M. Tuttle, relevant to the structural transitions involved in progres- Y.Yao, L.L. Tennant, J. Chung, C.L. Brooks, S.J. Benkovic,* sion through the catalytic cycle (Fig. 1). A. Holmgren,** E.A. Komives*** Dihydrofolate reductase is also the test system for * Pennsylvania State University, University Park, Pennsylvania a series of experiments to address the question, If all ** Karolinska Institutet, Stockholm, Sweden *** University of California, San Diego, California of the chemistry goes on at the active site, what is the purpose of the rest of the enzyme? We are using chimeric e use site-specific information on structure mutants, synthesized by our collaborator S.J. Benkovic, and dynamics obtained via nuclear magnetic Pennsylvania State University, by using a library approach. W resonance (NMR) to further the understand- The purpose of these experiments is to test the hypothesis ing of protein function. We focus on the mechanism of that local variations in amino acid sequence, 3-dimen- enzymes and the relationship between dynamics and sional structure, and polypeptide chain dynamics strongly function in a number of medically important systems. influence the local interactions that mediate enzyme DYNAMICS IN ENZYME ACTION catalysis and may constitute the essential circumstance Dynamic processes are implicit in the catalytic that allows enzymes to achieve high turnover rates as function of all enzymes. We use state-of-the-art NMR well as exquisite specificity in their reactions. A com- methods to elucidate the dynamic properties of several bination of NMR structure and dynamics measurements, enzymes. New methods have been developed for analysis single-molecule fluorescence measurements, and analy- of NMR relaxation data for proteins that tumble aniso- sis of the catalytic steps in these mutant proteins will tropically and for analysis of slow timescale motions. provide new insights into the role of the protein in Dihydrofolate reductase plays a central role in folate enzyme catalysis. metabolism and is the target enzyme for a number of STRUCTURE AND DYNAMICS OF PRION VARIANTS 15 antibacterial and anticancer agents. N relaxation exper- Onset of prion diseases is caused by conversion of iments on dihydrofolate reductase from Escherichia the cellular prion protein PrPC into an abnormally folded coli revealed a rich diversity of backbone dynamical isoform, PrPSc, that has the same primary structure as 184 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

an important biological interaction are unstructured or partly structured. In addition, even those interacting molecules that can be classified as “folded” have areas of mobility. Often, these areas are located precisely in the active site of an enzyme or in the binding site of an interacting molecule. A central molecular interaction in cellular control is the interaction between the nuclear transcription factor NF-κB and its inhibitor IκBα. IκBα consists of a series of ankyrin repeats, which appear to have differential mobility. Using hydrogen-deuterium exchange and mass spectrometry, our collaborator E.A. Komives, University of California, San Diego, found that the second, third, and fourth ankyrin repeats of IκBα are well folded,

Fig 1. Schematic diagram showing the energy landscape of dihy- whereas the fifth and sixth repeats, apparently with drofolate reductase catalysis. Ground state (larger) and higher energy exactly the same structure, are highly dynamic. These (smaller) structures of each intermediate in the cycle, modeled on observations prompt a number of questions: Are the published x-ray structures are shown. For each intermediate in the motions inferred from the hydrogen-deuterium mass catalytic cycle, the higher energy conformations detected in the spectrometry experiments also reflected in the back- relaxation dispersion experiments resemble the ‘ground-state’ conformations of adjacent intermediates. Rate constants for the intercon- bone and side-chain dynamics of the protein, as mea- version between the complexes, measured by pre–steady state enzyme sured by NMR relaxation? Are the motions still present kinetics at 298 K, pH6 are indicated with gray arrows, while the in the IκBα–NF-κB complex? Are they necessary for rates measured in relaxation dispersion experiments are shown with complex formation, so that if they are damped out, for black arrows. From Boehr et al., Science 313:1638, 2006. Reprinted example, by site-directed mutagenesis at appropriate with permission from AAAS. positions, is the formation of the complex disfavored? To PrPC but a totally different 3-dimensional conformation. answer these questions, we are doing a series of NMR The abnormally folded (“scrapie”) form of the protein is experiments on IκBα and its complexes with NF-κB. associated with several diseases, including scrapie in PUBLICATIONS sheep, bovine spongiform encephalopathy (mad cow Boehr, D.D., Dyson, H.J., Wright, P.E. An NMR perspective on enzyme dynamics. disease), and human Creutzfeldt-Jakob disease and Chem. Rev. 106:3055, 2006. other inherited prion diseases. We are gathering informa- Boehr, D.D., McElheny, D., Dyson, H.J., Wright, P.E. The dynamic energy land- tion on the mechanism of PrPSc formation that can be scape of dihydrofolate reductase catalysis. Science 313:1638, 2006. obtained from structural and dynamic studies of mutant prion proteins corresponding to inherited prion diseases. Individuals carrying familial mutations such as Ring Assemblies Mediating P102L (P101L in our study) are more susceptible than those without such mutations to prion disease. On the ATP-Dependent Protein Folding other hand, sheep or humans carrying Q167R and/or Q218K mutations are resistant to scrapie and Creutzfeldt- and Unfolding Jakob disease, respectively. We are using the protease- A.L. Horwich, W.A. Fenton, E. Chapman, E. Koculi, resistant cores of wild-type and mutant mouse prion J. Hinnerwisch proteins to study the structural and dynamic basis of PrPC-to-PrPSc conversion in inherited prion diseases. arge ring assemblies function in many cellular The core is sufficient to transmit infectivity. contexts as compartments within a compartment, DYNAMICS AND THE FUNCTION OF IΚ B α L where actions can be carried out on a substrate It is becoming increasingly clear that the function bound in the central space inside an oligomeric ring of many systems in living cells depends not only on the by a high local concentration of surrounding active sites. structures of the components but also on their flexibil- Both protein folding and unfolding are carried out in ity. Numerous examples exist in which components of an ATP-dependent fashion by such assemblies (Fig. 1). MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 185

with a similarly obtained GroEL-GroES-ADP structure (8.7-Å resolution), we discovered that the differences occur mainly in the trans (unliganded) ring. Information on the nucleotide state of the cis ring appears to be transmitted through the positioning of helix D, which extends from the nucleotide-binding site to the inter-ring interface, where it interacts with the corresponding helix in the trans ring. ATP binding Fig. 1. Protein folding and unfolding by chaperone ring assemblies. In protein folding mediated by the chaperonin GroEL (left), changes the relationship of these helices from that of the energy of binding ATP and the cochaperonin GroES is used to an unliganded ring, resulting in a series of changes in produce rigid body movements of a GroEL ring that eject a bound the interface and the position of the trans helix that nonnative substrate polypeptide into a GroES-encapsulated central lead to a closing of the nucleotide pocket and a reduc- cavity, switched from hydrophobic (shaded) to hydrophilic wall tion in exposure of the substrate-binding hydrophobic character, where productive folding proceeds. The free energy pro- residues in the trans apical domain. Hydrolysis of ATP vided by a set of hydrogen bonds formed between the γ-phosphate of ATP and the nucleotide pocket is critical to producing a power to ADP reverses these changes, opening both the trans stroke of apical domain movement that can eject the substrate apical domains and the trans nucleotide pocket to per- polypeptide into the folding chamber. In contrast, in ClpA-mediated mit ligand binding and the formation of a new folding unfolding (right), this chaperone seems to use ATP hydrolysis by its chamber on this ring. D2 ATPase domain to drive a forceful distalward movement of a THE TRAJECTORY OF PROTEIN FOLDING AT GroEL loop facing its central channel, exerting mechanical force on a Another question of interest regarding protein folding bound protein that is proposed to exert an unfolding action. by GroEL is whether, and how, GroEL affects the fold- We are studying the essential double-ring components ing pathway taken by a substrate protein. Most likely known as chaperonins that assist protein folding to the some action beyond simply preventing aggregation is native state. We are focusing on the bacterial chaper- occurring, because some substrate proteins cannot fold onin GroEL. We have also been examining an opposite spontaneously without GroEL and GroES function. In number, an “unfoldase,” the bacterial heat-shock protein collaboration with R. Horst and K. Wüthrich, Depart- 100 ring assembly known as ClpA. During the past year, ment of Molecular Biology, we are studying the folding we continued to investigate the structural correlates of pathways of human dihydrofolate reductase during both the ATPase cycle of GroEL and the polypeptide-binding spontaneous and GroEL-assisted folding. Using hydro- mechanisms of ClpA. gen-deuterium exchange during refolding of 15N-labeled G roEL STRUCTURE AND ALLOSTERY human dihydrofolate reductase and nuclear magnetic res- GroEL is an allosteric and highly cooperative machine onance analysis of the final native form, we are examin- with positive cooperativity of ATP binding among the ing pathways taken inside and outside the chaperonin. subunits of a ring and negative cooperativity of binding ROLE OF THE N-DOMAINS OF ClpA between the 2 rings. A long-standing question has been We and others have shown that the N-domains of how the information on nucleotide binding and ATP-vs- ClpA are not required for action on substrates bearing ADP state is transmitted from one ring to the other to the ssrA degradation tag, but removal of the N-domains produce these changes in affinity that lie at the heart slows the unfolding and degradation of such substrates. of the chaperonin protein-folding cycle. We explored this observation further and found that In collaboration with N.A. Ranson, University of whereas binding and unfolding of ssrA-tagged substrate Leeds, Leeds, England, and H.R. Saibil, University of are not affected by removal of the N-domains, removal London, London, England, we completed a electron of the domains results in diminished stability of a com- cryomicroscopy reconstruction of the transient complex plex composed of ClpA and ClpP, a double-ring protease composed of GroEL, its cochaperonin GroES, and ATP by that cooperates with ClpA to degrade certain proteins. taking advantage of a mutant form of GroEL (D398A). Consequently, proteins unfolded and translocated by The mutant form binds ATP normally but hydrolyzes it at ClpA interact with the protease component and hence about 2% of the normal rate, permitting the complex to escape degradation. In contrast, substrate proteins bear- be captured by rapid freezing on electron cryomicroscopy ing a RepA degradation tag appear to be completely grids. By comparing this structure (7.7-Å resolution) dependent on the N-domains for initial binding to ClpA. 186 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

LOOPS IN ClpA sequence specificity and perhaps decrease unwanted In collaboration with A. Deniz, Department of side effects while retaining the ability of the compound Molecular Biology, we are examining the role of the to kill cancer cells. We recently found that a specific recently identified loops in ClpA that mediate ssrA bind- polyamide-chlorambucil conjugate called 1R-Chl alters ing and substrate protein translocation and associated the morphology and growth characteristics of colon unfolding. We have designed a series of cysteine substi- carcinoma cells in culture and causes the cells to arrest tution variants of ClpA and ClpP that can be labeled with in the G2/M stage of the cell cycle, without any appar- fluorescent reporters and used in single-molecule experi- ent cytotoxic effects. ments designed to observe the putative motion of these Cells treated with 1R-Chl do not grow in soft agar loops during the substrate translocation/unfolding cycle and do not form tumors in nude mice, indicating that of ClpA. Such experiments may enable us to correlate polyamide-treated cells are no longer tumorigenic. The movement with ATP hydrolysis and to determine whether compound blocks proliferation of metastatic colon car- such movement is coordinated among the 6 subunits cinoma cells in immunocompromised mice, and no of a ClpA ring or is random. apparent toxic effects occur at doses required for a therapeutic effect. Importantly, this gene-targeted small PUBLICATIONS Hinnerwisch, J., Reid, B.G., Fenton, W.A., Horwich, A.L. Roles of the N-domains molecule requires no delivery vehicle because the mol- of the ClpA unfoldase in binding substrate proteins and in stable complex formation ecule is cell permeable and localizes in the nucleus of with the ClpP protease. J. Biol. Chem. 280:40838, 2005. various cancer cell lines. Using microarray analysis, we Horst, R., Wider, G., Fiaux, J., Bertelsen, E.B., Horwich, A.L., Wüthrich, K. Pro- found that the gene target of 1R-Chl is the gene for ton-proton Overhauser NMR spectroscopy with polypeptide chains in large structures. Proc. Natl. Acad. Sci. U. S. A. 103:15445, 2006. histone H4c, a member of the gene family that encodes

Ranson, N.A., Clare, D.K., Farr, G.W., Houldershaw, D., Horwich, A.L., Saibil, a critical component of cellular chromatin and a gene H.R. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes. Nat. Struct. that is highly expressed in a wide range of cancer cells. Mol. Biol. 13:147, 2006. Reduction in histone H4 protein by polyamide treatment was confirmed in cells treated with 1R-Chl, which caused chromatin decondensation. Chemical Regulation of To confirm that downregulation of histone H4c Gene Expression transcription is the primary event leading to cell-cycle arrest by 1R-Chl, we turned to short interfering RNAs D. Alvarez, R. Burnett, C.J. Chou, D. Herman, K. Jenssen, directed toward H4c mRNA. Unlike 1R-Chl, which

S. Ku, E. Soragni, J. Puckett,* S. Tsai,* M. Farkas,* arrests cells at the G2/M phase of the cell cycle, the P.B. Dervan,* J.M. Gottesfeld H4c short interfering RNA arrests cells at the G1/S * California Institute of Technology, Pasadena, California phase. However, G2/M arrest by 1R-Chl and downreg- he ability to control gene expression at will has ulation of the H4c gene can be confirmed in other been a longstanding goal in molecular biology and tumorigenic cell lines. We found that 1R-Chl causes T human medicine. We focus on pyrrole-imidazole extensive DNA damage in colon cancer cells, leading polyamides, a class of small molecules that can be pro- to phosphorylation of histone H2A.X at serine 139 and grammed by chemical synthesis to recognize a wide recruitment of the DNA repair protein Nbs1 to discrete range of DNA sequences. The following is a summary sites in the genome. These events are hallmarks of the of our recent efforts to develop polyamides as therapeu- cellular DNA damage response pathway. Control poly- tic agents for human disease and to identify another amide-Chl conjugates that lack binding sites in the H4c class of small molecules that offer promise in the treat- gene and have no antiproliferative effects by themselves ment of neurodegenerative diseases. can cause G2/M cell-cycle arrest when used in combination with short interfering RNAs to histone mRNAs. BLOCKING CANCER CELL PROLIFERATION WITH On the basis of these findings, we propose that A POLYAMIDE-CHLORAMBUCIL CONJUGATE The nitrogen mustard chlorambucil is a common 1R-Chl exerts its antiproliferative effect through a novel DNA alkylator used to treat a variety of lymphatic can- 2-hit mechanism. The highly transcribed H4c gene in cers. Because chlorambucil alkylates DNA at all poten- several cancer cell lines is a primary target for DNA tially available guanine residues in the genome, coupling alkylation by 1R-Chl, resulting in downregulation of of chlorambucil to a polyamide will increase the DNA- H4c transcription and histone H4 protein. Loss of his- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 187 tone protein leads to a transition from condensed to open tion methods to examine the chromatin structure of the chromatin, exposing otherwise hidden binding sites for gene for frataxin in normal cells and in cell lines derived 1R-Chl. These sites are then alkylated by 1R-Chl, caus- from patients with Friedreich’s ataxia. We found that gene ing widespread DNA damage and a cascade of events silencing at expanded frataxin alleles was accompa- leading to G2/M arrest and loss of tumorigenicity. nied by hypoacetylation of histones H3 and H4 and Our findings indicate how a single molecule can tar- methylation of histone H3 at lysine 9, consistent with a get cancer cells because of a specific gene expression heterochromatin-mediated repression mechanism. profile and block cancer cell proliferation. Ongoing stud- These findings suggest that histone deacetylase inhib- ies are aimed at the development of 1R-Chl as a poten- itors, compounds that reverse heterochromatin, might tial human cancer therapeutic agent. activate frataxin. We identified a commercial histone POLYAMIDES AS ACTIVATORS OF GENE EXPRESSION deacetylase inhibitor, BML-210, that partially reverses The neurodegenerative disease Friedreich’s ataxia silencing in the Friedreich’s ataxia cell line. On the basis is caused by gene silencing through expansion of GAA- of the structure of this compound, we synthesized and TTC triplet repeats in the first intron of a nuclear gene assayed a series of derivatives of BML-210 and identi- that encodes the essential mitochondrial protein frataxin. fied histone deacetylase inhibitors that reverse frataxin Normal frataxin alleles have 6–34 repeats whereas alle- silencing in primary lymphocytes from patients with les from patients with Friedreich’s ataxia have 66–1700 Friedreich’s ataxia. These molecules act directly on the repeats. Longer repeats cause a more profound frataxin histones associated with frataxin, increasing acetylation deficiency and are associated with earlier onset and at particular lysine residues on histones H3 and H4. increased severity of the disease. Two models have Unlike many triplet-repeat diseases (e.g., the polyglut- been proposed to account for gene silencing by expanded amine expansion diseases such as Huntington’s disease GAA-TTC repeats: unusual DNA structures and repres- and the spinocerebellar ataxias), expanded GAA-TTC sive heterochromatin. triplets do not alter the coding potential of frataxin. Molecules that reverse formation of unusual DNA Thus, gene activation would be of therapeutic benefit. structures and/or heterochromatin in the gene for frataxin Studies in animals are under way to explore the bioavail- most likely increase transcription through expanded ability and efficacy of these histone deacetylase inhibitors. GAA-TTC repeats, thereby relieving the deficiency in PUBLICATIONS frataxin mRNA and protein in cells from patients with Alvarez, D., Chou, C.J., Latella, L., Zeitlin, S.G., Ku, S., Puri, P.L., Dervan, P.B., Friedreich’s ataxia. We found that polyamides target- Gottesfeld, J.M. A two-hit mechanism for pre-mitotic arrest of cancer cell proliferation by a polyamide-alkylator conjugate. Cell Cycle 5:1537, 2006. ing GAA-TTC repeats partially alleviated transcription repression of frataxin in a cell line derived from white Burnett, R., Melander, C., Puckett, J.W., Son, L.S., Wells, R.D., Dervan, P.B., Gottesfeld, J.M. DNA sequence-specific polyamides alleviate transcription inhibi- blood cells from a patient with Friedreich’s ataxia. These tion associated with long GAA-TTC repeats in Friedreich’s ataxia. Proc. Natl. Acad. molecules also increased frataxin protein levels in these Sci. U. S. A. 103:11497, 2006. cells, and microarray studies showed that a limited Herman, D., Jenssen, K., Burnett, R., Soragni, E., Perlman, S.L., Gottesfeld, J.M. Histone deacetylase inhibitors reverse gene silencing in Friedreich’s ataxia. Nat. number of genes in the human genome were affected Chem. Biol. 2:551, 2006. by polyamides targeting GAA-TTC repeat DNA. Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, We hypothesize that polyamides might act as a ther- D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of modynamic “sink” and lock GAA-TTC repeats into double- 5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006. stranded B DNA. Such an event would disfavor duplex Trzupek, J.D., Gottesfeld J.M., Boger D.L. Alkylation of duplex DNA in nucleosome core particles by duocarmycin SA and yatakemycin. Nat. Chem. Biol. 2:79, 2006. unpairing, which is necessary for formation of the unusual DNA structures associated with expanded triplet repeats. Alternatively, polyamides may relieve heterochromatin-mediated repression by opening the chromatin Nucleic Acid Dynamics domain containing frataxin. To explore this last hypoth- D.P. Millar, J. Gill, G. Pljevalj˘ci´c, S. Pond, G. Stengel, esis, we turned to another class of small molecules. N. Tassew, E.J.C. Van der Schans HISTONE DEACETYLASE INHIBITORS THAT REVERSE FRATAXIN SILENCING he focus of our research is the assembly and We used antibodies to the various modification states conformational dynamics of nucleic acid–based of the core histones and chromatin immunoprecipita- T macromolecular machines and assemblies. We 188 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE use single-molecule fluorescence methods to investi- cules immobilized on a solid surface. We also use sin- gate a range of systems, including ribozymes, ribonu- gle-pair FRET to probe changes in the conformation of cleoprotein complexes, and DNA polymerases. Our the RRE during the assembly process. We are using the studies reveal the dynamic structural rearrangements results of these mechanistic studies to develop novel that occur during the assembly and function of these fluorescence-based methods for high-throughput screen- macromolecular machines. ing of libraries of chemical compounds. The new screen- RIBOZYMES ing tools are being used to identify small molecules that RNA conformation plays a central role in the mecha- block binding of Rev to the RRE or prevent the subse- nism of ribozyme catalysis. The hairpin ribozyme is a quent Rev-Rev oligomerization. small nucleolytic ribozyme that serves as a model sys- DNA POLYMERASES tem for studies of RNA folding and catalysis. The hair- DNA polymerases are remarkable for their ability to pin ribozyme consists of 2 internal loops, 1 of which synthesize DNA at rates approaching several hundred contains the scissile phosphodiester bond, displayed base pairs per second while maintaining an extremely on 2 arms of a 4-way multihelix junction. low frequency of errors. To elucidate the origin of poly- To attain catalytic activity, the ribozyme must fold merase fidelity, we are using single-molecule fluores- into a compact conformation in which the 2 loops cence methods to examine the dynamic interactions become connected by a network of tertiary hydrogen that occur between a DNA polymerase and its DNA bonds. We monitor the formation of this docked struc- and nucleotide substrates. The FRET method is being ture by using fluorescence resonance energy transfer used to observe conformational transitions of the (FRET) and ribozyme constructs labeled with donor and enzyme-DNA complex that occur during selection and acceptor dyes within the loop-bearing arms. By mea- incorporation of an incoming nucleotide substrate. suring FRET at the level of single ribozyme molecules, Our results reveal that binding of a correct nucleotide we reveal subpopulations of compact and extended con- substrate induces a slow conformational change within formers that are not detected in ensemble experiments. the polymerase, causing the “fingers” subdomain to close Using this approach, we found that the ribozyme pop- over the DNA primer terminus and incoming nucleotide. ulates an intermediate state in which the 2 loops are Our studies are providing new insights into the dynamic in proximity but tertiary interactions have yet to form. structural changes responsible for nucleotide recognition This quasi-docked state forms rapidly (submillisecond and selection by DNA polymerases. Single-pair FRET timescale), but the subsequent formation of the tertiary methods are also being used to monitor the movement contacts between the 2 loops occurs much more slowly. of the DNA primer/template between the separate poly- The hairpin ribozyme is an ideal system for exploring merizing and editing sites of the enzyme. This active-site this fundamental mechanism of the formation of RNA switching of DNA plays a key role in the proofreading tertiary structure. process used to remove misincorporated nucleotides RIBONUCLEOPROTEIN ASSEMBLY from the newly synthesized DNA. The advantage of The Rev protein from HIV type 1 is a key regulatory single-molecule observations is that they eliminate the protein that controls the transition from early to late need to synchronize a population of molecules, allow- patterns of viral gene expression. Rev binds to a highly ing these dynamic processes to be directly observed. structured region within the viral mRNA, known as the PUBLICATIONS Rev response element (RRE), where it forms an oligo- Bailey, M.F., Van der Schans, E.J.C., Millar, D.P. Dimerization of the Klenow fragment of Escherichia coli DNA polymerase I is linked to its mode of DNA binding. meric ribonucleoprotein complex. The formation of this Biochemistry, in press. complex inhibits splicing and facilitates export of the Tian, F., Debler, E.W., Millar, D. P., Deniz, A.A., Wilson, I.A., Schultz, P.G. Multi- viral RNA from the nucleus to the cytoplasm. Because color fluorescent antibodies. Angew. Chemie, in press. of its critical role in the viral life cycle, the Rev-RRE complex provides a novel target for the development of therapeutic drugs. To dissect the mechanism of assembly of ribonucleoprotein complexes, we use single-molecule fluorescence imaging methods to monitor the progressive formation of oligomeric complexes of Rev on individual RRE mole- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 189 Single-Molecule Biophysics imental repertoire, to further facilitate studies of molecular structure, folding, and function. A.A. Deniz, S.Y. Berezhna, J.P. Clamme, A.C.M. Ferreon, Finally, using high-sensitivity fluorescence imaging, Y. Gambin, E. Lemke, S. Mukhopadhyay, P. Zhu we are beginning to study and compare the pathways of nuclear and cytoplasmic RNA interference. In studies e develop and use state-of-the-art single- done in collaboration with P.G. Schultz, Department of molecule fluorescence methods to address Chemistry, our observations of the localization of small W key biological questions. Single-molecule and interfering RNA in live cells provide evidence for a yet- small-ensemble methods offer key advantages over tradi- to-be-determined mechanism that directs the RNA to tional measurements, allowing us to directly observe cellular compartments containing the target RNA. the behavior of individual subpopulations in mixtures of molecules and to measure kinetics of structural tran- PUBLICATIONS Berezhna, S.Y., Supekova, L., Supek, F., Schultz, P.G., Deniz, A.A. siRNA in sitions of stochastic processes under equilibrium condi- human cells selectively localizes to target RNA sites. Proc. Natl. Acad. Sci. U. S. A. tions. We use these methods to study multiple structural 103:7682, 2006. states or reaction pathways during the folding and Zhu, P., Clamme, J.-P., Deniz, A.A. Fluorescence quenching by TEMPO: a sub-30 assembly of biomolecules. Å single-molecule ruler. Biophys. J. 89:L37, 2005. A major goal is to apply single-molecule methods to studies of protein folding and aggregation. Using relatively simple model systems, we are addressing several Computer Modeling of Proteins fundamental questions about folding mechanisms. Par- tially folded or misfolded protein structures are also and Nucleic Acids thought to play important cellular roles, and these states also can be studied by using single-molecule methods. D.A. Case, M. Crowley, Q. Cui, F. Dupradeau,* S. Moon, In this context, we are examining the interplay between D. Nguyen, V. Pelmentschikov, D. Shivakumar, R.C. Walker, folding and aggregation of Sup35, a yeast prion protein, W. Zhang, J. Ziegler** in collaboration with S.L. Lindquist, Whitehead Institute * Université Jules Verne, Amiens, France for Biomedical Research, Cambridge, Massachusetts, ** Universität Bayreuth, Bayreuth, Germany and of α-synuclein, a protein implicated in the patho- omputer simulations offer an exciting approach to genesis of Parkinson’s disease and other neurodegen- the study of many aspects of biochemical inter- erative diseases. C actions. We focus primarily on molecular dynam- In addition, we have developed a single-molecule ics simulations (in which Newton’s equations of motions fluorescence quenching method that will be useful for are solved numerically) to model the solution behavior measuring distances shorter than 30 Å in proteins and of biomacromolecules. Recent applications include RNA, a scale at which the resolution of single-pair fluo- detailed analyses of electrostatic interactions in short rescence resonance energy transfer (FRET) is low. This peptides (folded and unfolded), proteins, and oligonu- method is being used to monitor structural properties of cleotides in solution. Sup35 as a function of the aggregation process. In addition, molecular dynamics methods are use- To better study the folding, assembly, and activity ful in refining solution structures of proteins by using of larger and multicomponent biological complexes, we constraints derived from nuclear magnetic resonance are developing new multicolor single-molecule FRET (NMR) spectroscopy, and we continue to explore new methods. As part of this continuing goal, we have been methods in this area. Our developments are incorporated improving our recently developed diffusion 3-color sin- into the Amber molecular modeling package, designed gle-molecule FRET method for simultaneously measur- for large-scale biomolecular simulations, and into other ing more than a single intramolecular or intermolecular software, including Nucleic Acid Builder, for developing distance. In collaboration with J.R. Williamson, Depart- 3-dimensional models of unusual nucleic acid structures; ment of Molecular Biology, we are using these novel SHIFTS, for analyzing chemical shifts in proteins and methods to study the detailed mechanisms of assembly nucleic acids; RNAmotif, for finding structural motifs of fragments of the bacterial ribosome. Most recently, in genomic sequence databases; and DOCK, for plac- we began adding microfluidics capabilities to our exper- ing inhibitors into enzyme active sites. 190 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

NMR AND THE STRUCTURE AND DYNAMICS OF native states through molecular dynamics simulations PROTEINS AND NUCLEIC ACIDS and the construction of models for molecular motion Our overall goal is to extract the maximum amount and disorder. of information about biomolecular structure and dynam- All of these modeling activities are based on molecu- ics from NMR experiments. To this end, we are studying lar mechanics force fields, which provide estimates of the use of direct refinement methods for determining energies as a function of conformation. We continue to biomolecular structures in solution, going beyond dis- work on improvements in force fields; recently, we tance constraints to generate closer connections between focused on adding aspects of electronic polarizability, calculated and observed spectra. We are also using going beyond the usual fixed-charge models, and on quantum chemistry to study chemical shifts and spin- methods for handling arbitrary organic molecules that spin coupling constants. Other types of data, such as might be considered potential inhibitors in drug discov- chemical shift anisotropies, direct dipolar couplings in ery efforts. Overall, the new models should provide a partially oriented samples, and analysis of cross-corre- better picture of the noncovalent interactions between lated relaxation, are also being used to guide structure peptide groups and the groups’ surroundings, leading refinement. In recent structural studies, we focused on ultimately to more faithful simulations. the binding of zinc finger proteins with RNA and on VIBRATIONAL ANALYSIS OF IRON-SULFUR CLUSTERS structural influences on amide proton chemical shifts. IN PROTEINS NUCLEIC ACID MODELING A wide variety of proteins contain iron-sulfur clusters Another project centers on the development of novel at their active sites; these proteins participate in electron- computer methods to construct models of “unusual” transport chains and in important enzymatic reactions nucleic acids that go beyond traditional helical motifs. such as the reduction of atmospheric nitrogen to ammo- We are using these methods to study circular DNA, small nia by nitrogenase. Advances in synchrotron radiation RNA fragments, and 3- and 4-stranded DNA complexes, sources now make it possible to probe the vibrational including models for recombination sites. We continue behavior of these clusters by using nuclear resonance to develop efficient computer implementations of contin- vibrational spectroscopy (NRVS). This technique senses uum solvent methods to allow simplified simulations the coupling of a nuclear (Mossbauer) excitation to that do not require a detailed description of the sol- molecular vibrations. The result is a set of vibrational fre- vent (water) molecules; this approach also provides a quencies and intensities that indicate what sorts of defor- useful way to study salt effects. mations can take place. When the molecular structure is Recent efforts have made second derivatives of these known, this information can contribute to the under- energies available, so that normal mode analyses of standing of oxidation-reduction behavior and electron nucleic acids with dozens to hundreds of nucleotides transfer kinetics. In situations in which the cluster struc- can be analyzed and the predictions compared with ture is not known, NRVS data might useful as a “finger- those of simpler, elastic continuum models. These print” to help identify the structure. efforts provide a new avenue for developing and test- We have been using quantum chemistry calcula- ing low-resolution models that can be used for large tions to help understand NRVS spectra. Figure 1 shows molecular assemblies. a early example, comparing calculated and experimen- DYNAMICS AND ENERGETICS OF NATIVE AND tal spectra for a simple iron-sulfur “cubane” structure, NONNATIVE STA TES OF PROTEINS a cluster type found in hundreds of known proteins. The Analysis methods similar to those described for calculations (shown as a dashed line) are in excellent nucleic acids are also being used to estimate thermo- agreement with experimental data (solid line), both in dynamic properties of “molten globules” and unfolded terms of frequencies and in terms of intensities. We are states of proteins. These studies are an extension of our extending these calculations to models for the active earlier work on the folding of peptide fragments of pro- site of nitrogenase, where the structure of the complex teins. A key feature is the development of computational is still uncertain. If calculations like these can be used methods that can be used to model pH and salt depen- to closely track the experimental results, NRVS will be dence of complex conformational transitions such as an important new tool for characterizing the active sites unfolding events. A second aspect of this research is a of metalloenzymes. detailed interpretation of NMR results for protein non- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 191

Rizzo, R.C., Aynechi, T., Case, D.A., Kuntz, I.D. Estimation of absolute free energies of hydration using continuum methods: accuracy of partial charge models and optimization of nonpolar contributions. J. Chem. Theory Comput. 2:128, 2006.

Steinbrecher, T., Case, D.A., Labahn, A. A multistep approach to structure-based drug design: studying ligand binding at the human neutrophil elastase. J. Med. Chem. 49:1837, 2006.

Wang, J., Wang, W., Kollman, P.A., Case, D.A. Automatic atom type and bond type perception in molecular mechanical calculations. J. Mol. Graphics Model. 25:247, 2006.

Xiao, Y., Fisher, K., Smith, M.C., Newton, W.E., Case, D.A., George, S.J., Wang, H., Sturhahn, W., Alp, E.E., Zhao, J., Yoda, Y., Cramer, S.P. How nitrogenase shakes: initial information about P-cluster and FeMo-cofactor normal modes from nuclear resonance vibrational spectroscopy (NRVS). J. Am. Chem. Soc. 128:7608, 2006.

Xiao, Y., Koutmos, M., Case, D.A., Coucouvanis, D., Wang H., Cramer, S.P. 2– Dynamics of an [Fe4S4(SPh)4] cluster via IR, Raman, and nuclear resonance vibrational spectroscopy (NRVS): analysis using 36S substitution, DFT calculations, and empirical force fields. Dalton Trans. 2192, 2006, Issue 18.

Quantum Chemistry of Redox- Active Metalloenzymes

L. Noodleman, D.A. Case, W.-G. Han, V. Pelmenschikov, J.A. Fee, L. Hunsicker-Wang,* T. Lovell,** T. Liu*** * Trinity University, San Antonio, Texas ** AstraZeneca R&D, Mölndal, Sweden *** University of Maryland, College Park, Maryland

Fig. 1. Calculated and experimental NRVS spectra for an iron- e use a combination of modern quantum sulfur cluster. chemistry (density functional theory, DFT) W and classical electrostatics to describe the PUBLICATIONS energetics, reaction pathways, and spectroscopic proper- Baker, N.A., Bashford, D., Case, D.A. Implicit solvent electrostatics in biomolecular simulation. In: New Algorithms for Macromolecular Simulation. Leimkuhler, B., ties of metalloenzymes. et al. (Eds.). Springer, New York, 2006, p. 263. Lecture Notes in Computational Critical biosynthetic and regulatory processes may Science and Engineering, Vol. 49. involve catalytic transformations of fairly small mole- Brown, R.A., Case, D.A. Second derivatives in generalized Born theory. J. Comput. Chem. 27:1662, 2006. cules or groups by transition-metal centers. The iron- molybdenum cofactor center of nitrogenase catalyzes Case, D.A., Cheatham, T.E. III, Darden, T., Gohlke, H., Luo, R., Merz, K.M., Jr., Onufriev, A., Simmerling, C., Wang, B., Woods, R. The Amber biomolecular simu- the multielectron reduction of molecular nitrogen to 2 lation programs. J. Comput. Chem. 26:1668, 2005. molecules of ammonia plus molecular hydrogen. We

Dixit, S.B., Beveridge, D.L., Case, D.A., Cheatham, T.E. III, Giudice, E., Lankas, are continuing our work on the catalytic cycle of this R., Lavery, R., Maddocks, J.H., Osman, R., Sklenar, H., Thayer, K.M., Varnai, P. enzyme, following up on our earlier work on the struc- Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides, II: sequence context effects on the dynamical structures of ture and oxidation state of the cofactor complex in the the 10 unique dinucleotide steps. Biophys. J. 89:3721, 2005. “resting enzyme” before multielectron reduction and Dupradeau, F.-Y., Case, D.A., Yu, C., Jimenez, R., Romesberg, F.E. Differential nitrogen binding. solvation and tautomer stability of a model base pair within the minor and major grooves of DNA. J. Am. Chem. Soc. 127:15612, 2005. On the basis of DFT calculated vs experimental physical properties, including redox potentials, cluster Lee, B.M., Xu, J., Clarkson, B.K., Martinez-Yamout, M.A., Dyson, H.J., Case, D.A., Gottesfeld, J.M., Wright, P.E. Induced fit and “lock and key” recognition of geometries, and Mössbauer isomer shifts, the core 5S RNA by zinc fingers of transcription factor IIIA. J. Mol. Biol. 357:275, 2006. cluster has a MoFe7S9X prismane active site, where Mathews, D.H., Case, D.A. Nudged elastic band calculation of minimal energy the central X most likely is nitride and the “resting pathways for the conformational change of a GG noncanonical pair. J. Mol. Biol. cluster oxidation state” is Mo(IV)Fe(II) Fe(III) . If the 357:1683, 2006. 4 3 central ligand is nitride, as we have proposed, this Moon, S., Case, D.A. A comparison of quantum chemical models for calculating ligand is not a substrate or a reaction product of the NMR shielding parameters in peptides: mixed basis set and ONIOM methods combined with a complete basis set extrapolation. J. Comput. Chem. 27:825, 2006. catalytic cycle. Instead, nitride is inserted into a cen- 192 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE tral vacancy site of a more open iron-molybdenum cofac- Noodleman, L., Han, W.-G. Structure, redox, pKa, spin: a golden tetrad for understanding metalloenzyme energetics and reaction pathways. J. Biol. Inorg. Chem. tor precursor, probably in a noncatalytic deamination 11:674, 2006. process that occurs before insertion of the cluster into the iron-molybdenum protein. Class I ribonucleotide reductases are aerobic enzymes that catalyze the reduction of ribonucleotides to deoxyri- bonucleotides, providing the required building blocks for DNA replication and repair. These ribonucleotide- to-deoxyribonucleotide reactions occur via a long-range radical (or proton-coupled electron transfer) propagation mechanism initiated by a fairly stable tyrosine radical, “the pilot light.” When this pilot light goes out, the tyrosine radical is regenerated by a high-oxidation-state Fe(III)-Fe(IV)-oxo enzyme called intermediate X. We are using DFT and electrostatics calculations in combination with analysis of Mössbauer, electron nuclear double resonance, and magnetic circular dichroism spectroscopies to search for a proper structural and electronic model for intermediate X. We have also examined the mechanism of formation of intermediate X, starting from an earlier Fe(III)2-µ- peroxo intermediate (Fig. 1). Spectroscopic and quantum chemical DFT evidence indicates that the formation of intermediate X is proton catalyzed. On the basis of calculations of spectroscopic parameters and energies, we propose that intermediate X contains a dioxo bridging the Fe(III)-Fe(IV) in an asymmetric diamond structure. The Fe(IV) site is farther from and the Fe(III) site is closer to the redox-active tyrosine 122. Figure 2 shows the molecular orbitals corresponding to the lowest energy Fe(IV) d→d optical excitation. The 3 Fe(IV) d→d bands that we predict on the basis of DFT vertical self-consistent reaction field methods are in excellent agreement with the bands observed by using magnetic circular dichroism spectroscopy. Further exploration of the tyrosine radical activation and subsequent catalytic cycle are planned.

PUBLICATIONS Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Active site structure of class I ribonucleotide reductase intermediate X: a density functional theory analysis of structure, energetics, and spectroscopy. J. Am. Chem. Soc. 127:15778, 2005.

Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Density functional theory study of Fe(IV) d-d optical transitions in active-site models of class I ribonucleotide reductase intermediate X with vertical self-consistent reaction field methods. Inorg. Chem. 45:8533, 2006.

Han, W.-G., Liu, T., Lovell, T., Noodleman, L. DFT calculations of 57Fe Mössbauer isomer shifts and quadrupole splittings for iron complexes in polar dielectric media: Fig. 1. A feasible path showing how ribonucleotide reductase applications to methane monooxygenase and ribonucleotide reductase. J. Comput. Chem. 27:1292, 2006. intermediate X is formed by the reaction of oxygen with the reduced ribonucleotide reductase–R2 di-iron center. Reproduced Han, W.-G., Liu, T., Lovell, T., Noodleman, L. Seven clues to the origin and struc- with permission from J. Am. Chem. Soc. 127:15778, 2005. ture of class-I ribonucleotide reductase intermediate X. J. Inorg. Biochem. 100:771, 2006. Copyright 2005 American Chemical Society. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 193

tum chemistry to aid in determining the parameters for the models. Calculation of thermodynamic properties requires the development and implementation of new theoretical and computational approaches that connect averages over atomistic descriptions to experimentally measurable thermodynamic and kinetic properties. Interpreting experimental results at more microscopic levels is fueled by the development and investigation of theoretical models for the processes of interest. Massive computational resources are needed to realize these objectives, and this motivates our efforts aimed at the efficient use of new computer architectures, including large supercomputers, Linux Beowulf clusters, computational grids, and Internet-based volunteer supercomputers. Each of the objectives and techniques mentioned Fig. 2. Our proposed model for the active site of class I ribonu- represents an ongoing development area within our cleotide reductase intermediate X. Molecular orbital plots show the lowest energy Fe(IV) d→d optical excitation. research program in computational biophysics. The following are highlights of a few specific projects. FOLDING, STRUCTURE, AND FUNCTION OF Theoretical and Computational MEMBRANE-BOUND PROTEINS Molecular Biophysics Folding, insertion, assembly, and stability of membrane proteins are directly governed by the unique C.L. Brooks III, C. An, R. Armen, I. Borelli, D. Bostick, hydrophilic and hydrophobic environment provided by D. Braun, L. Bu, J. Chen, M.F. Crowley, O. Guvench, R. Hills, biological membranes. Modeling this heterogeneous W. Im,* J. Khandogin, I. Khavrutskii, J. Lee, J. Magee,** environment is both an obstacle and an essential req- R. Manige, M. Michino, A. Mitsutake,*** H.D. Nguyen, uisite to experimental and computational studies of the S. Patel,**** D.J. Price, V. Reddy, H.A. Scheraga,***** structure and function of membrane proteins. Because C. Shepard, F. Tama,† I.F. Thorpe, M.C. Tripp, R. Wheeler,†† of the biological importance and marked presence of C. Wildman, K. Yoshimoto membrane proteins in known genomes (i.e., about 30% * Kansas University, Lawrence, Kansas of all proteins), one aim of modern molecular biophysics ** University of Manchester, Manchester, England should be the development of methods that can be used *** Kelo University, Tokyo, Japan in experimental studies to understand the structure and **** University of Delaware, Newark, Delaware function of these systems. We recently developed theoret- ***** Cornell University, Ithaca, New York ical methods that enable the exploration of protein inser- † University of Arizona, Tucson, Arizona tion and folding in membranes. These methods combine †† University of Oklahoma, Norman, Oklahoma the sampling methods of replica-exchange molecular nderstanding the forces that determine the dynamics with novel generalized Born implicit solvent/ structure of proteins, peptides, nucleic acids, implicit membrane continuum electrostatic theories. U and complexes containing these molecules and A key question these methods allow us to address the processes by which these structures are adopted is the association of integral membrane proteins to form is essential to complete our knowledge of the molecular oligomeric structures. Many important functional com- nature of structure and function. To address such ques- plexes of membrane proteins exist as oligomers, such as tions, we use statistical mechanics, molecular simula- the signal-transducing G protein–coupled receptors and tion, statistical modeling, and quantum chemistry. membrane-bound ion channels and transporters. Our Creating atomic-level models to simulate biophysi- recent approach provides a way to predict the structures cal processes (e.g., protein folding or binding of a ligand of these key oligomeric states. Figure 1 shows the pre- to a biological receptor) requires (1) the development dicted oligomeric structures of glycophorin A (function- of new potential energy functions that accurately rep- ally a dimer), the tetrameric M2 transmembrane peptide resent the atomic interactions and (2) the use of quan- proton channel, and the phospholamban pentameric 194 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

One recent advance came in exploring the structure of the ribosome in complex with the SecY protein-conducting channel (PCC). The translocation of secreted and membrane proteins across or into cell membranes occurs through PCCs. Using an electron cryomicroscopy reconstruction of the Escherichia coli PCC, which consisted of SecY complexed with the ribosome and a nascent chain containing a signal anchor, we observed the components of protein synthesis and translocation, including mRNA, 3 tRNAs, the nascent chain, and features of both a translocating PCC and a second, nontranslocating PCC bound to mRNA hairpins (Fig. 2). Fig. 1. The predicted structure of dimeric glycophorin A, a dominant structural component of red blood cells, indicates the “classic” GVXXGV helical interface. For the M2 proton channel involved in replication of the influenza virus, the structure of the functional tetrameric proton-conducting channel is shown. In phospholamban, which is localized in the membrane of the cardiac sarcoplasmic reticulum and involved in phosphorylation-controlled regulation of the cardiac calcium pump, the predicted pentameric structure selectively conducts calcium. oligomer. Our calculations provide detailed predictions of the protein-protein interfaces for these systems and may Fig. 2. Electron cryomicroscopy image of the ribosome with 2 be useful in elucidating the primary oligomerization bound PCCs obtained during the modeling of structural components states. The predicted models shown in the figure are in of the SecY dimer into the electron density for the nontranslocating excellent agreement with existing structural models (from and translocating PCCs. The figure on the lower right illustrates the experiments and other model building). structure of the SecY dimer fit into the experimental electron densi- LARGE-SCALE FUNCTIONAL DYNAMICS IN ty map by using normal mode flexible fitting. NNMF indicates normal mode flexible fitting. MOLECULAR ASSEMBLIES Many naturally occurring machines, such as ribo- Normal mode flexible fitting of the SecYEb structure somes, myosin, and viruses, require large-scale dynami- into the PCC electron microscopy densities favors a cal motions as a component of their normal functioning. front-to-front arrangement of 2 SecYEG complexes in These motions involve the “mechanical” reorganization the PCC and supports channel formation by the opening of major parts of the structure of the machine in response of 2 linked SecY halves during polypeptide transloca- to binding of effectors or the addition of energy in the tion. From the models elucidated by the combination form of thermal fluctuations or provided by chemical of electron cryomicroscopy and modeling based on catalysis. Exploring and understanding the character normal mode flexible fitting, we were able to develop and nature of such large-scale reorganization of biologi- a model for cotranslational protein translocation. cal machines are ongoing goals in our laboratory. Using PUBLICATIONS theoretical approaches derived from the treatment of Chen, J., Im, W., Brooks, C.L. III. Application of torsion angle molecular dynamics mechanoelastic materials, we developed new structure for efficient sampling of protein conformations. J. Comput. Chem. 26:1565, 2005. refinement methods to model large-scale macromolecular Chen, J., Im, W., Brooks, C.L. III. Balancing solvation and intramolecular interactions: toward a consistent generalized Born force field. J. Am. Chem. Soc. assemblies. The methods are based on atomic-level 128:3728, 2006. structures of the component macromolecules (e.g., Im, W., Chen, J., Brooks, C.L. III. Peptide and protein folding and conformational RNAs, DNAs, and proteins) or on single-particle or equilibria: theoretical treatment of electrostatics and hydrogen bonding with tomographic images from electron microscopy. Using implicit solvent models. Adv. Protein Chem. 72:173, 1005. these new methods, which we call normal mode flexi- Khandogin, J., Brooks, C.L. III. Constant pH molecular dynamics with proton tautomerism. Biophys. J. 89:141, 2005. ble fitting, we have collaborated with several colleagues Khavrutskii, I.V., Byrd, R.H., Brooks, C.L. III. A line integral reaction path approx- in elucidating new structural models for functionally imation for large systems via nonlinear constrained optimization: application to ala- important molecular assemblies. nine dipeptide and the β-hairpin of protein G. J. Chem. Phys. 124:194903, 2006. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 195

Konecny, R., Trylska, J., Tama, F., Zhang, D., Baker, N.A., Brooks, C.L. III, showed the effectiveness of 3-dimensional molecular McCammon, J.A. Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids. Biopolymers 82:106, 2005. models as a tangible human-computer interface in educational and research settings. Within our compo- Mitra, K., Schaffitzel, C., Shaikh, T., Tama, F., Jenni, S., Brooks, C.L. III, Ban, N., Frank, J. Structure of the E. coli protein-conducting channel bound to a trans- nent-based visualization environment, we continue to lating ribosome. Nature 438:318, 2005. develop methods for predicting biomolecular interactions, Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, John- analyzing biomolecular structure and function, and pre- son, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol. 3:809, 2005. senting the biomolecular world in education and outreach. We have applied these methods to several important Patel, S., Brooks, C.L. III. Fluctuating charge force fields: recent developments and applications from small molecules to macromolecular biological systems. Mol. systems in human health and welfare. In a novel distrib- Simul. 32:231, 2006. uted computing network, we continue the search for HIV

Patel, S., Brooks, C.L. III. Revisiting the hexane-water interface via molecular protease inhibitors to fight the growing problem of drug dynamics simulations using nonadditive alkane-water potentials. J. Chem. Phys. resistance in HIV disease. We used AutoDock, a suite 124:204706, 2006. of programs for predicting bound conformations and bind- Patel, S., Brooks, C.L. III. Structure, thermodynamics, and liquid-vapor equilib- ing energies for biomolecular complexes, in the virtual rium of ethanol from molecular-dynamics simulations using nonadditive interactions. J. Chem. Phys. 123:164502, 2005. screening of large databases of compounds and ultimately

Price, D.J., Brooks, C.L. III. Detailed considerations for a balanced and broadly identified new compounds for use in the treatment of applicable force field: a study of substituted benzenes modeled with OPLS-AA. J. cancer. We used methods for predicting protein inter- Comput. Chem. 26:1529, 2005. action to probe the mechanism of blood coagulation. Tama, F., Brooks, C.L. III. Symmetry, form, and shape: guiding principles for T ANGIBLE INTERFACES IN STRUCTURAL BIOLOGY robustness in macromolecular machines. Annu. Rev. Biophys. Biomol. Struct. 35:115, 2006. We have continued to develop autofabricated physical models (“solid printing”) of biological molecules and Tama, F., Brooks, C.L. III. Unveiling molecular mechanisms of biological functions in large macromolecular assemblies using elastic network normal mode analysis. the components and assemblies of the molecules; our In: Normal Mode Analysis: Theory and Applications to Biological and Chemical goal is to use the models in both research and educa- Systems. Cui, Q., Bahar, I. (Eds.). Chapman & Hall/CRC Press, Boca Raton, FL, 2006, p. 111. Mathematical and Computational Biology Series. tion. We integrated computer graphics and computation

Taufer, M., An, C., Kerstens, A., Brooks, C.L. III. Predictor@Home: a “protein with these physical models by using augmented reality structure prediction supercomputer” based on global computing. IEEE Trans. Paral- to create custom interfaces to facilitate exploration and lel Distributed Syst. 7:786, 2006. computation of molecular interactions. We have begun Thorpe, I.F., Brooks, C.L. III. Conformational substates modulate hydride transfer to use a self-assisted protein-folding model to teach ele- in dihydrofolate reductase. J. Am. Chem. Soc. 127:12997, 2005. ments of protein structure and assembly to our graduate Trylska, J., McCammon, J.A., Brooks, C.L. III. Exploring assembly energetics of students. We are continuing to develop the software that the 30S ribosomal subunit using an implicit solvent approach. J. Am. Chem. Soc. 127:11125, 2005. will enable the control of interactive computations through manipulation of the tangible models. Yadav, M.K., Leman, L.J., Price, D.J., Brooks, C.L. III, Stout, C.D., Ghadiri, M.R. Coiled coils at the edge of configurational heterogeneity: structural analyses of par- In collaboration with T. Herman, Milwaukee School allel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution. Biochemistry 45:4463, 2006. of Engineering, Milwaukee, Wisconsin, we created a model of the active site of acetylcholinesterase that can be opened to show the buried active site and bound sub- Computation and Visualization strates (Fig. 1). This model was used, along with an interactive Internet guide to the structure, as part of a in Structural Biology Waksman Challenge at Rutgers, the State University of New Jersey. Groups of teachers and students were asked A.J. Olson, D.S. Goodsell, M.F. Sanner, S. Dallakyan, to use the models and associated materials to explore A. Gillet, R. Harris, Y. Hu, R. Huey, J. Huntoon, S. Karnati, problems with insecticide resistance in compounds that W. Lindstrom, G.M. Morris, A. Omelchenko, M. Pique, act on mosquito acetylcholinesterase. B. Norledge, R. Rosenstein, M. Utsintong, G. Vareille, Recently, we worked with Biomedical Graphics at Q. Zhang, Y. Zhao Scripps Research to establish a solid-model printing n the Molecular Graphics Laboratory, we develop service for researchers at Scripps and elsewhere. This novel computational methods to analyze, understand, service is now in operation and has made a number of I and communicate the structure and interactions molecular models for scientists working with molecular of complex biomolecular systems. This past year, we structures. The users’ responses have been positive, and 196 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

mal binding site for ligands on the surface of a protein of known structure. The method identifies the contiguous constant-volume region with the most favorable binding affinity. The optimal binding sites identify regions of primary binding affinity and regions of suboptimal binding strength, which can be used to predict the function of proteins if the function is unknown or to identify target locations for the design of new inhibitors. We showed the usefulness of the method in the design of inhibitors for HIV type 1 protease, and we are applying the method to a large database of protein structures. Fig. 1. A tangible model of the active site of acetylcholinester- COMPONENT-BASED VISUALIZATION ENVIRONMENTS ase. The model separates into 4 pieces, allowing students to fit different substrates into the buried active site. To facilitate the integration and interoperation of computational models and techniques from a wide vari- the research community is beginning to see how a solid ety of scientific disciplines, we continue to expand our 3-dimensional model can provide tangible, multimodal component-based software environment. The environ- feedback that mouse, keyboard, and image behind a ment is centered on Python, a high-level, object-oriented, glass screen cannot provide. interpretive programming language. This approach allows ADVANCES IN COMPUTATIONAL DOCKING the compartmentalization and reuse of software com- We have just completed developing and testing a ponents. Python provides a powerful computation “glue” semiempirical free energy force field for use in AutoDock for assembling computational components and, at the and similar grid-based docking methods. The force field same time, a flexible language for the interactive script- is based on a comprehensive thermodynamic model that ing of new applications. allows incorporation of intramolecular energies into the We released version 1.4.1 of our software compo- predicted free energy of binding. The model also incorpo- nents in March 2006. This release contains substantial rates a charge-based method for evaluating desolvation enhancements, including a completely rewritten inter- designed to use a typical set of atom types. The method face to the adaptive Poisson-Bolzman solver APBS, was calibrated by using a set of 188 diverse protein- making it easy to produce high-quality pictures of electro- ligand complexes of known structure and binding energy static potentials on molecular surfaces. A new control and was tested by using a set of 100 complexes of panel provides a high-level interface for rapidly dis- ligands with retroviral proteases. Compared with the playing molecular models in a variety of representations. previous AutoDock force field, the new force field pro- This new release is also distributed with installer provides an improvement in redocking simulations. grams for computers running the Windows and Macin- AutoDock 4 has been modified to support more atom tosh OS X operating systems. We also added a parser types and to use an improved atom-typing mechanism. for macromolecular Crystallographic Information File Importantly, AutoDock 4 also now simulates the molecu- that allow users to read and write files in the macro- lar system being docked in the unbound state, by gener- molecular Crystallographic Information File format. This ating and evaluating the extended conformation of the addition helps overcome limitations in the Protein Data ligand and moving side chains in the receptor before Bank format such as maximum number of atoms or the docking occurs. The unbound state is now consid- chain IDs. ered in the calculation of the change in free energy upon MODELING PROTEIN FLEXIBILITY IN DOCKING binding. AutoDock’s companion graphical user interface, We have developed a hierarchical and multiresolution AutoDockTools, has been modified to support prepara- representation of the flexibility of biological macromole- tion of input files for AutoDock 4, in particular to allow cules that can be used in computational simulations. the definition of flexible side chains in macromolecules. This treelike structure enables the computationally AutoDockTools has also been made easier to use by tractable encoding of a small subset of a protein’s confor- simplifying the menus. mational subspace. After implementing the core infra- On the basis of the AutoDock force field, we devel- structure of the Flexibility Tree and developing intuitive oped a method for locating and characterizing the opti- graphical interfaces for building such trees, we have MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 197 started exploring the use of this data structure in the con- and has enabled us to compute a complete scan of the text of automated docking. We reproduced a cross-dock- National Cancer Institute diversity set (2000 compounds) ing experiment carried out earlier with AutoDock in which against a panel of 200 mutant HIV proteases in a mat- 20 inhibitors of HIV protease I where docked systemati- ter of 4 months. This computation required more than cally into the 20 conformations of the receptor. We 2 quadrillion energy evaluations of ligand vs protein. showed that by adding receptor flexibility, we could INTERACTIONS OF TISSUE FACTOR increase the rate of successful cross docking from We used our ligand-protein and protein-protein model 72% to 98%. to study the interactions of tissue factor (TF) in the PROTEIN DOCKING initiation of blood coagulation and the related regula- In collaboration with C. Bajaj, University of Texas, tory roles of the factor. TF plays a potential role in Austin, we are investigating a novel fast Fourier trans- metastasis, growth, and angiogenesis of tumor cells via form–based method for predicting the association of pro- 2 distinct mechanisms: interaction of the complex con- tein in complexes. In parallel with this docking method, sisting of TF and factor VIIa with protease-activated we evaluated the effect on blurring molecular surfaces receptor 2 (PAR2) and interaction of the complex con- on the shape complementarity at the interface between sisting of TF, factor VIIa, and factor Xa with PAR1 or proteins in a complex. We characterized the level of PAR2. However, no PAR structures are available for distortion introduced by blurring atomic spheres by studying these mechanisms. Because PAR2 is involved using gaussian distributions and determined an optimal in both pathways, we performed protein homology mod- blurring level for docking purposes. In addition, we eling studies of this receptor. added software components for the calculating the cur- We found 8 unique PAR2 sequences. For each unique vature of meshes that are used in the docking procedure. sequence, we searched its homology sequences in the Protein Data Bank and chose as the homology template FIGHTING DRUG RESISTANCE IN HIV DISEASE the sequence that has the highest-resolution x-ray crys- As part of a program project, we continue our work tal structure. Sequence alignment was then performed on inhibitors to fight drug resistance in the treatment of between the PAR2 sequence and the template sequence. AIDS. In collaboration with K.B. Sharpless and C.-H. The alignment was then input to MODELLER for build- Wong, Department of Chemistry, we have designed and ing 10 homology structures, from which we chose the optimized a series of inhibitors built around a triazole structure with the best quality as the homology model. formed in a click chemistry reaction. We are also explor- The homology models have enabled us to use AutoDock ing larger issues of resistance via docking experiments to perform docking studies of PAR2-activating peptides with large chemical databases and large sets of mutant and small molecules on PAR2. The discovered binding protease structures. These massive docking experiments modes are being confirmed by our collaborator, W. Ruf, are made possible by the resources available in the Department of Immunology. FightAIDS@Home distributed computing system. Figh- STRUCTURE-BASED DRUG DESIGN IN GAUCHER tAIDS@Home enlists the worldwide community in a DISEASE large computational effort to design effective therapeutic Gaucher disease is the most common lipid-storage agents to fight AIDS. Personal computers are used by disorder caused by activity-compromising mutations in the program when the computers are not in use by their glucosylceramidase and is the most common genetic owners, providing an enormous, and largely untapped, disease affecting Ashkenazi Jews. In addition to causing computational resource. The current goal is to identify great pain, anemia, and massive enlargement of the inhibitors that are effective against the wild-type virus liver and spleen, Gaucher disease can lead to neurologic and against common mutant forms of the virus. impairment or early demise. Among the most promis- In the past year, we moved FightAIDS@Home to the ing treatments, small-molecule chemical chaperones IBM World Community Grid. This transition involved can rescue the enzyme activity of the misfolded gluco- working closely with the team at IBM to board our sylceramidase. The experimentally identified deoxyno- automated docking software, AutoDock, to be able to jirimycin-type inhibitors have a narrow concentration run on the Windows United Devices client and the Linux range and cause a mild improvement in the activity of and Macintosh OS X Berkeley Open Infrastructure for the mutant enzyme. Network Computing clients. This move has increased We did a molecule fragment–based virtual screen- the number of available processors to about 300,000 ing with the National Cancer Institute diversity data set. 198 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

A total of 72 compounds identified as the best com- to science education and outreach with a combination pounds of interest by virtual screening were tested in of presentations, popular and professional illustrations the enzyme assays by our collaborator, J. Kelly, Depart- and animations, 3-dimensional tangible models, and a ment of Chemistry. A total of 13 compounds are insolu- presence on the Worldwide Web. In these projects, we ble in dimethyl sulfoxide at up to 10 mM; 25 precipitate use the diverse visualization tools developed in the in the assay buffer. Among the remaining 34 compounds Molecular Graphics Laboratory to disseminate results examined by using in vitro enzyme assays, 16 show that range from atomic structure to cellular function. significant inhibition of the enzyme. The top compounds We also continued several regular features that of interest have almost doubled the activities of the informally present molecular structure and function. mutant enzymes N370S and G202R in vivo. The “Molecule of the Month” at the Protein Data Bank PROTEIN PHOSPHATASE 2C INHIBITORS entered its seventh year of providing an accessible intro- In collaboration with P. Greengard, Rockefeller Uni- duction to the central database of biomolecular struc- versity, New York, New York, we used AutoDock to screen ture. Each month, a new molecule is presented with the National Cancer Institute diversity set against protein a description of the molecule’s structure, function, and phosphatase 2C (PP2C), an enzyme that must remain relevance to health and welfare (Fig. 2). Visitors are active for tumor growth in breast cancer. Several compounds were identified as inhibitors of PP2C in the computational screen. The compounds were ordered from the National Cancer Institute and were assayed experimentally. Several were inhibitory at micromolar concentrations; the potency of the best was between 5 and 10 µM. The lead compounds discovered in this study are the first nonphosphate-based PP2C inhibitors reported. MECHANISTIC STUDIES OF BIOCATALYSTS IN COCAINE ANTIBODIES Currently, no effective treatment of cocaine addiction approved by the Food and Drug Administration is available. One possible treatment based on receptor design entails thorough investigation of cocaine hydrolysis by catalytic antibodies. Between 2 possible reaction pathways (an oxyanion hole for carbonyl or an hydrogen-bond trap for hydroxide ion formed by tyrosines at positions H50 and L94), quantum mechanical, molecular docking, and molecular dynamics free-energy calculations have shown no conclusive evidence for a dominant pathway between 2 possible ones. An explanation for the Fig. 2. ATP synthase was presented as the Molecule of the Month in 2005. The illustration of this complex molecular machine low turnover rate of the antibody is the failure of the was constructed from 4 separate entries in the Protein Data Bank: antibody to promote any mechanism selectively because 1c17, 1e79, 2a7u, and 1l2p. of a homogeneous microenvironment generated by eliciting against a hapten with 2 equivalent phosphorus- then given suggestions about to how to begin their own oxygen bonds. Computational modeling would help exploration of the structures in the data bank. Other pro- improve hapten design and thus improve the efficiency jects include “The Molecular Perspective,” articles in of the catalytic antibody. the journal The Oncologist that present structures of VISUAL METHODS FROM ATOMS TO CELLS interest to clinical oncologists and provide a source of Understanding structural molecular biology is essen- continuing education for physicians; “Recognition in tial to foster progress and critical decision making among Action,” a new series at the Journal of Molecular Recog- students, policy makers, and the general public. In the nition; and work with the Nanoscale Informal Science past year, we continued our longstanding commitment Network supported by the National Science Foundation MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 199 to develop new materials for presenting the science of tating and characterizing the protein structures in terms nanotechnology. of their interaction interfaces and flexibility; predicting protein associations; modeling homologous structures PUBLICATIONS and membrane proteins; predicting conformational Beuscher, A., Olson, A.J., Goodsell, D.S. Identifying protein binding sites and optimal ligands. Lett. Drug Des. Discov. 2:483, 2005. rearrangements; and, finally, using ligand docking and

Cheng, T.-J., Goodsell, D.S., Kan, C.-C. Identification of sanguinarine as a novel virtual screening to detect inhibitors of specific molec- HIV protease inhibitor from high-throughput screening of 2,000 drugs and natural ular targets. This past year, our efforts in the last area products with a cell-based assay. Lett. Drug Des. Discov. 2:364, 2005. led to new or improved inhibitors against the receptor Dickerson, T.J., Beuscher, A.E. IV, Rogers, C.J., Hixon, M.S., Yamamoto, N., Xu, Y., for epidermal growth factor (EGFR), anthrax lethal factor, Olson, A.J., Janda, K.D. Discovery of acetylcholinesterase peripheral anionic site ligands through computational refinement of a directed library. Biochemistry dynamin, α1-antitrypsin, and the androgen receptor. 44:14845, 2005. BIOINFORMATICS AND CHEMINFORMATICS

Goodsell, D.S. Computational docking of biomolecular complexes with AutoDock. We helped G. Siuzdak and his group, Department In: Protein-Protein Interactions: A Molecular Cloning Manual, 2nd ed. Golemis, E., of Molecular Biology, build a cheminformatics system Adams, P. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2005, p. 885. for characterizing metabolites on the basis of liquid chromatography–mass spectrometry data. The result- Goodsell, D.S. The molecular perspective: c-Abl tyrosine kinase. Oncologist 10:758, 2005; Stem Cells 24:209, 2006. ing software (XCMS) incorporates novel nonlinear

Goodsell, D.S. The molecular perspective: cisplatin. Oncologist 11:316, 2006; retention time alignment, matched filtration, peak Stem Cells 24:514, 2006. detection, and peak matching and is freely available

Goodsell, D.S. The molecular perspective: double-stranded DNA breaks. Oncologist from http://metlin.scripps.edu/download/. The software 10:361, 2005; Stem Cells 23:1021, 2005. helps identify changes in specific endogenous metabo-

Goodsell, D.S. The molecular perspective: RAD51 and BRCA2. Oncologist lites, such as potential biomarkers. 10:555, 2005; Stem Cells 23:1434, 2005. We have proposed a method for sharing chemical Goodsell, D.S. The molecular perspective: tumor necrosis factor. Oncologist 11:83, information in conjunction with data on experimental 2006. compounds without revealing the identity of the com- Goodsell, D.S. Recognition in action: DNA mimicry. J. Mol. Recognit. 18:427, 2005. pounds. Privacy of chemical structure is of paramount

Goodsell, D.S. Representing structural information. In: Current Protocols in Bioin- importance in the industrial sector, and the proposed formatics. Baxeranis, A.D., Davison, D.B. (Eds.). Wiley & Sons, Hoboken, NJ, solution opens a way to transfer a rich knowledge base 2005, p. 5.4.1. from the pharmaceutical industry to academia. Huey, R., Morris, G.M., Olson, A.J., Goodsell, D.S. A semi-empirical free energy Finally, we collaborated with scientists at the force field with charge-based desolvation. J. Comput. Chem., in press. Structural Genomics Consortium, Oxford, England, to Rogers, J.P., Beuscher, A.E. IV, Flajolet, M., McAvoy, T., Nairn, A.C., Olson, A.J., improve the way the new structures are annotated, Greengard, P. Discovery of protein phosphatase 2C inhibitors by virtual screening. J. Med. Chem. 49:1658, 2006. distributed, and animated by using internal coordinates–based methods. Sanner, M., Stolz, M., Burkhard, P., Kong, X.-P., Min, G., Sun, T.-T., Driamov, S., Aebi, U., Stoffler, D. Nature at work from the nano to the macro scale. LIGAND DISCOVERY Nanobiotechnology 1:7, 2005. Small-molecule therapeutic agents can be discovered by using docking and virtual chemical library screening. The docking technology can also help in Predicting Protein Structure, understanding structural mechanisms of action of small molecules and rational design of better molecules. How- Association, and Inhibitors ever, modeling protein flexibility and ligand-induced conformational changes is a major challenge. We modeled R. Abagyan, J. An,* W. Bisson, A. Cheltsov, K. Hyun, the induced receptor rearrangements at several levels, J. Kovacs, I. Kufareva, P. Lam,** G. Nicola, A. Saldanha including relevant normal modes combined with full * Genome Sciences Centre, Vancouver, British Columbia side-chain sampling and “minus-one” calculations. In ** Molsoft L.L.C., La Jolla, California particular, we used the developed ligand-induced recep- oday the Protein Data Bank contains more than tor simulation techniques to identify new antagonists 37,000 structures and is growing at a rate of of the androgen receptor and the first small-molecule T 20 per day. These structures provide a unique inhibitors of α1-antitrypsin polymerization. opportunity for functional studies and rational design Our docking-based in silico chemical library screening of therapeutic agents. We continue to focus on anno- against the EGFR tyrosine kinase and the consequent 200 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE experimental validation allowed identification of several compounds with antiproliferative effects on cancer cells. Among them, a C(4)-N(1)-substituted pyrazolo[3,4- d]pyrimidine inhibits EGFR tyrosine kinase activity at micromolar concentrations. We screened a library of tyrphostins against the GTPase activity of dynamin I and performed optimization of discovered compounds. The results yielded a number of promising inhibitors that are effective at micromolar concentrations. Using a fragment-based approach, we developed inhibitors of the lethal factor metalloproteinase of Bacil- lus anthracis. The discovered compounds are highly potent and selective against lethal factor in in vitro assays, including cell-based assays. PEPTIDE DOCKING AND STRUCTURE PREDICTION Predicting partial protein structure or molecular Fig. 1. Predicting protein oligomerization geometry by using association is a critical task of computational biology, protein interface recognition. which remains a focus of our research. In particular, sensitive location on the biomolecule and assessing the we developed a method for ab initio prediction of pep- reliability of the local structure. This approach was tide-MHC binding geometry for diverse class I MHC applied to the Skp1-Cullin-F-box protein ubiquitin ligase allotypes. Such models are useful for predicting specific interface. It can be used before high-throughput or vir- ternary complexes with T-cell receptors and for designing tual library screening. new molecules that interact with these complexes. The CD59 is a membrane glycoprotein with therapeutic surprisingly accurate prediction (0.75-Å backbone root potential for treatment of inflammatory conditions. Using mean square deviation) that we achieved by using our scanning mutagenesis, refined nuclear magnetic reso- method for cross-docking of a highly flexible decapeptide, nance models, and additional site-specific mutations, dissimilar to the original bound peptide, and docking we identified a binding interface on CD59 that is much predictions with homology models for 2 allotypes with broader than previously thought. We identified substitu- mean backbone root mean square deviations of less tions that decreased CD59 activity and a surprising than 1.0 Å illustrate the effectiveness of the method. number of substitutions that enhanced it. On the basis PREDICTING FUNCTIONAL SITES of these findings, we prepared clinically relevant soluble Functional annotation of protein structures involves mutant CD59-based proteins that had up to a 3-fold identifying and characterizing protein-protein interfaces, increase in complement inhibitory activity. oligomerization states, and binding sites for small ligands. We developed a method called protein interface recog- PUBLICATIONS Abagyan, R. Problems in computational structural genomics. In: Structural Pro- nition that can be used to predict interfaces on the basis teomics. Sundstrom, M., Norin, M., Edwards, A. (Eds.). CRC Press, Boca Raton, of an isolated protein structure and does not depend on FL, 2006, p. 223. evolutionary information. The method was benchmarked Abagyan, R., Lee, W.H., Raush, E., Budagyan, L., Totrov, M., Sundstrom, M., by using a diverse set of 748 protein interfaces. The Marsden, B.D. Disseminating structural genomics data to the public: from a data dump to an animated story. Trends Biochem. Sci. 31:76, 2006. accuracy and efficiency make the method a suitable tool for automated high-throughput annotation of protein Bordner, A., Abagyan, R.A. Ab initio prediction of peptide-MHC binding geometry for diverse class I MHC allotypes. Proteins 63:512, 2006. structures discovered in structural proteomics studies Cardozo, T., Abagyan, R. Druggability of SCF ubiquitin ligase-protein interfaces. (Fig. 1). Methods Enzymol. 399:634, 2005. Some protein interfaces can safely be targeted for Cavasotto, C.N., Orry, A.J.W., Abagyan, R. Receptor flexibility in ligand docking. In: drug discovery. We developed a systematic approach Handbook of Theoretical and Computational Nanotechnology. Rieth, M., Schommers, to assessing the “druggability” of a protein interface. W. (Eds.). American Scientific Publishers, Stevenson Ranch, CA, 2006, Vol.6, p. 217. The approach includes detecting a suitable ligand-bind- Cavasotto, C.N., Orry, A.J.W., Abagyan, R.A. The challenge of considering receptor flexibility in ligand docking and virtual screening. Curr. Comput. Aided Drug ing pocket with maximal confidence in a functionally Des. 1:423, 2005. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 201

Cavasotto, C.N., Ortiz, M.A., Abagyan, R.A., Piedrafita, F.J. In silico identification of novel EGFR inhibitors with antiproliferative activity against cancer cells. Bioorg. Med. Chem. Lett. 16:1969, 2006.

Forino, M., Johnson, S., Wong, T.Y., Rozanov, D.V., Savinov, A.Y., Li, W., Fat- torusso, R., Becattini, B., Orry, A.J., Jung, D., Abagyan, R.A., Smith, J.W., Alibek, K., Liddington, R.C., Strongin, A.Y., Pellecchia, M. Efficient synthetic inhibitors of anthrax lethal factor. Proc. Natl. Acad. Sci. U. S. A. 102:9499, 2005.

Hill, T., Odell, L.R., Edwards, J.K., Graham, M.E., McGeachie, A.B., Rusak, J., Quan, A., Abagyan, R., Scott, J.L., Robinson, P.J., McCluskey, A. Small molecule inhibitors of dynamin I GTPase activity: development of dimeric tyrphostins. J. Med. Chem. 48:7781, 2005. Fig. 1. A novel nonlinear approach for correcting and analyzing

Huang, Y., Smith, C.A., Song, H., Morgan, B.P., Abagyan, R., Tomlinson, S. mass spectrometry data for characterization of metabolites. Insights into the human CD59 complement binding interface toward engineering VIRAL CHARACTERIZATION new therapeutics. J. Biol. Chem. 280:34073, 2005. We have developed novel methods for characterizing Kovacs, J.A., Cavasotto, C.N., Abagyan, R.A. Conformational sampling of protein flexibility in generalized coordinates: application to ligand docking. J. Comput. viruses that have applications to whole viruses, viral pro- Theor. Nanosci. 2:354, 2005. teins, and viral metabolites. Our results have enabled us Kufareva, I., Budagyan, L., Raush, E., Totrov, M., Abagyan, R. PIER: protein to examine both local and overall viral structure, gain- interface recognition for structural proteomics. Proteins, in press. ing insight into the dynamic changes of proteins on the Orry, A.J., Abagyan, R.A., Cavasotto, C.N. Structure-based development of target- viral surface and the changes that occur during viral specific compound libraries. Drug Discov. Today 11:261, 2006. infection (Fig. 2). Smith, C.A., O’Maille, G., Want, E.J., Qin, C., Trauger, S.A., Brandon, T.R., Cus- todio, D.E., Abagyan, R., Siuzdak, G. METLIN: a metabolite mass spectral database. Ther. Drug Monit. 27:747, 2005.

Smith, C.A., Want, E.J., O’Maille, G., Abagyan, R., Siuzdak, G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal. Chem. 78:779, 2006.

Tetko, I.V., Abagyan, R., Oprea, T.I. Surrogate data: a secure way to share corporate data. J. Comput. Aided Mol. Des. 19:749, 2005.

Mass Spectrometry

Fig. 2. A comprehensive approach for studying viral infection by G. Siuzdak, J. Apon, H.P. Benton, E. Go, K. Harris, L. Hoang, using a combination of mass spectrometry techniques. Three differ- R. Lowe, A. Meyers, H. Morita, A. Nordstrom, T. Northen, G. ent aspects of viral infection within an infected cell—the expression O’Maille, C. Qin, Z. Shen, C. Smith, M. Sonderegger, kinetics of the viral proteins, changes in the expression levels of cel- S. Trauger, W. Uritboonthai, E. Want, W. Webb, W. Wikoff, lular proteins, and changes in cellular metabolites—were monitored. D. Wong These analyses reveal the complexity of the protein and metabolite regulation involved in cellular transformations that occur during METABOLITE PROFILING viral infection. ndogenous small-molecule metabolites, ubiquitous

in biofluids, are crucial elements in understanding MASS SPECTROMETRY IN SILICO E living organisms whether in fundamental biochem- We are also developing ultra-high-sensitivity istry, disease diagnosis, or drug toxicity. The inherent approaches in mass spectrometry with a new strat- advantage of monitoring small molecules rather than pro- egy that involves pulsed laser desorption/ionization teins is the relative ease of quantitative analysis of the from a silylated silicon surface. In desorption/ionization molecules with mass spectrometry. We are implementing on silicon, silicon is used to capture analytes and laser novel mass spectrometry and bioinformatics techniques radiation is used to vaporize and ionize these mole- (Fig. 1) to investigate the profile of small-molecule meta- cules. Using this technology, we can analyze a wide bolites. Our purposes are to correlate metabolite activity range of molecules with unprecedented sensitivity, in with protein regulation and to develop metabolite analy- the yoctomole range. sis as a diagnostic method. Our ultimate goal is to create analytical and chemical technologies and data manage- PUBLICATIONS ment approaches to identify and structurally characterize Cohen, L., Go, E.P., Siuzdak, G. Small-molecule desorption/ionization mass analysis. In: A Practical Guide to MALDI MS: Instrumentation, Methods and Applica- metabolites of physiologic importance. tions. Hillenkamp, F., Peter-Katalinic, J. (Eds.). Wiley & Sons, New York, in press. 202 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Lee, J.-C.,Wu, C.-Y., Apon, J.V., Siuzdak, G., Wong, C.-H. Reactivity-based one- We using a wide variety of biophysical techniques to pot synthesis of tumor-associated antigen N3 minor octasaccharide for the development of a cleavable DIOS-MS sugar array. Angew. Chem. Int. Ed. 45:2753, 2006. study the mechanism of assembly of the 30S ribosome in vitro. Lowe, R., Tong, G., Voelcker, N.H., Siuzdak, G. Monitoring EDTA and endogenous metabolite from serum with mass spectrometry. Spectroscopy 19:137, 2005. The 30S ribosome can be reconstituted from purified components in vitro with extremely high efficiency, Luo, G., Chen, Y., Siuzdak, G., Vertes, A. Surface modification and laser pulse length effects on internal energy transfer in DIOS. J. Phys. Chem. B. 109:24450, 2005. a characteristic that has enabled detailed mechanistic

Nordstrom, A., Apon, J.V., Uritboonthai, W., Go, E.P., Siuzdak, G. Surfactant studies. The 30S ribosome is composed of a single enhanced desorption/ionization on silicon mass spectrometry. Anal. Chem. 78:272, RNA chain of approximately 1500 nucleotides and 20 2006. small proteins of 8–20 kD. Pioneering work by Nomura Nordstrom, A., He, L., Siuzdak, G. Desorption/ionization on silicon (DIOS). In: more than 30 years ago led to the development of an Hyphenation Methods. Niessen, W. (Ed.). Elsevier, St. Louis, in press. Encyclope- dia of Mass Spectrometry, Vol 8. Gross, M.L., Caprioli, R.M. (Eds. in Chief). assembly map that outlines the basic order of protein binding. However, the mechanistic basis for these early Nordstrom, A., O’Maille, G., Qin, C., Siuzdak, G. Nonlinear data alignment for UPLC-MS and HPLC-MS based metabolomics: quantitative analysis of endogenous observations was unknown. Using nuclear magnetic and exogenous metabolites in human serum. Anal. Chem. 78:3289, 2006. resonance, x-ray crystallography, calorimetry, and fluo- Siuzdak, G. The Expanding Role of Mass Spectrometry in Biotechnology, 2nd ed. rescence methods, we have studied the details of MCC Press, San Diego, CA, 2006. assembly of small RNA-protein complexes derived from Smith, C.A., O’Maille, G., Want, E.J., Qin, C., Trauger, S.A., Brandon, T.R., Cus- the 30S subunit to elucidate these molecular events. todio, D.E., Abagyan, R., Siuzdak, G. METLIN: a metabolite mass spectral database. Ther. Drug Monit. 27:747, 2005. A guiding principle for ribosome assembly is that each protein recognizes a small local region of the RNA Smith, C.A., Want, E.J., O’Maille, G., Abagyan, R., Siuzdak, G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, as its binding site. The protein cannot bind until the matching, and identification. Anal. Chem. 78:779, 2006. RNA structure in its binding site is properly folded. We

Talkington, M.W., Siuzdak, G., Williamson, J.R. An assembly landscape for the showed that the assembly reaction can be considered 30S ribosomal subunit. Nature 438:628, 2005. an alternating series of RNA conformational changes Want, E., Cravatt, B.F., Siuzdak, G. The expanding role of mass spectrometry in and protein binding events (Fig. 1). RNA helices must metabolite profiling and characterization. Chembiochem 6:1941, 2005.

Want, E.J., O’Maille, G., Smith, C.A., Brandon, T.R., Uritboonthai, W., Qin, C., Trauger, S.A., Siuzdak, G. Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry. Anal. Chem. 78:743, 2006.

Fig. 1. A model for ribosome assembly. The RNA chain is Assembly Landscape of the shown as cylinders representing helical regions of RNA structure. In the first step, the RNA changes conformation, which creates a 30S Ribosome protein-binding site for the protein S15. Next, a subsequent RNA folding event occurs, which in turn creates a binding site for the J.R. Williamson, F. Agnelli, A. Beck, C. Beuck, A. Bunner, proteins S6 and S18. Assembly appears to proceed as an alternat- A. Carmel, J. Chao, S. Edgcomb, M. Hennig, E. Johnson, ing series of folding and binding events. D. Kerkow, E. Kompfner, S. Kwan, P. Mikulecky, W. Ridgeway, be properly arranged to create the binding site for the H. Schultheisz, L.G. Scott, E. Sperling, B. Szymczyna first protein, which is protein S15 in Figure 1. Binding he ribosome is a large molecular machine that of S15 effectively consolidates the gains from RNA fold- is responsible for synthesis of all proteins in the ing in the previous step. Furthermore, after S15 bind- T cell. It is composed of 2 multicomponent subunits ing, the next RNA conformational change is facilitated; that bind mRNA, tRNAs, and other factors to carry out this change sets up the binding site for the next proteins, translation of the genetic code from RNA into protein which are S6 and S18 in Figure 1. product. In bacteria, the large, or 50S, subunit is respon- Thus, each protein serves as a local reporter for sible for catalyzing the formation of peptide bonds, RNA folding in a specific region of the 30S subunit. whereas the small, or 30S, subunit is responsible for The overall assembly reaction can be schematically reading out the genetic code. An elaborate process exists illustrated as shown in Figure 2, where an unfolded for biogenesis of the ribosome machinery in cells to RNA chain is combined with 20 different proteins, a assemble the ribosome from individual components. change that after a complex series of RNA conforma- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 203

a number of parallel pathways exist by which the ribosome can assemble. In addition to the functional restrictions placed on the sequence of the RNA, most likely the sequence is also selected under evolutionary pressure to fold efficiently under a variety of conditions encountered by bacteria in the environment.

PUBLICATIONS Chao, J.A.., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., Williamson, J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol. 12:952, 2005. Fig. 2. The 30S ribosome assembly reaction. The RNA chain is represented as a thin line that is disordered at the beginning of the Davis, J.H., Tonelli, M., Scott, L.G., Jaeger, L., Williamson, J.R., Butcher, S.E. RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-recep- experiment. The 20 small proteins that bind to the RNA are repre- tor complex [published correction appears in J. Mol. Biol. 760:742, 2006]. J. sented as circles. The final assembled subunit is composed of high- Mol. Biol. 351:371, 2005. ly folded RNA with each protein bound at a specific location. Hennig, M., Munzarova, M.L., Bermel, W., Scott, L.G., Sklenar, V., Williamson, J.R. Measurement of long-range 1H-19F scalar coupling constants and their glyco- tional changes and protein-binding events results in the sidic torsion dependence in 5-fluoropyrimidine-substituted RNA. J. Am. Chem. structured 30S subunit. Our previous analyses involved Soc. 128:5851, 2006. fragments of the overall structure, and we were inter- Scott, L.G., Williamson, J.R. The binding interface between Bacillus stearother- ested in monitoring the kinetics of binding and assem- mophilus ribosomal protein S15 and its 5′-translational operator mRNA. J. Mol. Biol. 351:280, 2005. bly of the intact 30S subunits. Monitoring the simultaneous binding of 20 differ- Talkington, M.T., Siuzdak, G., Williamson, J.R. An assembly landscape for the 30S ribosomal subunit. Nature 438:628, 2005. ent proteins to an RNA molecule is a serious technical challenge. To surmount this challenge, we developed an isotope-pulse chase assay in which mass spectrom- Development of the Genetic etry is used to indicate binding of the proteins to the RNA. Assembly is initiated by using a pulse of a mixture Code and Its Connection to of the 20 15N-labeled proteins; after a short assembly time, a mixture of 14N-proteins is added as the Human Disease chase. The fully assembled subunits are isolated, and the fraction of 15N for each protein is measured as a P. Schimmel, J. Bacher, K. Beebe, Z. Druzina, K. Ewalt, function of the pulse time by using quantitative mass M. Kapoor, E. Merriman, C. Motta, L. Nangle, F. Otero, spectrometry. In this way, the time course of binding J. Reader, R. Reddy, M. Swairjo, K. Tamura, E. Tzima, for each protein can be measured. The strength of the W. Waas, X.-L. Yang method is that the binding rates can all be measured he genetic code is thought to have developed in simultaneously. the putative RNA world and thereby enabled the Using this method, we can perform mechanistic T transition to the modern world of proteins. The experiments on 30S ribosome assembly by using the early code was primitive and over many eons was kinetic tools of physical chemistry. We have varied the refined. This refinement came from the acquisition of protein concentration to show that the binding rates new activities by a group of proteins known as amino- correspond to a bimolecular association, not to a rate- acyl-tRNA synthetases. These proteins established the limiting RNA conformational change. We have varied rules of the code through aminoacylation reactions, the magnesium ion concentration to show that ions can whereby each of the 20 amino acids is covalently joined play 2 opposing roles during assembly. Some parts of the to its cognate tRNA. The tRNA harbors the genetic code 30S subunit speed up at lower magnesium concentra- triplet associated with the specific amino acid that is tions, and different parts slow down. Perhaps most joined to the tRNA. important, we have measured the rates as a function Each amino acid has a single tRNA synthetase. The of temperature and performed Arrhenius analysis of the synthetases are thought to be among the earliest pro- activation energies for binding. teins, essential components of the translation appara- The main conclusion from these studies is that 30S tus that established the genetic code and that were assembly has no single global rate-limiting step. Rather, present in the last common ancestor of the universal 204 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE tree of life. As the tree developed and branched into Recently, we showed how the error-correction activity the 3 great kingdoms—archaebacteria, bacteria, and is essential for maintaining cell viability and how defects eukaryotes—the enzymes were incorporated into every in this activity can lead to disease. In collaboration with cell type of every organism. S.L. Ackerman, Jackson Laboratories, Bar Harbor, Maine, Detailed investigations of the structures and evolu- we found that a single point mutation in mice leads to tion of the aminoacyl-tRNA synthetases have provided neurodegeneration (Fig. 2). In particular, Purkinje cells a picture of the development of the genetic code and how the development was directed by the evolution of the synthetases and tRNAs. In previous research, we focused on the specifics of the molecular recognition of tRNAs and how the enzymes distinguish one tRNA from another to achieve accurate aminoacylation for a precise genetic code. During these studies, examination of a recent crystal structure of human tryptophanyl-tRNA synthetase (TrpRS) in complex with the tRNA for tryptophan (tRNATrp) revealed 2 states of the enzyme-tRNA complex (Fig. 1). In one state, the tRNA is entering the

Fig. 1. Crystal structures of uncharged tRNATrp associating with TrpRS (A) and charged tRNATrp dissociating from the enzyme (B). The location of the bound free amino acid (Trp) in the 2 active sites of the homodimer is indicated. The tRNA binds across both subunits. active site. In the other state, it has been charged (that is, tryptophan has been joined to tRNATrp in the amino- Fig. 2. Pathologic changes in sticky mutant mice (A). B-D, Cal- acylation reaction) and is dissociating from the enzyme. bindin D-28 (Calb) immunohistochemistry of sagittal sections of During the long evolutionary development of amino- cerebella from 3-week-old (B), 6-week-old (C), and 12-month-old (D) sti/sti mutant and 12-month-old wild-type (WT; E) mice. Cere- acyl-tRNA synthetases and their populating of every cell bellar lobules are indicated by roman numerals. F-H, Hematoxylin type, the enzymes adopted novel functions while keep- and eosin staining of Purkinje cells (arrowheads) in lobule II of ing their canonical role as determinants of the genetic cerebella from 1-month-old (F) or 12-month-old (G) sti/sti mutant code. Related to their central role, the enzymes acquired and 12-month-old wild-type (H) mice. I-N, Cleaved caspase 3 novel domains enabling them to correct errors of amino- (Casp3) immunohistochemistry (I-K) and TUNEL analysis (L-N) of acylation and thereby ensure the stringent accuracy of cerebella from 4-week-old mutant mice. Scale bars: For B-E, 500 µm; F-H, 50 µm; I-N, 10 µm. the code. Unrelated to the canonical activities of the enzymes in translation, the expanded functions include in the brain deteriorate and ataxia develops. This simple, regulation of transcription and translation in bacteria, heritable mutation is due to small amounts of misacyla- RNA splicing in fungal organisms, and cytokine signal- tion of alanine-specific tRNA (tRNAAla) to generate, for ing in mammalian cells. These novel functions con- example, serine attached to the tRNA. In this instance, nect translation to other central pathways that control small amounts of serine are incorporated in place of ala- growth, development, and regulation of all cell types. nine in the polypeptides that are produced. Thus, a mild MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 205 editing defect can lead to heritable neurologic disorders. Swairjo, M.A., Reddy, R.R., Lee, B., Van Lanen, S.G., Brown, S., de Crécy- Lagard, V., Iwata-Reuyl, D., Schimmel, P. Crystallization and preliminary x-ray A more severe defect in editing would doubtless be lethal characterization of the nitrile reductase QueF: a queosine-biosynthesis enzyme. and not sustained in the population. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61(Pt. 10):945, 2005.

In other collaborative studies with R. Burgess, Jack- Tamura, K., Schimmel, P.R. Chiral-selective aminoacylation of an RNA minihelix: son Laboratories, we established a connection between mechanistic features and chiral suppression. Proc. Natl. Acad. Sci. U. S. A. 103:13750, 2006. glycyl-tRNA synthetase and Charcot-Marie-Tooth disease. Tzima, E., Schimmel, P. Inhibition of tumor angiogenesis by a natural fragment of A single point mutation in the synthetase leads to the a tRNA synthetase. Trends Biochem. Sci. 31:7, 2006. disease. This mutation does not affect the aminoacylation Waas, W.F., de Crécy-Lagard, V., Schimmel, P. Discovery of a gene family critical activity of glycyl-tRNA synthetase. Instead, the results to wyosine base formation in a subset of phenylalanine-specific transfer RNAs. J. suggest that glycyl-tRNA synthetase has an additional Biol. Chem. 280:37616, 2005. function, possibly in neurologic development. Several Yang, X.-L., Otero, F.J., Ewalt, K.L., Liu, J., Swairjo, M.A., Kohrer, C., RajB- examples of Charcot-Marie-Tooth disease due to mutations handary, U.L., Skene, R.J., McRee, D., Schimmel, P. Two conformations of a crys- talline human tRNA synthetase-RNA complex: implications for protein synthesis. in glycyl-tRNA synthetase in humans have been found. EMBO J. 25:2919, 2006. To better understand the molecular origins of this disease, we obtained crystals of human glycyl-tRNA synthetase that diffract to about 3 Å. A structure is Mechanisms of RNA Assembly being determined, and mutations found in humans will be mapped on the structure. This information will and Catalysis be used in conjunction with other assays and experiments to understand the connection between neuro- M.J. Fedor, E.M. Calderon, J.W. Cottrell, C.P. Da Costa, logic development, Charcot-Marie-Tooth disease, and S. Daudenarde, J.W. Harger, Y.I. Kuzmin, E.M. Mahen, M. Roychowdhury-Saha glycyl-tRNA synthetase. These results with glycyl-tRNA synthetase support ur goal is to generate basic insights into catal- the hypothesis that aminoacyl-tRNA synthetases in ysis by RNA and RNA-protein enzymes, RNA mammals are not only components of the translation O folding, and RNA interactions with small mole- apparatus but also a reservoir of cytokines with activi- cules. In addition to contributing basic knowledge of ties that are unmasked by specific activation events, RNA structure and function in normal growth and devel- such as alternative splicing or generation of specific opment, results of our studies provide a framework for fragments by proteolysis. Examples we are studying developing technical and therapeutic applications involv- include tyrosyl- and tryptophanyl-tRNA synthetases. ing RNAs as targets and reagents. These are both procytokines that when split by alter- Apart from the ribosome, which catalyzes peptidyl ative splicing or natural proteolysis, result in fragments transfer, the naturally occurring ribozymes catalyze trans- that are active in signal transduction pathways. For fer of phosphate groups. The small RNA enzymes that example, a fragment of tryptophanyl-tRNA synthetase we study catalyze reversible phosphodiester cleavage is a potent angiostatic agent. reactions that generate 5′ hydroxyl and 2′,3′-cyclic phosphate termini (Fig. 1). Possible strategies for cataly- PUBLICATIONS sis of phosphoryl transfer reactions include aligning reac- Lee, J.W., Beebe, K., Nangle, L.A., Jang, J., Longo-Guess, C.M., Cook, S.A., Davisson, M.T., Sundberg, J.P., Schimmel, P., Ackerman, S.L. Editing-defective tive groups in an optimal orientation for an in-line attack tRNA synthetase causes protein misfolding and neurodegeneration in the sticky mechanism, general acid-base catalysis of proton transfer mouse. Nature 443:50, 2006. to activate nucleophilic oxygens or to stabilize oxyanion- Nangle, L.A., Motta, C.M., Schimmel, P. Global effects of mistranslation from an editing defect in mammalian cells. Chem. Biol. 13:1091, 2006. leaving groups, electrostatic stabilization of negative charge that accumulates in the transition state, and Reader, J.S., Ordoukhanian, P.T., Kim, J.-G., de Crécy-Lagard, V., Hwang, I., Farrand, S., Schimmel, P. Major biocontrol of plant tumors targets tRNA synthetase destabilizing the ground state. Our goal is to understand [published correction appears in Science 310:54, 2005]. Science 309:1533, 2005. which of these catalytic strategies RNA enzymes use.

Schimmel, P., Beebe, K. From the RNA world to the theatre of proteins. In: The In contrast to the chemical versatility of the amino RNA World, 3rd ed. Gesteland, R.R., Cech, T.R. Atkins, J.F. (Eds.). Cold Spring acid side chains that make up the active sites of pro- Harbor Laboratory Press, Cold Spring Harbor, NY, 2005, p. 227. tein enzymes, just 4 nucleotides are available for the Seburn, K.L., Nangle, L.A., Cox, G.A., Schimmel, P., Burgess, R.W. An active construction of ribozyme active sites. Nucleotides are dominant mutant of glycyl-tRNA synthetase causes neuropathy in Charcot-Marie- Tooth 2D mouse model. Neuron 51:715, 2006. well suited to faithful storage and transmission of genetic 206 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

test this model, we replaced guanine 8 with an abasic residue, a substitution that eliminates the nucleobase but leaves the phosphodiester backbone intact. However, this abasic variant had the same pH dependence as an unmodified ribozyme, arguing that the pH transition does not involve guanine 8. Replacing adenine 38 with an abasic residue, on the other hand, did eliminate the pH-dependent transition in activity, implicating adenine Fig. 1. Chemical mechanism of RNA cleavage mediated by the 38 in a catalytically important deprotonation. family of small catalytic RNAs that includes the hairpin ribozyme. These and other results are consistent with 2 models Cleavage of the phosphodiester bond occurs through an S 2-type N of the hairpin ribozyme catalytic mechanism in which mechanism that involves in-line attack of the 2′ oxygen nucleophile on the adjacent phosphorus to form a trigonal bipyramidal transi- adenine 38 contributes either general acid-base catal- tion state. Breaking of the 5′ oxygen-phosphorus bond generates ysis (Fig. 2A) or electrostatic stabilization of negative products with 5′ hydroxyl and 2′,3′-cyclic phosphate termini. information through complementary base pairing, but they are not particularly adept at catalytic chemistry. Protonation and deprotonation of nucleotides occur at high or low pH extremes, a situation that would make it difficult to mediate general acid or base catalysis at neutral pH. No positively charged nucleotide functional groups are expected to be available at neutral pH to function as Lewis acids to activate a nucleophile or stabilize an electronegative transition state or an oxyanion-leaving group. Recent high-resolution structures of self-cleaving RNAs lay the groundwork for experiments to probe fundamental questions about how RNA enzymes use their functional groups for catalysis. Like all enzymes, hairpin ribozymes combine several strategies to enhance catalytic rate. One important strategy, apparent from the Fig. 2. Two models of hairpin ribozyme catalysis. Results of mech- crystal structures, is the alignment of nucleophilic and anistic studies of the hairpin ribozyme are consistent with 2 models leaving-group oxygens in the optimal orientation for an in which the functional form of adenine 38 is either protonated or in-line SN2-type nucleophilic attack. The structure of the unprotonated. In the first model (A), protonated adenine 38 would hairpin ribozyme active site places guanine 8, adenine 9, act as a general acid by donating a proton to the 5′ oxygen, acting adenine 10, and adenine 38 nucleobases near the reac- in concert with hydroxide ion that activates the 2′ oxygen nucleophile tive phosphate. Guanine 8 and adenine 38 occupy posi- during cleavage, and unprotonated adenine 38 would act as a general base to activate the 5′ oxygen nucleophile during ligation. In the tions reminiscent of 2 histidine residues in the active site second model (B), unprotonated adenine 38 accepts a hydrogen bond of ribonuclease A, a protein enzyme that catalyzes the from the 5′ hydroxyl nucleophile during ligation and accepts a hydro- same reaction. Histidine residues perform general acid- gen bond from a protonated bridging 5′ oxygen during cleavage, pro- base catalysis during ribonuclease A catalysis, so the viding electrostatic stabilization to developing negative charge. In both similarity between hairpin ribozyme and ribonuclease A models, the amidine group of guanine 8, in its protonated form, donates active sites raised the possibility that guanine 8 and hydrogen bonds to the 2′ and phosphoryl oxygens that stabilize nega- adenine 38 nucleobases might perform functions similar tive charge that develops in the transition state and positions reactive groups in the orientation appropriate for an S 2 in-line nucleophilic to those of histidine residues. N attack. Reproduced with permission from Fedor, M.J., Williamson, J.R. Hairpin ribozyme activity increases with increasing The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6:399, 2005. pH, consistent with the notion that activity depends on Copyright 2005 Nature Publishing Group/Macmillan Magazines Ltd. the availability of the deprotonated form of guanine 8 to accept a proton from the 2′ hydroxyl nucleophile as charge that develops in the transition state as 5 elec- predicted by the general acid-base catalysis model. To tronegative oxygen atoms from transient bonds with MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 207 phosphorus (Fig. 2B) and guanine 8 donates hydrogen bonds to stabilize the transition state electrostatically.

Directed Evolution of Nucleic Acid Enzymes

G.F. Joyce, S.E. Hamilton, D.P. Horning, T.A. Jackson,

G.C. Johns, B.J. Lam, B.M. Paegel, G.G. Springsteen, Fig. 1. Composition of an RNA enzyme (A) and a DNA enzyme S.B. Voytek (B) related by evolutionary descent. Both enzymes contain about 50 nucleotides and catalyze the joining of 2 RNA substrates (S1 ll life known to exist on Earth today is based on and S2). The evolved DNA enzyme contains 10 mutations relative DNA genomes and protein enzymes, but most to the starting RNA enzyme (highlighted with black circles). A likely it was preceded by a simpler form of life based on RNA. This earlier era is referred to as the generations of evolution, we obtained a population of “RNA world.” During that time, genetic information DNA enzymes with the desired activity. A typical example resided in the sequence of RNA molecules and pheno- contains 10 mutations relative to the starting sequence type was derived from the catalytic behavior of RNA. and has a catalytic rate of 0.052 min–1 (Fig. 1B). When By studying the properties of RNA in the laboratory, this DNA enzyme was prepared as the corresponding especially with regard to the evolution of catalytic RNA enzyme, it had no detectable activity. Thus, the function, we can gain insight into the RNA world. In evolutionary transition from an RNA enzyme to a DNA addition, we can develop novel nucleic acid enzymes enzyme represents a switch in the chemical basis of that have applications in biology and medicine. catalytic function. CONVERTING AN RNA ENZYME TO A DNA ENZYME Evolutionary pathways such as this one for conver- The transfer of sequence information between 2 dif- sion of an RNA enzyme to a DNA enzyme may exist ferent classes of nucleic acid–like molecules, for example between other classes of nucleic acid–like molecules. between RNA and DNA, is straightforward because it The RNA world may have been preceded by a simpler relies on the 1-to-1 correspondence of Watson-Crick “pre-RNA world” based on a nucleic acid–like molecule pairing. Nearly 50 years ago, in articulating the cen- that would have occurred more readily on the primitive tral dogma of molecular biology, Francis Crick referred to Earth. Our findings suggest that the catalytic function of this property as “sequentialization.” Sequentialization a pre-RNA molecule might have been transferred to a also applies to the transfer of information from RNA to corresponding RNA enzyme through darwinian evolution. protein via the genetic code. The transfer of function, CONTINUOUS EVOLUTION OF RNA ENZYMES however, is more difficult because function is an overall Processes of darwinian evolution are fundamental property of a macromolecule and cannot be conveyed in to understanding biological form and function but are a sequential manner. There is no known example of an difficult to appreciate on the human timescale. During RNA enzyme that retains catalytic activity when prepared the past decade, we have developed methods for evolving as the corresponding DNA molecule, and vice versa. molecules rapidly and under controlled laboratory con- We used in vitro darwinian evolution to convert an ditions. One of the most powerful of these methods, and RNA enzyme to a DNA enzyme of the same function, the one that most closely resembles biological evolution, after the acquisition of a few critical mutations. The is a system for the continuous in vitro evolution of RNA starting RNA had the ability to join 2 RNA substrates enzymes. It involves a population of RNA enzymes that in a template-directed manner, with a catalytic rate of catalyze an RNA-joining reaction. Any molecule in the 0.14 min–1 (Fig. 1A). A corresponding DNA molecule population that performs the reaction becomes amplified in which ribose was replaced by deoxyribose and uracil to produce “progeny” molecules, which then have the was replaced by thymine had no detectable activity. The opportunity to perform the reaction again. The entire DNA molecule was used as a starting point to generate process takes place within a common reaction mixture trillions of randomized variants, which were selected for and can be continued indefinitely, so long as an ade- the ability to catalyze the RNA-joining reaction. After 10 quate supply of reaction materials is maintained. 208 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Recently, we developed a novel approach for the Nature’s strategies to prepare novel molecules that per- continuous evolution of RNA enzymes that uses micro- form specific functional tasks, such as regulating a gene, fluidic technology. With this approach, evolution can destroying cancer, or catalyzing a reaction with enzyme- be carried out in an automated fashion under computer like efficiency. We hope to apply these novel insights, control, with continuous monitoring of the population technologies, methods, and products to provide solutions size and precise control over critical parameters such to human diseases, including cancer, HIV disease, and as mutation frequency and selection pressure. We have genetic diseases. used the microfluidic device to conduct evolution experi- DIRECTING THE EVOLUTION OF CATALYTIC FUNCTION ments, beginning with a reaction mixture containing Using our concept of reactive immunization, we have about 1 billion RNA enzymes and carrying out repeated developed antibodies that catalyze aldol as well as rounds of RNA catalysis and selective amplification in retro-aldol reactions of a wide variety of molecules. The an automated fashion. The amount of RNA is monitored catalytic proficiency of the best of these antibodies continuously by using a confocal laser fluorescence approaches 1014, a value 1000 times that of the best microscope. When a predetermined threshold concen- catalytic antibodies reported to date and overall the tration is reached, the computer initiates an automated best of any synthetic protein catalyst. We have shown dilution and provides a fresh supply of reagents. This the efficient asymmetric synthesis and resolution of a process of selective amplification and dilution was car- variety of molecules, including tertiary and fluorinated ried out for 70 successive dilutions of 10-fold each dur- aldols, and have used these chiral synthons to synthesize ing a period of 6.5 hours. The microfluidic system is natural products (Fig. 1). The results highlight the poten- now being used to address fundamental questions of macromolecular evolution, such as the role of genetic diversity in escaping evolutionary bottlenecks and the maximum frequency of mutation that can be tolerated by an evolving population.

PUBLICATIONS Joyce, G.F., Orgel, L.E. Progress toward understanding the origin of the RNA world. In: The RNA World, 3rd ed. Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2006, p. 23.

Oberhuber, M., Joyce, G.F. A DNA-templated aldol reaction as a model for the formation of pentose sugars in the RNA world. Angew. Chem. Int. Ed. 44:7580, 2005.

Paul, N., Springsteen, G., Joyce, G.F. Conversion of a ribozyme to a deoxyri- bozyme through in vitro evolution. Chem. Biol. 13:329, 2006.

Studies at the Interface of

Molecular Biology, Chemistry, Fig. 1. A variety of compounds synthesized with the world’s first commercially available catalytic antibody, 38C2, produced at and Medicine Scripps Research.

C.F. Barbas III, M. Ahmad, K. Albertshofer, tial synthetic usefulness of catalytic antibodies as artificial L. Asawapornmongkul, N.S. Chowdari, S. Eberhardy, enzymes in addressing problems in organic chemistry R. Fuller, B. Gonzalez, R. Gordley, J. Guo, D.H. Kim, that are not solved by using natural enzymes or more R.L. Lerner, C. Lund, J. Mandell, S. Mitsumori, W. Nomura, traditional synthetic methods. M. Popkov, D.B. Ramachary, S.S.V. Ramasastry, Other advances in this area include the development L.J. Schwimmer, D. Shabat,* F. Silva, J. Suri, F. Tanaka, of the first peptide aldolase enzymes. By using both U. Tschulena, N. Utsumi, Y. Ye, Y. Yuan, H. Zhang design and selection, we have created small peptide * Tel Aviv University, Tel Aviv, Israel catalysts that recapitulate many of the kinetic features e are concerned with problems in molecular of large enzyme catalysts. These smaller enzymes allow biology, chemistry, and medicine. Many of us to address the relationship between the size of nat- W our studies involve learning or improving on ural proteins and the proteins’ catalytic efficiency. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 209

ORGANOCATALYSIS: A BIOORGANIC APPROACH TO CATALYTIC ASYMMETRIC SYNTHESIS To further explore the principles of catalysis, we are studying amine catalysis as a function of catalytic scaffold. Using insights garnered from our studies of aldolase antibodies, we determined the efficacy of simple chiral amines and amino acids for catalysis of aldol and related imine and enamine chemistries such as Michael, Mannich, Knoevenagel, and Diels-Alder reactions. Although aldolase antibodies are superior catalysts in terms of the kinetic parameters, these more simple catalysts are enabling us quantify the importance of pocket sequestration in catalysis.

Furthermore, many these catalysts are cheap, envi- Fig. 2. Design of the new catalyst (3R,5R)-5-methyl-3-pyrro- ronmentally friendly, and practical for large-scale syn- lidinecarboxylic acid (right) allows efficient access to anti-Mannich thesis. With this approach, we showed the scope and products not accessible through proline catalysis (left). usefulness of the first efficient amine catalysts of direct cines. Laboratories and pharmaceutical companies asymmetric aldol, Mannich, Diels-Alder, and Michael around the world now apply the phage display technol- reactions. The organocatalyst approach is a direct out- ogy that we developed for antibody Fab fragments. In come of our studies of catalytic antibodies and provides our laboratory, we are targeting cancer and HIV disease. an effective alternative to organometallic reactions that One of our antibodies, IgG1-b12, protects animals use severe reaction conditions and often-toxic catalysts. against primary challenge with HIV type 1 (HIV-1) and We think that our discovery that simple naturally has been further studied by many researchers. We occurring amino acids such as L-proline and other amines improved this antibody by developing in vitro evolution can effectively catalyze a variety of enantioselective inter- strategies that enhanced its neutralization activity. By molecular reactions will change the way many reactions coupling laboratory-evolved antibodies with potent tox- will be performed. As a testament to the mild nature of ins, we showed that immunotoxins can effectively kill this approach, we developed the first catalytic asym- infected cells. metric Aldol, Mannich, Michael, and fluorination reac- We are also developing genetic methods to halt HIV tions involving aldehydes as nucleophiles. Previously, by gene therapy. We created unique human antibodies such reactions were considered out of the reach of tra- that can be expressed inside human cells to make the ditional synthetic methods. cells resistant to HIV infection. In the future, these anti- In extensions of these concepts, we designed novel bodies might be delivered to the stem cells of patients amino acid derivatives that direct the stereochemical infected with HIV-1, allowing the development of a outcome of reactions in ways not possible with proline disease-free immune system that would obviate the (Fig. 2). In other studies, we created the first asymmetric intense regimen of antiviral drugs now required to treat small-molecule aldol catalysts that are highly effective HIV disease. with water and seawater as solvent. We think that our Using our increased understanding of antibody-anti- results are also relevant to the prebiotic synthesis of the gen interactions, we extended our efforts in cancer ther- molecules of life. For example, we have shown that our apy and developed rapid methods for creating human amino acid strategy can be used to synthesize carbo- antibodies from antibodies derived from other species. hydrates directly, thereby providing a provocative pre- We produced human antibodies that should enable us biotic route to the sugars essential for life. to selectively starve a variety of cancers by inhibiting THERAPEUTIC ANTIBODIES, IN AND OUT OF CELLS angiogenesis and antibodies that will be used to deliver We developed the first human antibody phage dis- radionuclides to colon cancers to destroy the tumors. play libraries and the first synthetic antibodies and We hope that these antibodies will be used in clinical methods for the in vitro evolution of antibody affinity. trials done by our collaborators at the Sloan-Kettering The ability to manipulate large libraries of human anti- Cancer Center in New York City. bodies and to evolve such antibodies in the laboratory On the basis of our studies on HIV-1, we used intra- provides tremendous opportunities to develop new medi- cellular expression of antibodies directed against angio- 210 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE genic receptors to create a new gene-based approach our approach can become a key tool in selective che- to cancer. Our studies indicate that this type of gene motherapeutic strategies. To see a movie illustrating therapy can be successfully applied to the treatment this approach, visit http://www.scripps.edu/mb/barbas/ of cancer. antibody/antibody.mov. THERAPEUTIC APPLICATIONS OF CATALYTIC ADAPTOR IMMUNOTHERAPY: THE ADVENT OF ANTIBODIES CHEMOBODIES The development of highly efficient catalytic anti- We think that combining the chemical diversity of bodies opens the door to many practical applications. small synthetic molecules with the immunologic char- One of the most fascinating is the use of such antibodies acteristics of antibody molecules will lead to therapeutic in human therapy. We think that use of this strategy can agents with superior properties. Therefore, we developed improve chemotherapeutic approaches to diseases such a conceptually new device that equips small synthetic as cancer and AIDS. Chemotherapeutic regimens are molecules with both the immunologic effector functions typically limited by nonspecific toxic effects. To address and the long serum half-life of a generic antibody mol- this problem, we developed a novel and broadly applic- ecule. For a prototype, we developed a targeting device able drug-masking chemistry that operates in conjunc- based on the formation of a covalent bond of defined tion with our unique broad-scope catalytic antibodies. stoichiometry between (1) a 1,3-diketone derivative of This masking chemistry is applicable to a wide range an arginine–glycine–aspartic acid peptidomimetic that of drugs because it is compatible with virtually any targets the integrins αvβ3 and αvβ5 and (2) the reactive heteroatom. We showed that generic drug-masking lysine of aldolase antibody 38C2 (Fig. 4). The resulting groups can be selectively removed by sequential retro- aldol–retro-Michael reactions catalyzed by antibody 38C2 (Fig. 3). This reaction cascade is not catalyzed by any known natural enzyme.

Fig. 3. Targeting cancer and HIV with prodrugs activated by catalytic antibodies. A bifunctional antibody is shown targeting a cancer cell for destruction. A nontoxic analog of doxorubicin, prodoxorubicin, Fig. 4. Designed small-molecule targeting agents (SCS-873 as is being activated by an aldolase antibody to the toxic form of the drug. shown) program the specificity of the antibody 38C2 (A). The resulting chemobodies (cp38C2, B) have characteristics that are Application of this masking chemistry to the anti- often superior to either those of either the small molecule or the cancer drugs doxorubicin, camptothecin, and etoposide antibody alone. produced prodrugs with substantially reduced toxicity. complex spontaneously assembled in vitro and in vivo, These prodrugs are selectively unmasked by the cata- selectively retargeted antibody 38C2 to the surface of lytic antibody when the antibody is applied at thera- cells expressing integrins αvβ3 and αvβ5, dramatically peutically relevant concentrations. The efficacy of this increased the circulatory half-life of the peptidomimetic, approach has been shown in in vivo models of cancer. and effectively reduced tumor growth in animal models Currently, we are developing more potent drugs and novel of human Kaposi sarcoma, colon cancer, and melanoma. antibodies that will allow us to target breast, colon, and ZINC FINGER GENE SWITCHES prostate cancer as well as cells infected with HIV-1. The solution to many diseases might be simply turn- On the basis of our preliminary findings, we think that ing genes on or off in a selective way. In order to pro- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 211

duce switches that can turn genes on or off, we are expression of a protein known as CCR5, which is a key studying molecular recognition of DNA by zinc finger to infection of human cells by HIV-1. We developed an proteins and methods of creating novel zinc finger DNA- HIV-1–targeting transcription factor that strongly sup- binding proteins (Fig. 5). Because of their modularity presses HIV-1 replication. Genetic diseases such as sickle cell anemia are also being targeted with this approach. Using a library of transcription factors, we developed a strategy that effectively allows us to turn on and off every gene in the genome. With this powerful new strategy, we can quickly regulate a target gene or discover other genes that have a key role in disease. In the future, we hope to use novel DNA-modifying enzymes directed by zinc fingers to manipulate chromosomes themselves.

PUBLICATIONS Fig. 5. A designed polydactyl zinc finger binds 18 bp of DNA. A Alwin, S., Gere, M.B., Guhl, E., Effertz, K., Barbas, C.F. III, Segal, D.J., Weitz- single zinc finger domain is highlighted. With this approach, we man, M.D., Cathomen, T. Custom zinc-finger nucleases for use in human cells. Mol. Ther. 12:610, 2005. can now construct more than a billion gene switches and use them to specifically turn genes on or off in multiple organisms. Further Blancafort, P., Chen, E.I., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D., elaboration of the approach allows every gene in the genome to be Brachat, A., Brakenhoff, R.H., Quigley, J.P., Erdmann, D., Barbas, C.F. III. Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc. either turned on or upregulated or downregulated, providing a new Natl. Acad. Sci. U. S. A. 102:11716, 2005. approach to probe gene function across the genome. Blau, C.A., Barbas, C.F. III, Bomhoff, A.L., Neades, R., Yan, J., Navas, P.A., Peterson, K.R. γ-Globin gene expression in chemical inducer of dimerization (CID)- and well-defined structural features, zinc finger proteins dependent multipotential cells established from human β-globin locus yeast artifi- are particularly well suited for use as DNA-binding pro- cial chromosome (β-YAC) transgenic mice. J. Biol. Chem. 280:36642, 2005. teins. Each finger forms an independently folded domain Cheong, P.H.-Y., Zhang, H., Thayumanavan, R., Tanaka, F., Houk, K.N., Barbas, C.F. III. Pipecolic acid-catalyzed direct asymmetric Mannich reactions. Org. Lett. that typically recognizes 3 nucleotides of DNA. We 8:811, 2006. showed that proteins can be selected or designed that Corte-Real, S., Collins, C., Aires da Silva, F., Simas, P., Barbas, C.F. III, Chang, contain zinc fingers that recognize novel DNA sequences. Y., Moore, P., Goncalves, J. Intrabodies targeting the Kaposi sarcoma-associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells. Blood These studies are aiding the elucidation of rules for 106:3797, 2005. sequence-specific recognition within this family of pro- Dreier, B., Fuller, R.P., Segal, D.J., Lund, C.V., Blancafort, P., Huber, A., Koksch, B., teins. We selected and designed specific zinc finger Barbas, C.F. III. Development of zinc finger domains for recognition of the 5′-CNN- 3′ family DNA sequences and their use in the construction of artificial transcription domains that will constitute an alphabet of 64 domains factors. J. Biol. Chem. 280:35588, 2005. that will allow any DNA sequence to be bound selec- Eberhardy, S.R., Goncalves, J., Coelho, S., Segal, D.J., Berkhout, B., Barbas, tively. The prospects for this “second genetic code” are C.F. III. Inhibition of human immunodeficiency virus type 1 replication with artifi- fascinating and promise a major impact on basic and cial transcription factors targeting the highly conserved primer-binding site. J. Virol. 80:2873, 2006. applied biology. Lund, C.V., Popkov, M., Magnenat, L., Barbas, C.F. III. Zinc finger transcription We showed the potential of this approach in mul- factors designed for bispecific coregulation of ErbB2 and ErbB3 receptors: insights tiple mammalian and plant cell lines and in whole organ- into ErbB receptor biology. Mol. Cell. Biol. 25:9082, 2005. isms. With the use of characterized modular zinc finger Mandell, J., Barbas, C.F. III. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 34(Web server domains, polydactyl proteins capable of recognizing an issue):W516, 2006. 18-nucleotide site can be rapidly constructed. Our results Mase, N., Nakai, Y., Ohara, H., Yoda, H., Takabe, K., Tanaka, F., Barbas, C.F. III. suggest that zinc finger proteins might be useful as Organocatalytic direct asymmetric aldol reactions in water. J. Am. Chem. Soc. genetic regulators for a variety of human aliments and 128:734, 2006. provide the basis for a new strategy of gene therapy. Mitsumori, S., Zhang, H., Cheong, P.H.-Y., Houk, K.N., Tanaka, F., Barbas, C.F. III. Direct asymmetric anti-Mannich-type reactions catalyzed by a designed amino Our goal is to develop this class of therapeutic proteins acid. J. Am. Chem. Soc. 128:1040, 2006. to inhibit or enhance the synthesis of proteins, provid- Nathan, S., Rader, C., Barbas, C.F. III. Neutralization of Burkholderia pseudoma- ing a direct strategy for fighting diseases of either somatic llei protease by Fabs generated through phage display. Biosci. Biotechnol. or viral origin. Biochem. 69:2302, 2005. We are also developing proteins that will inhibit Popkov, M., Rader, C., Gonzelez, B., Sinha, S.C., Barbas, C.F. III. Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody the growth of tumors and others that will inhibit the effector. Int. J. Cancer 119:1194, 2006. 212 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Suri, J.T., Mitsumori, S., Albertshofer, K., Tanaka, F., Barbas, C.F. III. Dihydroxy- original tautomerization reaction via a general acid-base acetone variants in the organocatalytic construction of carbohydrates: mimicking tagatose and fuculose aldolases. J. Org. Chem. 71:3822, 2006. mechanism and the decarboxylation of oxaloacetate via a nucleophilic mechanism. Suri, J.T., Steiner, D.D., Barbas, C.F. III. Organocatalytic enantioselective synthesis of metabotropic glutamate receptor ligands. Org. Lett. 7:3885, 2005. We also showed that the electrostatic manipulation of an enzyme’s active site can alter the substrate speci- Swan, C.H., Buhler, B., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther. ficity of the enzyme in a predictable way. We replaced 13:1408, 2006. 1, 2, or all 3 active-site arginine residues with citrulline Tan, W., Dong, Z., Wilkinson, T.A., Barbas, C.F. III, Chow, S.A. Human immuno- analogs to maintain the steric features of the active site deficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA of 4-oxalocrotonate tautomerase while changing its elec- into a predetermined region in human cells. J. Virol. 80:1939, 2006. tronic properties. These synthetic changes revealed that

Tanaka, F., Barbas, C.F. III. Enamine-based reactions using organocatalysts: from the wild-type enzyme binds the natural substrate pre- aldolase antibodies to small amino acid and amine catalysts. J. Synth. Org. Chem. dominantly through electrostatic interactions. This and Jpn. 63:27, 2005. other mechanistic insights led to the design of a modi- Tanaka, F., Fuller, R., Barbas, C.F. III. Development of small designer aldolase fied enzyme that was specific for a new substrate that enzymes: catalytic activity, folding, and substrate specificity. Biochemistry 44:7583, 2005. had different electrostatic properties and that bound the

Weinstain, R., Lerner, R.A., Barbas, C.F. III, Shabat, D. Antibody-catalyzed asym- enzyme via hydrogen-bonding complementarity rather metric intramolecular Michael addition of aldehydes and ketones to yield the disfa- than electrostatic interactions. This research on synthetic vored cis-product. J. Am. Chem. Soc. 127:13104, 2005. enzymes is being done in collaboration with P.E. Dawson, Department of Cell Biology. CATALYTIC ANTIBODIES Synthetic Enzymes, Catalytic Engineering herbicide resistance in crops facilitates control of weed species, particularly weeds that are Antibodies, Ozone Scavengers in genetically related to the crop, and may be useful in Asthma, Organometallic selecting lines that have undergone multiple transformation events. We showed that herbicide-resistant plants Chemistry, and Biomolecular can be engineered by designing both a herbicide and a catalytic antibody that destroys the herbicide within Computing the plants. First, we developed a carbamate herbicide that can be catalytically destroyed by the aldolase anti- E. Keinan, O. Reany, C.H. Lo, S. Bauer, N. Metanis, body 38C2. Then we targeted the light chain and half E. Kossoy, M. Soreni, R. Piran, M. Sinha, I. Ben-Shir, of the heavy chain (Fab) of the catalytic antibody to the T. Ratner, T. Shekhter, T. Mejuch, E. Solel endoplasmic reticulum in 2 lines of Arabidopsis thaliana e focus on synthetically modified enzymes, transformants. Finally, we crossed the 2 transgenic plants

antibody-catalyzed reactions, anticancer and to produce a herbicide-resistant F1 hybrid (Fig. 1). Our W antiasthma agents, and biomolecular compu- results suggest that in vivo expression of catalytic anti- tation, as illustrated in the following examples. bodies could become a general strategy to achieve SYNTHETIC ENZYMES phenotype modifications not only in plants but also Efforts to generate new enzymatic activities from in other organisms. existing protein scaffolds may not only provide bio- OZONE SCAVENGERS AND ANTIASTHMA ACTIVITY technologically useful catalysts but also lead to better A new hypothesis we proposed on the mechanism understanding of the natural process of evolution. We of asthmatic inflammation has led to an ozone-scaveng- profoundly changed the catalytic activity and mecha- ing compound that prevents bronchial obstruction in rats nism of the enzyme 4-oxalocrotonate tautomerase by with asthma. Previously, scientists at Scripps Research means of rationally designed synthetic mutations. For discovered that ozone can be generated not only via the example, a single amino acid substitution that corre- antibody-mediated water oxidation pathway but also sponds to a mutation in a single base pair led to a by antibody-coated activated white blood cells during dramatic change in the catalytic activity. Although the inflammatory processes. This finding led us to specu- wild-type enzyme catalyzes only the tautomerization of late that the pulmonary inflammation in asthma might 4-oxalocrotonate, the mutant P1A catalyzes both the be caused by ozone production by white blood cells in MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 213

Fig. 1. Influence of herbicide (1) on the rooting and development Fig. 2. The catalytic cycle of the rhenium oxide–catalyzed heteroa- of seedlings of F1 hybrids and control A thaliana plants. The control cylative dimerization of tetrahydrofuran (THF), which is proposed on plants are shown in A and C; the hybrid plant lines (F1) expressing the basis of isotope-labeling experiments, starts with an attack of a both light and heavy chains of the catalytic antibody 38C2, in B rhenium oxo ligand on a coordinated tetrahydrofuran, then an attack and D. Plantlets grown on medium without the herbicide are shown of the resultant alkoxide ligand on a second coordinated tetrahydrofu- in A and B; those grown with the herbicide are shown in C and D. ran, nucleophilic addition of the resultant alkoxide ligand to the lungs and that inhalation of electron-rich olefins, which coordinated carboxylic acid, and finally, electrophilic cleavage of are known ozone scavengers, might have antiasthmatic the other coordinated alkoxide by trifluoroacetic anhydride (TFAA). effects. In experiments in rats, inhalation of such a BIOMOLECULAR COMPUTING DEVICES compound, limonene, caused a significant improvement Previously, we described a programmable finite in signs of asthma. These results could have conse- automaton with 2 symbols and 2 states that computed quences in the management of asthma. autonomously. All of the components of the device, ORGANOMETALLIC CHEMISTRY including hardware, software, input, and output, were Rhenium oxide, which is known primarily as a strong biomolecules mixed together in solution. The hardware oxidant, is a highly selective Lewis acid catalyst that consisted of a restriction nuclease and a ligase; the affects the heteroacylative dimerization of tetrahydro- software (transition rules) and the input were double- furan at room temperature. This multicomponent reac- stranded DNA oligomers. Computation was carried out tion, which involves tetrahydrofuran, trifluoroacetic by processing the input molecule via repetitive cycles anhydride, and a carboxylic acid, produces a nonsym- of restriction, hybridization, and ligation reactions to metrical diester (compound 3 in Fig. 2) in high yields. produce a final-state output in the form of a double- The proposed catalytic cycle (Fig. 2) involves a multi- stranded DNA molecule. step sequence of nucleophilic attacks, metal-oxygen More recently, we markedly increased the levels of bond metathesis, and electrophilic cleavage by trifluo- complexity and mathematical power of these automata roacetic anhydride. This synthetically useful reaction by the design of a 3-state–3-symbol automaton, thus highlights the unique, frequently avoided Lewis acidity increasing the number of syntactically distinct programs of transition-metal oxides. from 765 to 1 billion. We have further amplified the In study with the platinum complex TpPt(CO)CH 3 applicability of this design by using surface-anchored (Tp = hydridotrispyrazolylborate), we found that the input molecules and surface plasmon resonance tech- proton exchange between water and the methyl group nology to monitor the computation steps in real time. involves the formation and deprotonation of a “sticky” This technology allowed parallel computation with DNA σ-methane ligand. The efficiency of this nontrivial process is attributed to the spatial orientation of functional chips that carry multiple input molecules and can be groups that operate in concert to achieve a multistep used as pixel arrays for image encryption. proton walk. The key role played by the free pyrazolyl PUBLICATIONS nitrogen, acting as a proton carrier, is reminiscent of Lo, H.C., Han, H., D‚Souza, L.J., Sinha, S.C., Keinan, E. Rhenium(VII) oxide-cat- the dual functionality of the histidine in the catalytic alyzed heteroacylative ring-opening dimerization of tetrahydrofuran. J. Am. Chem. Soc., in press. triad of natural serine proteases. 214 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Lo, H.C., Iron, M.A. Martin, J.M.L., Keinan, E. Proton walk in the aqueous plat- ious biological systems and have resulted in the iden- inum complex TpPtMeCO via a sticky σ-methane ligand. Chem. Eur. J., in press. tification and characterization of proteases within those Metanis, N., Keinan, E., Dawson, P.E. Synthetic seleno-glutaredoxin 3: highly reduc- systems. For example, a cysteine protease from the ing oxidoreductases with enhanced catalytic efficiency. J. Am. Chem. Soc., in press. house dust mite Dermatophagoides pteronyssinus Tuttle, T., Keinan, E., Thiel, W. Understanding the enzymatic activity of 4-oxalocro- was identified by using a 4000-member PNA-encoded tonate tautomerase and its mutant analogues: a computational study. J. Phys. Chem. B Condens. Matter Mater. Surf. Interfaces Biophys. 110:19685, 2006. inhibitor library. The identified protease plays a key

Weiss, Y., Rubin, B., Shulman, A., Ben Shir, I., Keinan, E., Wolf, S. Determina- role in allergic hypersensitivity through the selective tion of plant resistance to carbamate herbicidal compounds inhibiting cell division degradation of CD25 from T cells. and early growth by seed and plantlets bioassays. Nat. Protoc., in press. Another example of functional characterization of Weiss, Y., Shulman, A., Ben Shir, I., Keinan, E., Wolf, S. Herbicide-resistance protein activity is the profiling of the substrate specific- conferred by expression of a catalytic antibody in Arabidopsis thaliana. Nat. Biotechnol. 24:713, 2006. ity of proteases from Dengue virus, the etiologic agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Using 2 substrate libraries of approx- Functional Characterization of imately 160,000 members, we characterized the structure-activity relationship of the NS3 protease from the Proteases via Combinatorial 4 Dengue virus serotypes and facilitated the develop- Libraries ment of inhibitors of the virus. PUBLICATIONS J.L. Harris, J. Alves* Harris, J.L. Protease substrate profiling. In: Enzyme assays: high-throughput screening, genetic selection and fingerprinting. Reymond, J.-L. (Ed.). Wiley-VCH, New York, 2006, p. 303. ith the complete sequencing of genomes from multiple organisms, information on the Harris, J.L., Winssinger, N. PNA encoding (PNA = peptide nucleic acid): from solution-based libraries to organized microarrays. Chem. Eur. J. 11:6792, 2005. W repertoire of genes can be readily established. Li, J., Lim, S.P., Beer, D., Patel, V., Wen, D., Tumanut, C., Tully, D.C., Williams, However, large gaps still remain in our knowledge of J.A., Jiricek, J., Priestle, J.P., Harris, J.L., Vasudevan, S.G. Functional profiling of the biological role of most genes. These gaps are mainly recombinant NS3 proteases from all four serotypes of Dengue virus using tetrapep- tide and octapeptide substrate libraries. J. Biol. Chem. 280:28766, 2005. due to the fact that most biological functions are regulated not at the gene or transcript level, but at the Petrassi, H.M., Williams, J.A., Li, J., Tumanut, C., Ek, J., Nakai, T., Masick, B., Backes, B.J., Harris, J.L. A strategy to profile prime and non-prime proteolytic posttranslational level. In contrast to the situation in substrate specificity. Bioorg. Med. Chem. Lett. 15:3162, 2005. genomics, in which the changes in the content or amount Winssinger, N., Harris, J.L. Microarray-based functional protein profiling using of cellular DNA or RNA can be readily examined, moni- peptide nucleic acid-encoded libraries. Expert Rev. Proteomics 2:937, 2005. toring translational and posttranslational dynamics of functional proteins on a genome-wide level is more difficult. Progress in our current understanding of bio- Organic Synthesis and Selective logical processes is limited by the available tools that can be used to probe function at the posttranslational Drug Delivery level. We are developing and applying technologies S.C. Sinha, R.A. Lerner, Z. Chen, S. De, S. Das, S. Abraham, based on small-molecule protein modifiers to profile F. Guo the active state of enzymes. In collaboration with N. Winssinger, Université ur main research interests are synthesis of biolog- Louis Pasteur, Strasbourg, Germany, we have devel- ically important natural and nonnatural molecules, oped an encoding strategy that uses peptide nucleic O synthetic methods, and antibody catalysis in acid (PNA) sequences. Encoding combinatorial libraries organic synthesis and selective drug delivery. During with PNA tags allows not only for the synthetic history the past year, we focused on 3 different classes of of the library to be captured in the resulting molecule compounds: the anticancer adjacent bis-tetrahydrofuran but also for spatial deconvolution of the molecules on annonaceous acetogenins, the antibacterial macrocyclic DNA microarrays. lactones sorangiolides, and nonnatural small-molecule Using this technology, we have created encoded drugs that target G protein–coupled receptors. In our protease inhibitor and substrate libraries of thousands work on antibody catalysis, we developed a proadapter of molecules. These libraries have been applied to var- approach for production of the chemically programmed MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 215 aldolase antibody 38C2 and new doxorubicin prodrugs oped new doxorubicin prodrugs that are not only more that are catalyzed by antibody 38C2 faster than the stable than the previously reported analogous prodrugs previously reported drugs are. but also activated faster by using antibody 38C2. We SELECTIVE CHEMOTHERAPY WITH CATALYTIC also produced 38C2-antagonist conjugates. The conju- ALDOLASE ANTIBODIES gates bound efficiently to MDA-MB-231 cells expressing

For selective chemotherapy, we intend to develop αvβ3 and αvβ5. We also found that the modified anti- drug conjugates and prodrugs that will target cell-sur- body can activate new doxorubicin prodrugs. face receptors, such as the glycoprotein integrins αvβ3 Therefore, we have all the tools to investigate pro- and αvβ5. These integrins are directly implicated in drug therapy in animal models. The selective chemo- tumor angiogenesis; they are overexpressed in the vas- therapy studies are carried out in collaboration with culature of angiogenic tumors and in numerous cancer C.F. Barbas, Department of Molecular Biology, and cells but are less expressed on quiescent blood vessels. B. Mueller, La Jolla Institute for Molecular Medicine, Using antibody 38C2 and small-molecule antagonists San Diego, California. of αvβ3 and αvβ5 (or a targeting agent), we developed SYNTHESIS OF NATURAL AND NONNATURAL SMALL antagonist-38C2 conjugates, also known as chemically MOLECULES programmed 38C2 (Fig. 1). Total synthesis of naturally occurring and biologically important compounds is important not only for confirming their structures but also for producing the compounds and their analogs for comprehensive biological evaluations. To synthesize these compounds, we are developing methods that involve both antibody catalysis and common synthetic routes. In the past year, in addition to synthesizing sorangiolides, which are naturally occurring macrocyclic lactones, and the library of Fig. 1. Schematic drawings of the diketone and the proadapter bis-tetrahydrofuran annonaceous acetogenins, we focused strategies used to produce chemically programmed antibody con- on small-molecule nonnatural ligands of G protein–cou- structs that target cells expressing the integrins αvβ3 and αvβ5. Abbreviations: Ab, antibody; TA, targeting agent. pled receptors. The studies on the synthesis of these ligands are carried out in collaboration with E. Roberts, The conjugation between the targeting agents and Department of Chemistry. the antibody takes place in the binding sites though Sorangiolides (Fig. 2) are weakly active antibacter- the diketone or vinyl ketone linkers. Because the vinyl ial compounds. Our goal is to synthesize the highly ketones are highly reactive, we used the corresponding acetone adduct as the prolinker, which undergoes 38C2- catalyzed reaction to produce the active linker before the active linker reacts. This strategy has been termed the proadapter approach. The conjugates prepared by both approaches bound efficiently to cells expressing

αvβ3 and αvβ5, including human breast cancer cell lines MDA-MB-435 and MDA-MB-231, and inhibited the growth of both the primary tumors and secondary Fig. 2. Structure of sorangiolides A and B (top) and a general metastasis in distant organs. structure of bis-tetrahydrofuran annonaceous acetogenins (bottom). Development of these strategies for the formation of antibody constructs can have a large effect on the active sorangiolide analogs. Thus, we have developed treatment of various diseases, including cancer. synthetic routes that can provide the macrocyclic struc- In the alternative approach, we are developing pro- ture of sorangiolides. Using an intermediate, we will drugs that can be efficiently activated by the aldolase synthesize both the natural and nonnatural molecules. antibodies 38C2 and 93F3. These antibodies will be For other bis-tetrahydrofuran acetogenins, which are targeted to tumor cells or the tumor vasculature by using among the most active cancer agents and are toxic to antagonists of αvβ3 and αvβ5. In the past year, we devel- several human cancer cell lines at much lower concen- 216 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE trations than doxorubicin is, we developed methods that C. Potter, V. Reddy, A. Schneemann, G. Siuzdak, J.R. can provide all the stereoisomers of asimicin and bullat- Williamson, and M.J. Yeager, and a variety of groups acin. The new methods involve a bidirectional approach. outside of Scripps. Now, we are pursuing synthesis of the 64 stereoiso- DOUBLE-STRANDED DNA VIRUSES mers of asimicin and bullatacin. HK97 is a double-stranded DNA virus similar to phage λ. It undergoes a remarkable morphogenesis in PUBLICATIONS its assembly and maturation, and this process can be Das, S., Li, L.-S., Abraham, S., Chen, Z., Sinha, S.C. A bidirectional approach to the synthesis of a complete library of adjacent-bis-THF annonaceous acetogenins. recapitulated in vitro. We determined the atomic resolu- J. Org. Chem. 70:5922, 2005. tion structure of the 650-Å mature, head II particle and Li, L.-S., Babendure, J.L., Sinha, S.C., Olefsky, J.M., Lerner, R.A. Synthesis and discovered the mechanism used to concatenate the sub- evaluation of photolabile insulin prodrugs. Bioorg. Med. Chem. Lett. 15:3917, 2005. units of the particle into a chain-mail fabric similar to Popkov, M., Rader, C., Gonzalez, B., Sinha, S.C., Barbas, C.F. III. Small molecule that seen in armor of medieval knights. In the past year, drug activity in melanoma models may be dramatically enhanced with an antibody effector. Int. J. Cancer 119:1194, 2006. we focused on the dynamics of maturation. Prohead II is a 500-Å metastable intermediate at pH 7 that can be induced to begin maturation by lower- Structure, Function, and ing the pH to 4. Solution x-ray scattering and single-molecule fluorescence showed that the initial transition to Applications of Virus Particles a particle of about 560 Å occurs as a highly cooperative, stochastic event with no detectable intermediates J.E. Johnson, M. Banerjee, A. Chatterji, Z. Chen, I. Gertsman, that takes place in less than 1 second for an individ- R. Huang, R. Khayat, G. Lander, J. Lanman, K.K. Lee, ual particle. A quorum of cross-links must form in this T. Matsui, P. Natarajan, A. Odegard, J. Speir particle to generate the second expansion intermediate (about 650 Å), which also forms cooperatively with no e investigate model virus systems that provide detectable intermediates. At pH 4, formation of cross- insights for understanding assembly, matu- links continues, with 360 formed per particle. Limited ration, entry, localization, and replication. We W pentamer dynamics (established from crystallography have also developed viruses as reagents for applications and electron cryomicroscopy) prevents the last 60 crossin nanomedicine, chemistry, and biology. We investi- links from forming, but pentamer trajectories extend gate viruses that infect bacteria, insects, plants, and at pH 7, allowing these cross-links to form, complet- the extreme thermophile Sulfolobus. These viruses ing maturation. have genomes of single-stranded RNA and double- Bacteriophage P22 is the prototype of the Podoviri- stranded DNA. dae, which are characterized by a T = 7 capsid with We use a variety of physical methods to investigate a short tail structure incorporated into a unique 5-fold structure-function relationships, including single-crystal vertex. We determined an asymmetric reconstruction and static and time-resolved solution x-ray diffraction, of this particle that revealed spooled DNA, the dode- electron cryomicroscopy and image reconstruction, mass cameric portal, and the location of the 9 gene products spectrometry, structure-based computational analyses, known to be in the particle. and methods associated with thermodynamic charac- Sulfolobus turreted icosahedral virus is an archaeal terization of virus particles and their transitions. Bio- virus isolated from Sulfolobus, which grows in the acidic logical methods we use include genetic engineering of hot sulfur springs (pH 2–4, 72°C–92°C) in Yellowstone viral genes and their expression in Escherichia coli, National Park. An electron cryomicroscopy reconstruction mammalian cells, insect cells, and yeast and the char- of the virus showed that the capsid has pseudo T = 31 acterization of these gene products by physical methods. quasi symmetry and is 1000 Å in diameter, including For cytologic studies of viral entry and infection, we the pentons. We solved the x-ray structure of the major use fluorescence and electron microscopy and particles capsid protein of the virus, and it revealed a fold nearly assembled in heterologous expression systems. Our identical to the major capsid proteins of the eukaryotic studies depend on extensive consultations and collab- adenoviruses and PRD-1, a virus that infects bacteria. orations with others at Scripps Research, including These findings indicate a virus phylogeny that spans groups led by C.L. Brooks, D.A. Case, B. Carragher, the 3 domains of life. Difference electron density maps M.G. Finn, M. Manchester, D.R. Millar, R.A. Milligan, in which the x-ray model is subtracted from the elec- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 217

tron cryomicroscopy density clearly shows an internal Ochoa, W., Chatterji, A., Lin, T., Johnson, J.E. Generation and structural analysis of reactive empty particles derived from an icosahedral virus. Chem. Biol. 13:771, 2006. membrane in which the capsid proteins are anchored. SINGLE-STRANDED RNA VIRUSES Prasad, T., Turner, M., Falkner, J., Mittleman, D., Johnson, J.E., Lin, T., Colvin, V. Nanostructured virus crystals for x-ray optics. IEEE Trans. Nanotechnol. 5:93, 2006. Flock House virus is a T = 3, single-stranded RNA virus that infects Drosophila. We are studying viral entry Reddy, V.S., Johnson, J.E. Structure-derived insights into virus assembly. Adv. Virus Res. 64:45, 2005. and early expression and assembly of the capsid protein. Recently, studies on viral entry indicated the presence Sapsford, K.E., Soto, C.M., Blum, A.S., Chatterji, A., Lin, T., Johnson, J.E., Ligler, F.S., Ratna, B.R. A cowpea mosaic virus nanoscaffold for multiplexed anti- of an “eluted” particle early in infection that has initiated body conjugation: application as an immunoassay tracer. Biosens. Bioelectron. its disassembly program but is then eluted back into the 21:1668, 2006. medium. We did a phenotypic characterization of the Shepherd, C.M., Borelli, I.A., Lander, G., Natarajan, P., Siddavanahalli, V., Bajaj, C., Johnson, J.E., Brooks, C.L. III, Reddy, V.S. VIPERdb: a relational database for particles, and we are using electron cryomicroscopy structural virology. Nucleic Acids Res. 34:386, 2006. to study them. For studies on the expression and assem- Soto, C.M., Blum, A.S., Vora, G.J., Lebedev, N., Meador, C.E., Won, A.P., Chat- bly of the capsid protein, we are using tags inserted terji, A., Johnson, J.E., Ratna, B.R. Fluorescent signal amplification of carbocyanine genetically in the capsid protein that allow the freshly dyes using engineered viral nanoparticles. J. Am. Chem. Soc. 128:5184, 2006.

made proteins to be optically visualized with a fluoro- Speir, J.A., Bothner, B., Qu, C., Willits, D.A., Young, M.J., Johnson, J.E. Enhanced phore and in the electron microscope with photoconver- local symmetry interactions globally stabilize a mutant virus capsid that maintains infectivity and capsid dynamics J. Virol. 80:3582, 2006. sion of the fluorophore. Recently, high-pressure freezing of infected cells revealed exceptionally detailed features Tang, J., Johnson, J.M., Dryden, K.A., Young, M.J., Zlotnick, A., Johnson, J.E. The role of subunit hinges and molecular “switches” in the control of viral capsid of viral entry and regions of replication within the cell. polymorphism. J. Struct. Biol. 154:59, 2006. Refined atomic models of tetravirus structures and Tang, L., Gilcrease, E.B., Casjens, S.R., Johnson, J.E. Highly discriminatory binding structure-based mutagenesis combined with highly of capsid-cementing proteins in bacteriophage L. Structure 14:837, 2006. sensitive assays for defining phenotypes have revealed Taylor, D.J., Speir, J.A., Reddy, V., Cingolani, G., Pringle, F.M., Ball, L.A., Johnson, the electrostatic principals of maturation for the J.E. Preliminary x-ray characterization of authentic providence virus and attempts to T = 4 tetraviruses. express its coat protein gene in recombinant baculovirus. Arch. Virol. 151:155, 2006. Cowpea mosaic virus is a 30-nM reagent that we Walukiewicz, H.E., Johnson, J.E., Schneemann, A. Morphological changes in the T = 3 capsid of Flock House virus during cell entry. J. Virol. 80:615, 2006. use for chemistry and nanomedicine. We found that particles of the virus with doxorubicin bound internally Wikoff, W.R., Conway, J.F., Tang, J., Lee, K.K., Gan, L., Cheng, N., Duda, R.L., Hendrix, R.W., Steven, A.C., Johnson, J.E. Time-resolved molecular dynamics of can be specifically targeted to tumor cells via peptides HK97 capsid maturation interpreted by electron cryo-microscopy and x-ray crystal- on the viral surface that recognize receptors for vascular- lography. J. Struct. Biol. 153:300, 2006. ization signals that are highly expressed on tumor cells.

PUBLICATIONS Nanomanufacturing on an du Plessis, L., Hendry, D.A., Dorrington, R.A., Hanzlik, T.N., Johnson, J.E., Appel, M. Revised RNA2 sequence of the tetravirus nudaurelia capensis ω virus (NωV): annotated sequence record. Arch. Virol. 150:2397, 2005. Icosahedral Scaffold and

Khayat, R., Tang, L., Larson, E.T., Lawrence, C.M., Young., M., Johnson, J.E. Struc- ture of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and Neutralization of Avian H5N1 bacterial viruses. Proc. Natl. Acad. Sci. U. S. A. 102:18944, 2005. Influenza Viruses Lander, G.C., Tang, L., Casjens, S.R., Gilcrease, E.B., Prevelige, P., Poliakov, A., Potter, C.S., Carragher, B., Johnson, J.E. The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 312:1791, 2006. T. Lin, J.E. Johnson, A. Censullo, A. Chatterji

Lee, K.K., Tsuruta, H., Hendrix, R.W., Duda, R.L., Johnson, J.E. Cooperative reor- MOLECULAR ELECTRONICS ON AN ICOSAHEDRAL ganization of a 420 subunit virus capsid. J. Mol. Biol. 352:723, 2005. SCAFFOLD Lin, T., Lomonossoff, G.P., Johnson, J.E. Structure-based engineering of an icosa- Molecular manufacturing, the essence of nanotech- hedral virus for nanomedicine and nanotechnology. In: Nanotechnology in Biology nology, involves the manipulation of molecules as the and Medicine: Methods, Devices, and Applications. Vo-Dinh. T. (Ed.). CRC Press, Boca Raton, FL, in press. self-assembling components at the nanometer scale to

Medintz, I.L., Sapsford, K.E., Konnert, J.H., Chatterji, A., Lin, T., Johnson, J.E., build devices in mesoscale. Although small molecules Mattoussi, H. Decoration of discretely immobilized cowpea mosaic virus with lumi- with novel electronic properties can be synthesized, mak- nescent quantum dots. Langmuir 21:5501, 2005. ing functional connectivity among the different compo- Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, John- nents in designed patterns is generally difficult. In son, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol. 3:809, 2005. contrast, biological macromolecules are more amenable 218 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE for self-assembly because of their versatility, program- analysis of escape mutants in conjunction with the mability through genetic engineering, and propensity vaccine development. Structural studies of antibody to form arrays and can be used either directly as devices interactions with the H5N1 viruses are also being car- or as scaffolds for patterning small molecules. ried out to shed light on the mechanism of neutraliza- We have shown that cowpea mosaic virus (CPMV), tion of the viruses. an icosahedral plant virus, can be used as the template for nanochemistry by introducing unique cysteine residues PUBLICATIONS Medintz, I.L., Sapsford, K.E., Konnert, J.H., Chatterji, A., Lin, T., Johnson, J.E., and exploiting the native lysine residues. In collaborative Mattoussi, H. Decoration of discretely immobilized cowpea mosaic virus with lumi- studies with B.R. Ratna, Naval Research Laboratory, nescent quantum dots. Langmuir 21:5501, 2005. Washington, D.C., the virus capsid was exploited as a Prasad, T., Turner, M., Falkner, J., Mittleman, D., Johnson, J.E., Lin, T., Colvin, V. Nanostructured virus crystals for x-ray optics. IEEE Trans. Nanotechnol., in press. nano circuit board, and the reactive groups were used as anchoring points for the assembly of the electronic molecules oligophenylene-vinylene and 1,4-C6H4[trans-(4- Design and Informatics in AcSC6H4≡CPt(Pbu3)2≡C]2. The establishment of the molecular network was shown by measuring electronic Structural Virology conductance with scanning tunnel microscopy.

NEUTRALIZATION OF AVIAN H5N1 INFLUENZA V.S. Reddy, S. Kumar, M. Tripp, P. Singh, R. Mannige, VIRUSES I. Borelli, J. Loo, C.L. Brooks III, J.E. Johnson, Influenza is one of the most important viral dis- M. Manchester, G. Nemerow, A. Schneemann eases in humans. It has caused morbidity and mortality in millions of people in frequent epidemics and pan- e are interested in understanding the structural demics throughout the centuries. Human influenza virus underpinnings and requirements for formation is typically associated with 3 H subtypes: H1, H2, and W and function of viral capsids and in designing H3. In recent years, an avian H5 (H5N1) influenza novel protein shells that polyvalently display molecules virus crossed the species barrier to infect humans with of interest. To this end, we use structural, computa- high virulence. To date, the avian virus has not been tional, informatics, and genetic methods. efficient in transmission from human to human, and Viruses are highly evolved macromolecular machines the disease has not spread in the human population. that perform a variety of functions during their life cycle, However, the continuous circulation and spreading of including selective packaging of the genome, self-assem- H5N1 viruses in avian species across the globe leads bly into uniform capsids, binding to host cells, and deliv- to more human infections and increases the likelihood ery of the genome to the targeted cells. Simple viruses, that the virus will acquire the necessary characteris- such as nonenveloped viruses, form closed protein shells tics for efficient human-to-human transmission through of uniform size and character by the self-association of genetic mutation or reassortment with a prevailing structural and functional components: proteins and the human influenza A virus. nucleic acid genome. Hence, these viruses are useful for The possible emergence of an H5N1 virus highly structural and functional analyses. contagious to humans is a serious pandemic threat. In collaboration of with G.R. Nemerow, Department Therefore, producing effective vaccines to counter the of Immunology, we are using x-ray crystallographic threat posed by the H5N1 viruses is important. CPMV methods to determine the structure of the entire is an effective scaffold for the development of subunit human adenovirus particle, currently at about 9-Å reso- vaccines. We are developing a novel combinatorial strat- lution. We are continuing to collect diffraction data egy in which the CPMV system is used to identify vac- at higher resolution. We continue to maintain and cine candidates. expand the virus structure database, namely VIPERdb In another study in collaboration with scientists in (http://viperdb.scripps.edu), where the coordinates of the Hong Kong and Southern China, the epicenter of the characterized spherical capsid structures are stored and influenza outbreaks, we have produced more than 100 organized in terms of viral taxonomy and capsid archi- monoclonal antibodies against the avian influenza viruses tecture. We are developing structural analysis tools to and have shown that many of these antibodies are neu- “mine” the capsid structures in terms of protein-protein tralizing. These neutralizing antibodies are used in the interactions, contacting residue pairs, association ener- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 219 gies, contributions of individual residues, and surface we are interested in the determinants that endow a characteristics. VIPERdb is being developed as part polypeptide chain with such versatility. We seek to of the Multiscale Modeling Tools for Structural Biology, harness this versatility for novel applications of viruses a National Institutes of Health research resource headed in biotechnology and nanotechnology. by C.L. Brooks, Department of Molecular Biology. In We focus on a structurally and genetically well-char- addition, on the basis of the structural similarity that acterized virus family, the T = 3 nodaviruses. Noda- occurs within a virus family, we are building homology viruses are composed of 180 copies of a single coat models for the uncharacterized members of virus fam- protein and 2 strands of positive-sense RNA. Currently, ilies. These models will be useful for molecular virol- we are elucidating the mechanism by which the 2 ogists investigating structural and functional relationships genomic RNAs are packaged into a single virion. Our in viruses. long-term goal is to develop nodaviruses as RNA pack- We are generating decoys of pathogenic molecules aging and delivery vectors. Our data indicate that the on the surfaces of viral capsids that can be used as 2 viral RNAs are recognized separately, but it is not yet vaccines against cytotoxins such as ricin. Currently, known whether packaging occurs sequentially and tomato bushy stunt virus–like capsids are our display whether one or more coat protein subunits are involved platform of choice; the platform consists of multiple in this process. Interestingly, we found that RNA copies of a 2-domain capsid protein subunit with the genome packaging is coupled to genome replication, C-terminal P-domain exposed on the surface. Such a suggesting potential approaches for packaging of for- unique subunit structure is useful for attaching peptides eign RNAs. or proteins of interest at the end of the C terminus of In other studies, we are investigating the mechanism the capsid protein or for replacing the external P-domain by which nodaviral protein B2 suppresses RNA silencing with the proteins of interest rather than inserting them in infected cells. In collaboration with J.R. Williamson, in a loop. Department of Molecular Biology, we showed that B2 binds to double-stranded RNA in a sequence-indepen- PUBLICATIONS Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A., dent manner and that it interferes with cleavage of Reddy, V.S. Characterization of polymorphism displayed by the coat protein double-stranded RNA substrates by the cellular protein mutants of tomato bushy stunt virus. Virology 349:222, 2006. Dicer. Moreover, in collaboration with J.L. Imler, Uni- Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, John- versity of Strasbourg, Strasbourg, France, we showed son, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol. 3:809, 2005. that B2 is critical for nodaviral infection of Drosophila and that Dicer plays an essential role in host defense Shepherd, C.M., Borelli, I.A., Lander, G., Natarajan, P., Siddavanahalli, V., Bajaj, C., Johnson, J.E., Brooks, C.L. III, Reddy, V.S. VIPERdb: a relational database for against nodaviruses in vivo. structural virology. Nucleic Acids Res. 34(Database Issue):D386, 2006. We are also collaborating with several investigators Taylor, D.J., Speir, J.A., Reddy, V., Cingolani, G., Pringle, F.M., Ball, L.A., John- at Scripps Research, the Salk Institute, and Harvard son, J.E. Preliminary x-ray characterization of authentic providence virus and attempts to express its coat protein gene in recombinant baculovirus. Arch. Virol. University to develop nodaviruses as platforms for deliv- 151:155, 2006. ery of anthrax antitoxins. To this end, we are using particles to display the VWA domain of capillary morphogenesis protein 2, the cellular receptor for anthrax Biology and Applications of toxin, in a multivalent fashion on the surface of the virion. Two insertion sites yielding different patterns of Icosahedral Virus Capsids 180 copies of the VWA domain were selected on the basis of computational modeling of the high-resolution A. Schneemann, B. Groschel, C. Hsu, J. Lee, D.J. Manayani, crystal structure of the insect nodavirus Flock House D. Marshall, J.E. Petrillo, M.E. Siladi, P.A. Venter virus. The resulting chimeric viruslike particles func- oat proteins of nonenveloped, icosahedral viruses tioned as a potent anthrax antitoxin in cell culture and perform multiple functions during the course of protected rats from challenge with lethal toxin. This C viral infection, including capsid assembly, specific research is important because it shows that protein encapsidation of the viral genome, binding to a cellu- domains containing more than 150 amino acids can be lar receptor, and uncoating. In some viruses, a single displayed on Flock House virus in a biologically func- type of protein is sufficient to carry out these functions; tional form, suggesting numerous additional applications. 220 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Flock House virus particles are also good candidates an AIDS-like syndrome in domestic cats and has struc- for novel materials in nanotechnology applications. The tural and functional similarities to HIV, the causative particles are stable, easily manipulated, biocompati- agent of AIDS in humans. Discovery of ways to inter- ble, and nontoxic in vivo and can be produced easily fere with FIV infection may ultimately result in devel- and in high quantities. The high-resolution x-ray struc- opment of treatments for infections in both cats and ture of the virus revealed the potential for using chem- humans. In recent studies, we continued to focus on ical approaches to attach ligands to the surface of the the molecular characterization of receptor interactions virus and for using genetic strategies to modify the and the molecular basis for the development of drug capsid. In collaboration with M. Manchester, Depart- resistance in the aspartic protease encoded by FIV. ment of Cell Biology, and M. Ozkan, University of Cali- RECEPTOR STUDIES fornia, Riverside, we used conjugation chemistry to Like many strains of HIV, FIV uses the chemokine couple inorganic nanotubes and quantum dots to Flock receptor CXCR4 to enter the primary target cell, the House virus particles to produce an array of novel hybrid CD4+ T cell. However, unlike HIV, FIV does not use structures. This approach may one day be used to fab- the cell-surface protein CD4 as a primary binding ricate unique materials for a variety of applications, receptor. Rather, the feline lentivirus uses the activa- including biofilms with tunable pore size, 3-dimensional tion antigen CD134 to initially bind to CD4+ T cells. scaffolds for production of nanoelectronic devices, and CD134 is expressed on activated CD4+ T cells, a drug delivery. finding that explains why FIV can infect and kill CD4+ T cells, even though the virus does not bind CD4. PUBLICATIONS Chao, J.A., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., Williamson, As reported last year, we showed that interaction J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. of the FIV surface glycoprotein gp95 with a soluble Struct. Mol. Biol. 12:952, 2005. version of CD134 allows productive infection of cells Destito, G., Schneemann, A., Manchester, M. Biomedical nanotechnology using that bear the entry receptor CXCR4 but lack cell-sur- virus-based nanoparticles. Curr. Top. Microbiol. Immunol., in press. face CD134. This finding is consistent with the notion Galiana-Arnoux, D., Dostert, C., Schneemann, A., Hoffmann, J.A., Imler, J.L. that binding of CD134 causes a conformational change Essential function in vivo for Dicer-2 in host defense against RNA viruses in Drosophila. Nat. Immunol. 7:590, 2006. in gp95, which in turn increases the affinity of interaction with CXCR4 to facilitate infection of the target Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A., Reddy, V.S. Characterization of polymorphism displayed by the coat protein cell. These effects are similar to the effects of binding mutants of tomato bushy stunt virus. Virology 349:222, 2006. of soluble CD4 by gp120, the surface glycoprotein of Schneemann, A. The structural and functional role of RNA in icosahedral virus HIV and indicate that although different primary recep- assembly. Annu. Rev. Microbiol. 60:51, 2006. tors are involved, the actual mechanism of infection Singh, P., Destito, G., Schneemann, A., Manchester, M. Canine parvovirus-like of FIV and HIV is strikingly similar. We speculate that particles, a novel nanomaterial for tumor targeting. J. Nanobiotechnol. 4:2, 2006. the benefit of this type of binding cascade is to limit Walukiewicz, H.E., Johnson, J.E., Schneemann, A. Morphological changes in the exposure of critical regions of the surface glycoproteins T = 3 capsid of Flock House virus during cell entry. J. Virol. 80:615, 2006. to the immune system until the primary binding event has already occurred, thus reducing the likelihood of virus neutralization. Molecular Biology of Using chimeric proteins consisting of feline and Retroviruses human CD134 (the human homolog does not bind FIV glycoprotein) and site-directed mutagenesis, we have J.H. Elder, A.P. de Parseval, Y.-C. Lin, S. de Rozieres, M. mapped regions of feline CD134 involved in interaction Sundstrom, K. Tam, M. Giffin,* H. Heaslet,* C.D. Stout, with gp95. The results indicated that as few as 3 amino B.E. Torbett* acids in the C-terminal part of outer domain 1 of feline * Department of Molecular and Experimental Medicine, Scripps Research CD134 are sufficient to impart FIV gp95 binding and receptor function to human CD134. Studies are in prog- ur research centers on the molecular character- ress to map the regions of gp95 that bind CD134. ization of retroviruses, with emphasis on feline Importantly, we have now a panel of antibodies immunodeficiency virus (FIV) and development O that bind and neutralize FIV only after CD134 is of ways to interfere with the viral life cycle. FIV causes bound; we have used peptides to map the region in MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 221 which these CD134-dependent neutralizing antibodies de Parseval, A., Bobardt, M.D., Chatterji, A., Chatterji, U., Elder, J.H., David, G., Zolla-Pazner, S., Farzan, M.R., Lee, T.-H., Gallay, P.A. A highly conserved arginine react. These studies effectively map regions of the viral in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs. glycoprotein critical for CD134 interaction. Cocrystalliza- J. Biol. Chem. 280:39493, 2005. tion studies are under way to determine the structure de Parseval, A., Grant, C.K., Sastry, K.J., Elder, J.H. Sequential CD134-CXCR4 interactions in feline immunodeficiency virus (FIV): soluble CD134 activates FIV of the region surrounding the antibody-binding site. Env for CXCR4-dependent entry and reveals a cryptic neutralization epitope. J. These experiments will contribute to our understand- Virol. 80:3088, 2006. ing of the nature of receptor binding and will define Gonzalez-Lira, B., Rueda-Orozco, P.E., Galicia, O., Montes-Rodriguez, C.J., Guz- targets for vaccine development. man, K., Guevara-Martinez, M., Elder, J.H., Prospero-Garcia, O. Nicotine prevents HIVgp120-caused electrophysiological and motor disturbances in rats. Neurosci. PROTEASE DRUG RESISTANCE Lett. 394:136, 2006. The aspartic protease of lentiviruses is responsible Heaslet, H., Kutilek, V., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout for processing the viral Gag and Pol polyproteins into C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease the final gene products required for viral replication and NL4-3. J. Mol. Biol. 356:967, 2006. must function efficiently to generate infectious virus. Liang, F.-S., Brik, A., Lin, Y.-C., Elder, J.H., Wong, C.-H. Epoxide in water and screening in situ for rapid discovery of enzyme inhibitors in microtiter plates. Drugs against HIV protease are keys to the success of Bioorg. Med. Chem. 14:1058, 2006. highly active antiretroviral therapy used to treat, but Whiting, M., Muldoon, J., Lin, Y.-C., Silverman, S.M., Lindstrom, W., Olson, A., not cure, patients infected with HIV. The substrate and Kolb, H.C., Finn, M.G., Sharpless, K.B., Elder, J.H., Fokin, V.V. Inhibitors of HIV-1 inhibitor specificities of FIV differ from those of HIV. protease by using in situ click chemistry. Angew. Chem. Int. Ed. 45:1435, 2006. We investigated the nature of these differences to better understand the structural basis of development of resistance to therapy, an ongoing problem with current Metalloenzyme Engineering drugs used to treat HIV disease. In certain instances, similarities exist between amino D.B. Goodin, C.D. Stout, S. Vetter, E.C. Glazer, R.F. Wilson, acid positions that dictate differences in substrate speci- A. Annalora, A.-M. Hays ficity between FIV and HIV aspartic protease and those ur goals are to understand the diverse reactivity that mutate in response to drug treatment. Mutations in of heme enzymes and to use that information these sites increase the dissociation constant for a O to generate engineered forms with novel catalytic given drug, but at a cost in catalytic efficiency for the properties. The primary hypothesis that has driven these viral protease. Compensatory amino acid substitutions studies is that the chemical reactivity displayed by can then occur that increase the catalytic efficiency of these enzymes resides partially within the heme cofac- the drug-resistant protease, thus increasing expression tor. One role of the protein is to limit or direct the access of virus despite drug treatment. of substrates to the active site in ways that result in We prepared mutants of FIV protease in which amino specific catalysis. In addition, many important exam- acids found in drug-resistant HIV protease were placed in ples exist in which the protein directly modulates the the equivalent positions in the FIV enzyme. Then, using activity of the heme. Thus, our goals are to delineate cells transduced with gag/pol gene expression vectors the boundaries between these 2 roles for the protein encoding HIV-FIV hybrid proteases, we tested the mutants and then use this information to introduce sites where for relative drug sensitivity. We found that the Gag/Pol nonnative substrates interact with the heme cofactor in polyproteins are processed by the hybrid proteases and ways that will induce new catalytic reactions. We use have drug sensitivity profiles similar to those of HIV a number of techniques in structural biology and spec- protease. However, the order of site cleavage, which is troscopy and strategies of rational protein redesign and critical to generation of infectious virus, is altered by molecular evolution. these specific changes. Studies are under way to estab- One area of emphasis is the basic physical, spec- lish a structural basis for this phenomenon. The findings troscopic, and functional properties of heme enzymes. highlight yet another potential approach to interrupting For example, the FeIII/FeII and FeII/FeI redox couples the viral life cycle. of inducible nitric oxide synthase have recently been measured by using direct cyclic voltammetry in organic PUBLICATIONS Brik, A., Alexandratos, J., Lin, Y.-C., Elder, J.H., Olson, A.J., Wlodawer, A., Good- films on graphite electrodes. These studies allow easy sell, D.S., Wong, C.-H. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis measurement of electron transfer between the enzyme of HIV-1 protease inhibitors. Chembiochem 6:1167, 2005. and the electrode surface and have revealed the inter- 222 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE conversion of several coordination states of the heme. Control of Cell Division The results will complement ongoing studies in which the enzymes are directly and homogeneously coupled S.I. Reed, C. Baskerville, L.-C. Chuang, S. Ekholm-Reed, to electrode surfaces by using molecular wires. M. Henze, J. Keck, V. Liberal, K. Luo, B. Olson, S. Rudyak, In other research, we used cavity complementation D. Tedesco, F. van Drogen, J. Wohlschlegel to introduce small-molecule binding sites near the active site of a protein environment. This approach has pro- iological processes of great complexity can be vided new ways to test ideas about the diversity of the approached by beginning with a systematic functions of heme enzymes and is a useful tool for test- B genetic analysis in which the relevant compo- ing predictions of protein-ligand interactions. For exam- nents are first identified and the consequences of selec- ple, in a collaboration with B. Shoichet, University of tively eliminating the components via mutations are California, San Francisco, we completed a study in which investigated. We have used yeast, which is uniquely compounds in a database were docked into a buried tractable to this type of analysis, to investigate control engineered cavity that has an unusual specificity for of cell division. In recent years, it has become appar- charged ligands. Using x-ray crystallography, we veri- ent that the most central cellular processes throughout fied the accuracy of the docking predictions for 15 of the eukaryotic phylogeny are highly conserved in terms of the top 16 compounds. both the regulatory mechanisms used and the proteins In other studies, we used synthetic molecular wires, involved. Thus, it has been possible in many instances substrate analogs linked to photochemical or redox-active to generalize from yeast cells to human cells. sensitizers, to bind at the active site of cytochrome CONTROL IN YEAST P450, peroxidases, and nitric oxide synthase. These In recent years, we have focused on the role and wires will be useful as reporters of the active-site envi- regulation of the Cdc28 protein kinase (Cdk1). Initially ronment and as triggers to study reaction mechanisms. identified by means of a mutational analysis of the yeast In collaboration with H.B. Gray, California Institute of cell cycle, this protein kinase and its analogs are ubiq- Technology, Pasadena, California, we recently solved uitous in eukaryotic cells and are central to a number of aspects of control of cell-cycle progression. structures of cytochrome P450cam bound to 2 such wires. Marked changes in the protein structure occurred near One current area of interest is regulation of cellular 2 helices that are similar to structural variations seen morphogenesis by Cdk1. The activity of Cdk1 driven by in mammalian P450s, suggesting that the degree of mitotic cyclins modulates polarized growth in yeast cells. structural plasticity in prokaryotic P450s is similar to Specifically, these activities depolarize growth by alter- that of mammalian forms. ing the actin cytoskeleton. We found that several proteins In other research, we removed the proposed elec- that modulate actin structure are targeted by Cdk1, and tron-transfer pathway from a peroxidase and replaced we are investigating whether these phosphorylation events it with a solvent-filled channel. We have designed sur- control actin depolarization. rogate molecular wires to replace the native pathway, A second major area of interest is in the regulation and we have shown that one of these binds the chan- of mitosis. A key aspect of mitotic regulation in yeast nel in a mode that is completely analogous to the native is the accumulation of Cdc20, which triggers the tran- structure. This technique will provide new ways to test sition from metaphase to anaphase. Cdc20 is an essen- proposals about the specificity and structural require- tial cofactor of the protein-ubiquitin ligase known as the ments of this important structural element. Finally, we anaphase-promoting complex or APC/C. It is through the are designing and synthesizing a class of cofactor-linked ubiquitin-mediated proteolysis of a specific anaphase ruthenium-diamine photosensitizers that are designed inhibitor, securin (Pds1 in yeast), that anaphase is initi- to specifically target nitric oxide synthase at the pterin- ated. We found that cells are prevented from entering binding site, allowing the role of the cofactor in cataly- mitosis when DNA replication is blocked by the drug sis to be probed by direct-charge injection-withdrawal hydroxyurea, which causes the destabilization of Cdc20 through the wire. and inhibition of Cdc20 translation. While investigating mitosis, we found that a Cks1, PUBLICATIONS small Cdk1-associated protein, appears to regulate the Brenk, R., Vetter, S., Boyce, S.E., Goodin, D.B., Shoichet, B. Probing molecular docking in a charged model binding site. J. Mol. Biol. 357:1449, 2006. proteasome. Proteasomes are complex proteases that tar- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 223 get ubiquitylated proteins, including important cell-cycle We showed that phosphorylation-dependent proteolysis regulatory proteins. Surprisingly, we found that Cks1 reg- of cyclin E depends on a protein-ubiquitin ligase known as ulates a nonproteolytic function of proteasomes, the tran- SCFhCdc4. The F-box protein hCdc4 is the specificity scriptional activation of Cdc20. Specifically, Cks1 is factor that targets phosphorylated cyclin E. We are required to recruit proteasomes to the gene CDC20 for investigating how ubiquitylation of cyclin E is coordi- efficient transcriptional elongation. Our investigations of nated with other processes required for its degradation, CDC20 have led to the conclusion that Cks1 is required including prolyl isomerization. We are also investigat- for recruitment of proteasomes to and transcriptional ing SCFhCdc4 ubiquitylation of other important cellu- elongation of many other genes as well. Currently, we are lar proteins. elucidating the mechanism whereby Cks1 recruits protea- Recently, we began determining the role of SCFhCdc4 somes and facilitates transcriptional elongation. Our most in neurodegenerative disease. We found that parkin, a recent results suggest that Cks1 and proteasomes in con- protein often mutated in hereditary Parkinson’s disease, junction with Cdk1 mediate remodeling of chromatin. regulates the stability of hCdc4, possibly leading to neu- CONTROL IN MAMMALIAN CELLS ropathologic changes. Consistent with this idea, we We showed previously that the human homologs found that SCFhCdc4 targets peroxisome proliferator–acti- of the Cdc28 protein kinase are so highly conserved, vated receptor γ coactivator-1α which protects neurons structurally and functionally, relative to the yeast pro- from oxidative damage. In addition, we showed that tein kinase, that they can function and be regulated SCFhCdc4 regulates the turnover of presenilins in the properly in a yeast cell. Analyzing control of the cell brain, proteins strongly implicated in Alzheimer’s disease. cycle in mammalian cells, we produced evidence for Another area of interest is the role of Cks proteins in the existence of regulatory schemes, similar to those mammals, complementing our research in yeast. Mam- elucidated in yeast, that use networks of both positive mals express 2 orthologs of yeast Cks1, known as Cks1 and negative regulators. and Cks2. Experiments in mice lacking the gene for A principal research focus is the positive regulator Cks1 and Cks2 revealed that each ortholog has a spe- of Cdk2, cyclin E. Cyclin E is often overexpressed cialized function. Cks1 is required as a cofactor for and/or deregulated in human cancers. Using a tissue Skp2-mediated ubiquitylation and turnover of inhibitors culture model, we showed that deregulation of cyclin p21, p27, and p130. Cks2 is required for the transi- E confers genomic instability, probably explaining the tion from metaphase to anaphase in both male and link to carcinogenesis. The observation that deregula- female meiosis I. Nevertheless, mice nullizygous at the tion of cyclin E confers genomic instability has led us individual loci are viable. However, doubly nullizygous to hypothesize a mechanism of cyclin E–mediated car- mice have not been observed because embryos die at cinogenesis based on accelerated loss of heterozygosity the morula stage, a finding consistent with an essen- at tumor suppressor loci. We are testing this hypothe- tial redundant function. We found that this function most sis in transgenic mouse models. We showed that a likely is involved in regulation of transcription and is cyclin E transgene expressed in the mammary epithe- linked to chromatin remodeling, as in yeast. lium markedly increases loss of heterozygosity at the PUBLICATIONS p53 locus, leading to enhanced mammary carcinogen- Jackson, L.P., Reed, S.I., Haase, S.B. Distinct mechanisms control the stability of esis. We are extending these investigations by using the related S-phase cyclins Clb5 and Clb6. Mol. Cell. Biol. 26:2456, 2006. mouse prostate, testis, and skin models. Reed, S.I. Skp’n with Cks1: revelations from the Skp1-Skp2-Cks1-p27 structure. In an attempt to understand cyclin E–mediated Mol. Cell 20:1, 2005. genomic instability, we are investigating how deregula- Reed, S.I. The ubiquitin-proteasome pathway in cell cycle control. Results Probl. Cell Differ. 42:147, 2006. tion of cyclin E affects both S phase and mitosis. Recent data suggest that deregulation of cyclin E impairs DNA Smith, A.P.L., Henze, M., Lee, J.A., Osborn, K.G., Keck, J., Tedesco, D., Bortner, D.M., Rosenberg, M.P., Reed, S.I. Deregulated cyclin E promotes p53 loss of het- replication by interfering with assembly of the prerepli- erozygosity and tumorigenesis in the mouse mammary gland. Oncogene, in press. cation complex. Cyclin E deregulation also impairs the Spruck, C., Sun, D., Fiegl, H., Marth C., Mueller-Holzner, E., Goebel, G., Wid- transition from metaphase to anaphase by promoting schwendter, M., Reed, S.I. Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer. Cancer Res. 66:7355, 2006. the accumulation of inhibitors of anaphase. Our interest in cyclin E deregulation in cancer led van Drogen, F., Sangfelt, O., Malyukova, A., Matskova, L., Yeh, E., Means, A.R., Reed, S.I. Ubiquitylation of cyclin E requires the sequential function of SCF com- us to investigate the pathway for turnover of cyclin E. plexes containing distinct hCdc4 isoforms. Mol. Cell 23:37, 2006. 224 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Wittenberg, C., Reed, S.I. Cell cycle-dependent transcription in yeast: promoters, expressed as an MBF target during late G phase, Nrm1 transcription factors, and transcriptomes. Oncogene 24:2746, 2005. 1 associates with MBF at target promoters and represses Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Improved identifica- expression as cells enter S phase (Fig. 1). Similarly, the tion of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J. Proteome Res. 5:761, 2006. Nrm1 homolog, SpNrm1, in the fission yeast Schizosac-

charomyces pombe regulates its only G1-specific transcription factor, MBF. Transcriptional and Proteolytic Control of Cell Proliferation and Adaptation to Environmental Stimuli

C. Wittenberg, M. Ashe, R. de Bruin, B.-K. Han. M. Guaderrama, T. Kalashnikova Fig. 1. Transcriptional circuitry regulating G1-specific gene expression. G1-specific transcription in S cerevisiae is regulated by 2 het- ellular decision making and coordination of cel- erodimeric transcription factors: SBF and MBF. Both transcription fac-

lular events often involves the differential regu- tors are bound to promoters during G1 phase before commitment to a C lation of the expression of genes. Recently, we new cell cycle. Commitment occurs when their target genes are acti- have focused on the mechanisms through which cells vated by the action of the cyclin-dependent protein kinase Cln3/CDK1. For SBF, Cln3/CDK1 activates transcription by phosphorylation and exert control over gene expression to regulate cell pro- inactivation of the SBF-specific repressor Whi5. Once activated, G1- liferation and the response to changes in environmen- specific transcription leads to the accumulation of many proteins, tal conditions. including those that promote repression of G1-specific transcription. REGULATION OF CELL PROLIFERATION Nrm1 is an MBF-specific corepressor encoded by an MBF target In most cells, commitment to a new round of cell gene. Together, these regulatory proteins can explain the confine- ment of G1-specific transcription to the G1 phase. division during the G1 phase of the cell cycle is accompanied by the activation of a large family of genes that The G1-specific transcriptional machinery is regu- encode activities involved in the duplication and segre- lated by checkpoints that monitor the integrity of cellu- gation of cellular components. G1-specific genes also lar structures and processes. When replication forks are encode regulatory factors that promote subsequent stalled during S phase in the fission yeast, repression cell-cycle events. In the budding yeast Saccharomyces of MBF-regulated transcription is disrupted. We have cerevisiae, G1-specific genes are regulated by 2 tran- shown that that response, which requires the Rad3 scription factors: SBF and MBF. Using mass spectrom- (ATM) and Cds1 (Chk2) checkpoint protein kinases, etry–based multidimensional protein identification leads to the phosphorylation of SpNrm1 and dissocia- technology, we have identified novel regulators of these tion from MBF-regulated promoters. Unexpectedly, transcription factors. according to the literature, derepression of MBF target SBF acts as a transcriptional activator and promotes genes also occurs via regulation of Nrm1 in response expression of its targets specifically during the G1 to activation of the DNA replication checkpoint in bud- interval. We established that promoter-bound SBF ding yeast. Consequently, replication stress appears to associates with the Whi5 repressor during early G1 be associated with genomic instability in the absence phase and that Whi5 is inactivated via phosphorylation of Nrm1. by a G1-specific cyclin-dependent protein kinase, thereby ADAPTATION TO ENVIRONMENTAL STIMULI activating transcription (Fig. 1). This regulation is anal- Adaptation to environmental changes generally ogous to the regulation of E2F by the tumor suppressor involves remodeling of the gene expression program. Rb in metazoans. We have studied the regulation of the HXT genes, which MBF, in conjunction with specific corepressors, acts encode hexose permeases, in response to extracellular primarily as a transcriptional repressor and limits glucose. Those genes are induced by glucose and are transcription of target genes to the G1 phase. We identi- repressed for most other carbon sources. Extracellular fied Nrm1, a novel MBF-associated corepressor. When glucose interacts with the Snf3 and Rgt2 receptors, MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 225 initiating a signaling cascade that culminates with the CHECKPOINTS activation of HXT gene transcription. We have shown The DNA replication and damage checkpoints pre- that signaling leads to the phosphorylation-dependent vent the onset of mitosis when DNA replication is destruction of a transcriptional corepressor, Mth1, by the interrupted or when DNA is damaged. A single double- E3 ubiquitin ligase SCFGrr1. Destruction of Mth1 leads strand break is sufficient to arrest the cell cycle. One to the phosphorylation of the transcriptional repressor aim of our studies is to understand how cells detect Rgt1 and its dissociation from HXT gene promoters. Con- DNA damage and transmit a checkpoint signal that versely, repression of HXT gene expression requires Mth1 arrests the cell cycle. and is associated with Rgt1 dephosphorylation. We Chk1 is the effector kinase of the DNA damage recently identified a type 2A protein phosphatase complex checkpoint. It regulates the activities of Cdc25 and involved in Rgt1 dephosphorylation and are actively pur- Mik1/Wee1 proteins, which modulate the inhibitory suing the protein kinase involved in Rgt1 phosphorylation. phosphorylation of the cyclin-dependent kinase Cdc2. Interestingly, SCFGrr1, the E3 ubiquitin ligase required Chk1 activation by Rad3 requires the adaptor protein for HXT gene induction, is also important for destruc- Crb2. Crb2 is rapidly recruited to double-strand breaks tion of phosphorylated G1 cyclins, critical regulators of in DNA. Rad3 and Tel1 (the ATM homolog in fission cell-cycle initiation. We are investigating the basis for yeast) stimulate Crb2 recruitment by phosphorylating discrimination between targets by SCFGrr1. We found a serine residue near the C terminus of histone H2A in that basic residues in the leucine-rich repeat and parts the vicinity of double-strand breaks. of the carboxy terminus of the F-box protein Grr1 are Our data indicate that tandem C-terminal BRCT important for recognition of phosphorylated substrates. domains in Crb2 associate directly with phosphorylated Our recent identification of additional substrates and histone H2A. Crb2 recruitment to double-strand breaks additional characterization of Grr1 are facilitating also requires the constitutive methylation of lysine at those studies. position 20 in histone H4. This step most likely involves a direct interaction with a Tudor motif in Crb2 that is PUBLICATIONS located to the N-terminal side of the BRCT motifs. We de Bruin, R., Kalashnikova, T.I., Chawan, C., McDonald, W.H., Wohlschlegel, J.A., Yates, J. III, Russell, P., Wittenberg, C. Constraining G1-specific transcription to recently found that these 2 histone modifications late G1 phase: the MBF-associated corepressor Nrm1 acts via negative feedback. Mol. Cell 23:483, 2006. cooperate in a nonredundant mechanism to promote recruitment of Crb2 to double-strand breaks (Fig. 1). Remarkably, neither histone modification is required for Cell-Cycle Checkpoints, recruitment of Crb2 to sustained double-strand breaks that cannot be repaired by homologous recombination. DNA Damage, and Oxidative We recently discovered that the histone modification– independent recruitment of Crb2 to double-strand breaks Stress Responses involves association between phosphorylated threonine- 215 in Crb2 and another checkpoint protein known as P. Russell, C. Chahwan, C. Dovey, L.-L. Du, P.-H. Gaillard, Cut5. In future studies, we will determine whether the V. Martin, B.A. Moser, T.M. Nakamura, mechanisms that regulate Crb2 in fission yeast are M.A. Rodríguez-Gabriel, J. Williams, Y. Yamada conserved for the analogous proteins in human cells. NA damage and oxidative stress elicit cellular DNA REPAIR responses that are highly conserved throughout Bloom, Warner, and Rothmund-Thomson syndromes D eukaryotic evolution. Consequently, studies of in humans are typified by predisposition to cancer or genetically tractable microorganisms such as the fission premature aging. These syndromes, which all result yeast Schizosaccharomyces pombe can provide a use- from defects in DNA helicases, are characterized by ful framework for the design and interpretation of experi- genomic instability arising from inappropriate homolo- ments with more complex multicellular organisms. We gous recombination. To better understand this process, use S pombe to study cell-cycle checkpoints, DNA repair, we used a 2-hybrid screen to identify novel proteins and stress response mechanisms. Defects in these that associate with Srs2 DNA helicase in fission yeast. mechanisms underlie a number of human diseases, We discovered a previously uncharacterized protein including cancer. that promotes the formation of toxic recombination struc- 226 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

current studies are aimed at providing a deeper biochemical and structural understanding of the SWS1- XRCC2-RAD51D complex. OXIDATIVE STRESS RESPONSE Oxidative stress caused by reactive oxygen species can be highly toxic, causing damage to proteins, lipids, and nucleic acids. Oxidative stress elicits a complex gene expression response that is orchestrated in large part by MAP kinase cascades. The fission yeast Spc1 MAP kinase pathway is homologous to the p38 pathway in humans. We recently discovered Csx1, a protein that collaborates with Spc1 to control gene expression in response to oxidative stress. Csx1 is an RNA-binding protein that mediates overall control of gene expression

Fig. 1. Parallel mechanisms of recruiting the DNA damage check- in response to oxidative stress by binding and stabiliz- point protein Crb2 to sites of DNA damage. Top, A proposed mech- ing mRNA that encodes Atf1, a transcription factor anism of how Crb2 associates with double-strand breaks. One mode that is also regulated by Spc1. of association involves interactions with modified histones. The Most recently, we have focused on a newly discov- tandem C-terminal BRCT domains associate with the phosphory- ered family of proteins that interact with Csx1. We have lated C-terminal region of histone H2A. The Tudor domain interacts named these proteins Cip1 and Cip2 (for Csx1-inter- with the constitutive methylation of histone H4 on lysine at position 20. These modes of interaction are not redundant. A third mode acting proteins 1 and 2). Remarkably, elimination of of interaction involves the phosphorylated threonine at position Cip1 or Cip2 results in substantial recovery of the 215 (T215) region of Crb2 and the Cut5. Cut5 is proposed to spe- sensitivity of Csx1 mutant cells to oxidative stress, cifically bind to the single-stranded DNA region near the end of the suggesting that Cip1 and Cip2 are part of a mecha- double-strand break. This binding might involve the association of nism that degrades Atf1 mRNA. Cut5 with other proteins that bind to single-stranded DNA. Bottom,

Fission yeast cells that express Crb2 were tagged with yellow fluo- PUBLICATIONS rescent protein (YFP) and Cut5 tagged with cyan fluorescent pro- Cavero, S., Chahwan, C., Russell, P. Xlf1 is required for DNA repair by nonhomol- tein (CFP). The cells were engineered to express the HO endonucle- ogous end-joining in Schizosaccharomyces pombe. Genetics, in press. ase and to contain a single HO cleavage site. The YFP-Crb2 and Coulon, S., Noguchi, E., Noguchi, C., Du, L.-L., Nakamura, T.M., Russell, P. Cut5-CFP foci indicate large-scale accumulation of these proteins Rad22Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4 at the site of the double-strand break created by HO endonuclease. endonuclease. Mol. Biol. Cell 17:2081, 2006.

de Bruin, R.A.M., Kalashnikova, T.I., Chahwan, C., McDonald, W.H., tures in yeast mutants that lack one or more DNA heli- Wohlschlegel, J., Yates III, J.R., Russell, P., Wittenberg, C. Constraining G1-spe- cases. This protein, which we christened Sws1 because cific transcription to late G1-phase: The MBF-associated corepressor Nrm1 acts via negative feedback. Mol. Cell. 23:483, 2006. it has a SWIM-type zinc finger, is conserved from yeast to humans. In collaborative mass spectrometry and pro- Du, L.-L., Nakamura, T.M., Russell, P. Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand teomics studies with J.R. Yates, Department of Cell breaks. Genes Dev. 20:1583, 2006. Biology, we found that Sws1 forms a complex with 2 Martin, V., Chahwan, C., Gao, H., Blais, V., Wohlschlegel, J., Yates, J.R. III, other proteins known as Rlp1 and Rdl1. Bioinformatic McGowan, C.H., Russell, P. Sws1 is a conserved regulator of homologous recombi- analysis revealed that these 2 proteins are Rad51 par- nation in eukaryotic cells. EMBO J. 25:2564, 2006. alogs (i.e., homologous sequences derived from gene Martín, V., Rodríguez-Gabriel, M.A., McDonald, W.H., Watt, S., Yates, J.R. III, Bähler, J., Russell, P. Cip1 and Cip2 are novel RNA-recognition-motif proteins that duplication) that promote the formation of the Rad51 counteract Csx1 function during oxidative stress. Mol. Biol. Cell 17:1176, 2006. nucleoprotein filament during homologous recombina- Matsumoto, S., Ogino, K., Noguchi, E., Russell, P., Masai, H. Hsk1-Dfp1/Him1, tion. Rlp1 and Rdl1 are equivalent to human XRCC2 the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a com- and RAD51D, proteins implicated in other human dis- ponent of the replication fork protection complex. J. Biol. Chem. 280:42536, 2005. eases characterized by genomic instability. In collabo- Nakamura, T.M., Moser, B.A., Du, L.-L., Russell, P. Cooperative control of Crb2 by ration with C.H. McGowan, Department of Molecular ATM family and Cdc2 kinases is essential for the DNA damage checkpoint in fission yeast. Mol. Cell. Biol. 25:10721, 2005. Biology, we found that using small interfering RNA to silence the gene for human SWS1 reduced the occur- Rodríguez-Gabriel, M.A., Russell, P. Distinct signaling pathways respond to arsen- ite and reactive oxygen species in Schizosaccharomyces pombe. Eukaryot. Cell rence of homologous recombination structures. Our 4:1396, 2005. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 227

Rodriguez-Gabriel, M.A., Watt, S., Bähler, J., Russell, P. Upf1, an RNA helicase conserved DNA repair protein that has homology to the required for nonsense-mediated mRNA decay, modulates the transcriptional response to oxidative stress in fission yeast. Mol. Cell. Biol. 26:6347, 2006. xeroderma pigmentosum F family of endonucleases. Xeroderma pigmentosum is a cancer-prone disorder that results from a failure to appropriately repair dam- DNA Damage Responses in aged DNA. Biochemical analysis shows that Mus81-Eme1 has Human Cells associated endonuclease activity against structure-specific DNA substrates, including Holliday junctions. Enzy- C.H. McGowan, V. Blais, M. Duquette, E. Langley, matic analysis, immunofluorescence studies, and the A. MacLaren, J. Scorah, D. Slavin, E. Taylor use of RNA interference have all contributed to the conclusion that Mus81-Eme1 is required for recombination omplex multicellular organisms, such as humans, repair in human cells. We are also using gene targeting have large numbers of mitotically competent cells to study the function of the Mus81-Eme1 endonuclease C that are capable of renewal, repair, and, to some in mice. Inactivation of Mus81 in mice increases geno- extent, regeneration. The advantages of being able to mic instability and sensitivity to DNA damage but does replace damaged or aged cells are off set by the inher- not promote tumorigenesis. In addition, we showed that ent susceptibility of mitotic cells to acquiring mutations Mus81-Eme1 is specifically required for survival after and becoming cancerous. DNA is inherently vulnerable exposure to cisplatin, mitomycin C, and other commonly to many sorts of chemical and physical modification; used anticancer drugs. As a point of interaction between thus, as cells duplicate and divide, they can acquire checkpoint control and DNA repair, the relationship mutations. Both spontaneous and induced DNA dam- between Mus8-Eme1 and Chk2 most likely provides age must be repaired with minimal changes if growth, information critical to understanding the response to renewal, and repair are to be successful. Our overall DNA damage as a whole. objective is to understand how mammalian cells protect Anticancer therapy is largely based on the use of themselves from DNA damage and thus from develop- genotoxic agents that damage DNA and thus kill dividing cancer. ing cells. Coordination of cell-cycle checkpoints and Eukaryotic cells have evolved with a complex net- DNA repair is especially important when unusually high work of DNA repair processes and cell-cycle checkpoint amounts of DNA damage occur after radiation or geno- responses to ensure that damaged DNA is repaired toxic chemotherapy. Hence, a detailed understanding of before it is replicated and becomes fixed in the genome. cellular responses to DNA damage is essential in under- These pathways are highly conserved throughout evo- standing both the development and the treatment of lution, and much information about human responses disease in humans. to DNA damage has been gained from studies of sim- PUBLICATIONS ple, genetically tractable organisms such as yeast. We Martin, V., Chawan, C., Gao, H., Blais, V., Wohlschlegel, J., Yates, J.R. III, use a combination of molecular, cellular, and genetic McGowan, C.H., Russell, P. Sws1 is a conserved regulator of homologous recombination in eukaryotic cells. EMBO J. 25:2564, 2006. techniques to determine how these pathways operate in human cells. Checkpoints control the order and timing of events in the cell cycle; they ensure that biochemically inde- DNA Repair and the pendent processes are coupled so that a delay in a Maintenance of Genomic critical cell-cycle process will cause a delay in all other aspects of progression of the cycle. In addition, check- Stability points also coordinate repair with delays in progression of the cell cycle and promote the use of the most appro- M.N. Boddy, S. Pebernard, J. Prudden priate repair pathway. We used genetic models to identify NA repair pathways have evolved to protect 2 checkpoint kinases in humans that limit progression the genome from ever-present genotoxic agents. of the cell cycle when DNA is damaged. One of these D Highlighting the importance of the pathways, kinases, Chk2, is activated in response to DNA dam- defects in DNA repair mechanisms strongly predispose age. Chk2 physically interacts with Mus81-Eme1, a the host to cancer and to neurologic and developmental 228 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE disorders. The DNA repair systems we study in fission cation of target proteins with ubiquitin and the small yeast are evolutionarily conserved, and therefore our ubiquitin-like protein SUMO. Such protein modifications studies provide a valuable framework for understand- play roles in DNA repair and chromatin remodeling. ing genome maintenance in human cells. We have carried out detailed genetic and biochem- Although many DNA repair mechanisms have been ical analyses of the Nse5-Nse6 heterodimer. Nse5 and described, information on how they are coordinated with Nse6 are not essential for growth; however, cells lack- necessary changes in chromatin structure is limited. We ing either protein have high levels of spontaneous genome are studying the essential structural maintenance of damage and are hypersensitive to ultraviolet light and chromosomes (SMC) complex Smc5-Smc6. The molecular other genotoxic agents. An important discovery is that functions of Smc5-Smc6 are unknown, but the complex Nse5-Nse6 prevents the deleterious engagement of an is related to the SMC complexes that hold replicated sis- ordinarily beneficial DNA repair pathway called homol- ter chromatids together (cohesin) and condense chro- ogous recombination. Our studies indicate that Nse5- matin before its segregation at mitosis (condensin). Nse6, and by extension the Smc5-Smc6 complex, acts In collaboration with J.R. Yates, Department of Cell either to prevent initiation of homologous recombination Biology, we purified the Smc5-Smc6 complex and deter- or to separate physically linked chromosomes that arise mined the identity of the core components. The holo- late in this process (Fig. 1B). Abrogating homologous complex consists of the Smc5-Smc6 heterodimer and 6 recombination by deleting a pivotal factor required for additional non-SMC elements, Nse1–Nse6. We expressed the process (called Rad51) reduces the sensitivity of and purified individual components of the complex and Nse5-Nse6 mutant cells to DNA damage. The sponta- determined the architecture of the holocomplex (Fig. 1). neous DNA damage observed in Nse5-Nse6 mutant cells is due to the attempted separation of chromosomes into daughter cells while the chromosomes are still physically linked. Such defective chromosome separation in humans could result in cancer and other diseases.

PUBLICATIONS Pebernard, S., Wohlschlegel, J., McDonald, W.H., Yates, J.R. III, Boddy, M.N. The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex [published correction appears in Mol. Cell. Biol. 26:3336, 2006]. Mol. Cell. Biol. 26:1617, 2006.

Raffa, G.D., Wohlschlegel, J., Yates, J.R. III, Boddy, M.N. SUMO-binding motifs mediate the Rad60-dependent response to replicative stress and self-association. J. Biol. Chem. 281:27973, 2006.

Fig. 1. Architecture and function of the Smc5-Smc6 holocomplex. A, Nse1, Nse3, and Nse4 form a stable heterotrimer that associates with Smc5. Nse2 interacts directly with Smc5 in the absence of the Delineating Oncogenic and other Nse proteins. Smc6 interacts directly with Smc5 but with none of the other components. Nse5 and Nse6 form a stable heterodimer Tumor-Suppressing Signal that also binds directly to Smc5. Double-headed arrows indicate interactions between subcomplexes. Nse5-Nse6 may recruit the holo- Transduction Pathways complex to stalled replication forks and certain DNA damage sites. B, Nse5-Nse6 might act to prevent the initiation of homologous P. Sun, Q. Deng, C. Kannemeier, R. Liao, A. Seit-Nebi, recombination catalyzed by a number of factors, including Rad51. N. Yoshizuka Nse5-Nse6 could be involved in the separation or “resolution” of physi- evelopment of cancer is a result of multiple onco- cally linked chromosomes that can result from homologous recombination. Evidence suggests that Nse5-Nse6 and Smc5-Smc6 perform genic genetic alterations, including activation such functions at replication forks and DNA double-strand breaks. D of oncogenes and inactivation of tumor suppressors. Despite the essential roles of these mutations in Nse1–Nse4 are essential for growth, and hypomorphic tumor formation, normal cells usually respond to these mutants of these proteins cause cellular sensitivity to oncogenic changes by initiating tumor-suppressing genotoxic agents such as ultraviolet light and x-rays. defense mechanisms such as premature senescence and Notably, Nse1 and Nse2 contain certain zinc finger apoptosis. Our main interests are delineating the signal domains that implicate these 2 elements in the modifi- transduction pathways that mediate these tumor-sup- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 229 pressing responses and determining how oncogenes allow inactivation of the tumor suppressor protein p53. How- a cell to evade the regulation by these cellular defense ever, we found that MDM2 confers resistance to a mechanisms to cause cancer. Currently, we are focusing growth-inhibitory cytokine, transforming growth factor β, on 2 well-known oncogenes: ras and mdm2. through a p53-independent mechanism. We are delin- The oncogene ras encodes a family of small GTP- eating this p53-independent activity of MDM2, which binding proteins that transduce mitogenic signals from may play an important role in tumorigenesis. We have extracellular growth factors. Constitutive activation of ras identified several MDM2 domains and activities that is common in tumors and contributes to tumor develop- are essential for the ability of MDM2 to mediate resis- ment. In normal cells, however, the initial response to tance to the growth factor. ras activation is a stable growth arrest called premature In other research, we are systematically searching senescence. As a result, the senescence response trig- for genetic alterations that contribute to specific tumor- gered by ras must be evaded before transformation can associated phenotypes, such as drug resistance, cellu- occur. We showed that ras induces senescence through lar immortalization, and metastasis. For these studies, sequential activation of 2 MAP kinase pathways. Initially, we are using cDNA expression libraries or libraries of ras activates the MAP kinase kinase (MEK)–extracellu- short interfering RNAs. lar signal–regulated kinase (ERK) pathway. Sustained activation of MEK-ERK turns on the stress-induced p38 PUBLICATIONS Lin, S., Xiao, R., Sun, P., Xu, X., Fu, X.D. Dephosphorylation-dependent sorting of pathway, which subsequently causes senescence. SR splicing factors during mRNP maturation. Mol. Cell 20:413, 2005. These studies have revealed a novel, tumor-suppressing function of p38, in addition to its known roles in inflammation and stress responses. In other studies, Hypocretins in Arousal, Feeding we identified additional signaling components, either upstream or downstream of p38, that mediate prema- Behavior, and Motivation ture senescence. We found that 1 of the 4 isoforms of p38 contributes to ras-induced senescence by activat- J.G. Sutcliffe, L. de Lecea ing the p53 tumor suppressor protein. In addition, a he 2 C terminally amidated hypocretin neuropep- serine/threonine protein kinase, which is a direct sub- tides (also called orexins) are produced from a strate of p38, also plays an essential role in ras-induced precursor whose expression in rats is restricted senescence. Inactivation of this protein kinase disrupts T to a few thousand neurons of the lateral hypothalamus. ras-induced senescence and promotes tumorigenesis These neurons are active during wakefulness but are both in vitro and in vivo. Our results have confirmed the tumor-suppressing function of the p38 pathway. quiescent during various phases of sleep. Two G pro- To determine how premature senescence is bypassed tein–coupled hypocretin receptors have different distri- in tumors, we dissected the functions of an adenovirus- butions within the CNS. encoded oncoprotein, E1A, that can rescue ras-induced The hypocretins are found in secretory vesicles at senescence. Our results indicated that bypassing of synapses of fibers that project to areas within the pos- senescence requires binding of the cellular proteins Rb terior part of the hypothalamus that are implicated in and p300/CBP by E1A. Although interference with the feeding behaviors and hormone secretion. Hypocretin p16INK4A/Rb pathway or p300/CBP functions alone did fibers also project to diverse targets in other brain regions not result in bypassing of senescence, these 2 types of and the spinal cord, including several areas implicated genetic alterations cooperated to rescue cells from ras- in cardiovascular function and sleep-wake regulation. induced senescence and lead to cellular transformation. The peptides are excitatory when applied directly in These results indicate that p300 and CBP are integral vivo. Most humans with narcolepsy have greatly reduced components of the senescence pathway. Both p300 and levels of hypocretin peptides in their cerebral spinal fluid CBP have tumor-suppressing functions. The critical role and no or barely detectable hypocretin neurons in their of p300 and CBP in the senescence response has pro- hypothalami, findings suggestive of autoimmune attack. vided a mechanistic basis for the tumor-suppressing func- Hypocretin peptides excite noradrenergic neurons tion of these proteins. in the locus coeruleus and serotonergic neurons in the Another focus of our research is mdm2, an onco- dorsal raphe to elevate muscle tone and histaminergic gene that can mediate transformation primarily through tuberomammillary neurons to promote wakefulness. 230 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

These components of the ascending reticular activat- nia of short, intermediate, and long duration and from ing system, and the hypocretin neurons themselves, the prefrontal cortex of matched control subjects with- project to and stimulate thalamic and basal forebrain out schizophrenia. Among many genes and pathways neurons, and all of these groups contribute to the depo- revealed as significantly altered in schizophrenia, we larization of the cerebral cortex. Arousal-related signal- are focusing on those related to glycosphingolipid metab- ing occurs through both hypocretin receptors. olism and myelination. A total of 40 genes with altered These peptides have diverse effects on brain reward expression in patients with schizophrenia were related and autonomic systems related to stress that increase to these systems. motivated behaviors, including feeding. The relation to To assess the effects of treatment with antipsy- feeding is complex. Acute administration of hypocretin chotic drugs on a subset of genes that encode struc- peptides to sleeping rats increases food consumption. tural components of myelin, we treated groups of mice However, patients and animals with impaired hypocre- with haloperidol, a widely prescribed “typical” antipsy- tin signaling have an increased likelihood of being obese chotic drug. Chronic haloperidol treatment caused sig- despite reduced daily calorie intake. nificant decreases in the expression levels of at least 8 myelin-related genes in several white matter regions of PUBLICATIONS de Lecea L., Sutcliffe, J.G. The hypocretins and sleep. FEBS J. 272:5675, 2005. mouse brain as revealed by in situ hybridization analysis. In other studies, we are investigating the molecu- Desplats, P.A., Kass, K.E., Gilmartin, T., Stanwood, G.D., Woodward, E.L., Head, S.R., Sutcliffe, J.G., Thomas, E.A. Selective deficits in the expression of striatal- lar basis for heterogeneity in schizophrenia by identi- enriched mRNAs in Huntington’s disease. J. Neurochem. 96:743, 2006. fying genes that have similar expression profiles in

Hedlund, P.B., Huitrón-Reséndiz, S., Henriksen, S.J., Sutcliffe, J.G. 5-HT7 recep- subsets of patients with the disorder. Using weighted gene tor inhibition and inactivation induce antidepressantlike behavior and sleep pattern. coexpression network analyses, we identified distinct Biol. Psychiatry 58:831, 2005. subtypes in our schizophrenia cohort, most notably, Hedlund, P.B., Sutcliffe, J.G. 5-HT7 receptors as favorable pharmacological targets dramatic differences in the expression profiles between for drug discovery. In: The Serotonin Receptors: From Molecular Pharmacology to Human Therapeutics. Roth, B.L. (Ed.). Humana Press, Totowa, NJ, 2006, p. 517. patients with short versus long duration of illness. We

Hilbush, B.S., Morrison, J.H., Young, W.G., Sutcliffe, J.G., Bloom, F.E. New are exploring subtype-specific pathways associated with prospects and strategies for drug target discovery in neurodegenerative disorders. these subgroups. NeuroRx 2:627, 2005. TRANSCRIPTIONAL DYSREGULATION IN Sutcliffe, J.G. de Lecea, L. The hypocretin/orexin system. In: Handbook of Con- HUNTINGTON’S DISEASE: STRIAT AL SPECIFICITY temporary Neuropharmacology. Sibley, D.R., et al. (Eds.). Wiley-InterScience, Hoboken, NJ, in press. Much evidence supports a role for transcriptional dysregulation in Huntington’s disease. Of particular Sutcliffe, J.G., de Lecea, L. Hypocretins/orexins in brain function. In: Handbook of Neurochemistry and Molecular Neurobiology: Neuroactive Proteins and Peptides, 3rd interest is how these disturbances may be specifically ed. Lim, R. (Volume Ed.), Lajtha, A. (Series Ed.). Springer, New York, 2006, p. 499. manifested in the striatum, the primary region of neurodegeneration in Huntington’s disease. Using microarray analysis and a transgenic mouse of Huntington’s Molecular Neurobiology of disease, we identified a cluster of striatal-enriched genes that was downregulated in the mice. The cluster included CNS Disorders the genes FoxP1, Bcl11b, and DRRF, which encode zinc finger–containing transcription factors, and RARB E.A. Thomas, J.G. Sutcliffe, P.A. Desplats, S. Narayan, and RXRG, which encode nuclear receptors. Real-time K.E. Kass, T. Gilmartin, L. Schaffer, S.R. Head polymerase chain reaction validated 57% and 40% GENE PROFILING IN SCHIZOPHRENIA reductions in levels of Bcl11b and FoxP1 mRNA, respec- chizophrenia is a life-long, heterogeneous men- tively, in the striatum of symptomatic transgenic mice tal illness with variable expression and unknown and a 73% decrease in the expression of FoxP1 in S etiology. We are interested in the molecular fac- human caudate from patients with Huntington’s disease. tors that influence the course of illness in schizophre- Transcripts for both of these factors are expressed in nia and how treatment modifies these factors. Using medium spiny projection neurons, which selectively oligonucleotide microarrays, we generated gene expres- degenerate in Huntington’s disease. Further colocaliza- sion profiles from tissue samples obtained at autopsy tion and coimmunoprecipitation studies have suggested from the prefrontal cortex of patients with schizophre- that Bcl11b and FoxP1 interact with polyglutamine- MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 231 expanded N-terminal huntingtin. Sequestration of these at the 5-HT7 receptor. Thus, both blockade and inacti- factors into nuclear aggregates in Huntington’s disease vation of the 5-HT7 receptor yield the same result. resulting in loss of function may contribute to specific Sleep disturbances are common in depression. dysregulation of striatal gene expression. This mecha- Increased amounts of REM sleep are a frequent find- nism may explain, in part, the specificity of the patho- ing. Compared with mice that have the 5-HT7 recep- logic changes associated with Huntington’s disease. tor mice lacking the receptor spend less time in REM sleep without alteration of other sleep parameters, fur- PUBLICATIONS Desplats, P.A., Kass, K.E., Gilmartin, T., Stanwood, G.D., Woodward, E.L., Head, ther establishing the antidepressant-like profile of the S.R., Sutcliffe, J.G. Thomas. E.A. Selective deficits in the expression of striatal- animals that lack the receptor. enriched mRNAs in Huntington’s disease. J. Neurochem. 96:743, 2006. Taken together our results suggest an important role Thomas, E.A. Apolipoprotein D and arachidonic acid interactions in the treatment for the 5-HT7 receptor in depression, and antagonists and pathology of schizophrenia. In: Fatty Acids and Oxidative Stress in Neuropsy- chiatric Disorders. Yao, J.K. (Ed.). Nova Science Publishers, Inc., Hauppauge, NY, to this receptor should be evaluated as a treatment 2006. for depression.

Narayan, S., Kass, K.E., Thomas, E.A. Chronic haloperidol treatment results in a OBSESSIVE-COMPULSIVE DISORDER decrease in the expression of myelin/oligodendrocyte-related genes in the mouse Obsessive-compulsive disorder is related to depres- brain. J. Neurosci. Res., in press. sion, at least to the extent that antidepressants are Thomas, E.A., Yao, J.K. Clozapine specifically alters the arachidonic acid pathway commonly used to treat both disorders. In an animal in mice lacking apolipoprotein D. Schizophr. Res., in press. model of obsessive-compulsive disorder (marble burying), Thomas, E.A. Molecular profiling of antipsychotic drug function: convergent mechanisms. In: The Pathology and Treatment of Psychiatric Disorders. Molecular Neu- we showed that blockade or inactivation of the 5-HT7 robiology, in press. receptor results in less compulsive behavior. Thus, the

Thomas, E.A. Striatal specificity of gene expression dysregulation in Huntington's 5-HT7 receptor might be of interest as a putative tar- disease. J. Neurosci. Res. 84:1151, 2006. get for treatment of obsessive-compulsive disorder.

SCHIZOPHRENIA Prepulse inhibition (PPI) of the acoustic startle The 5-HT7 Receptor in reflex is a well-characterized animal model of schizophrenia. The model is especially relevant because Neuropsychiatric Disorders similar responses can be observed in patients with schizophrenia. We showed that PPI per se is not altered P.B. Hedlund, P.E. Danielson, S. Huitrón-Reséndiz, in mice lacking the 5-HT receptor, but that when PPI S.J. Henriksen, S. Semenova, M.A. Geyer, A. Markou, 7 is disrupted by phencyclidine, the mice are significantly J.G. Sutcliffe less affected than are mice that have the receptor.

nterest in the serotonin 5-HT7 receptor as a putative Phencyclidine-induced disruption involves a glutamatergic target in neuropsychiatric disorders has been grow- component of PPI that is relevant for the action of atypi- I ing continually. The interest was prompted by the cal antipsychotics such as clozapine. Clozapine is a finding that several classes of drugs used to treat dis- drug with relatively high affinity for the 5-HT7 receptor. orders such as depression and schizophrenia have high PUBLICATIONS affinity for the 5-HT7 receptor. We have established Hedlund, P.B., Huitrón-Reséndiz, S., Henriksen, S.J., Sutcliffe, J.G. 5-HT7 recep- evidence that supports a role for this receptor in depres- tor inhibition and inactivation induce antidepressantlike behavior and sleep pattern. sion, obsessive-compulsive disorder, and schizophrenia. Biol. Psychiatry 58:831, 2005.

DEPRESSION Hedlund, P.B., Sutcliffe, J.G. 5-HT7 receptors as favorable pharmacological targets for drug discovery. In: The Serotonin Receptors: From Molecular Pharmacology to The forced swim test and the tail suspension test Human Therapeutics. Roth, B.L. (Ed.). Humana Press, Totowa, NJ, 2006, p. 517. are animal models of behavioral despair that have high Hedlund, P.B., von Euler, G. Z-analysis: a new approach to analyze stimulation value for predicting the antidepressant efficacy of drugs. curves with intrinsic basal stimulation. Biochem. Pharmacol. 70:170, 2005. The tests can also be used to characterize animals in which genes have been deleted. Using both of these tests, we showed that mice lacking the 5-HT7 receptor have a behavioral profile similar to that of mice treated with antidepressants. We replicated these findings by using a compound that acts as a selective antagonist 232 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE Lysophospholipid Signaling and Neural Aneuploidy

J. Chun, S. Appadurai, B. Almeida, B. Anliker, E. Birgbauer, A. Dubin, S. Gardell, D. Herr, G. Kennedy, M. Kingsbury, C.W. Lee, M. Lu, M. McCreight, C. Paczkowski, S. Peterson, S. Rehen, R. Rivera, A.H. Yang, X.Q. Ye, Y. Yung, L. Zhu

n the past year, we gained significant new insights into both lysophospholipid signaling and neural ane- I uploidy. First, we discovered that receptor-mediated lysophosphatidic acid (LPA) signaling, mediated by the cognate receptor known as LPA3, is essential for normal implantation of embryos in the uterine wall, a find- Fig. 1. Location of implantation sites in uteri at embryonic days ing that may be relevant to the treatment of female 4.5 (E4.5) and 5.5 (E5.5). Bands indicate implantation sites. Mice infertility. Second, we acquired new data that indicate lacking the gene for LPA3 have delayed implantation and at later the potential function of genomically nonidentical brain times have reduced and abnormally spaced implantation (arrows). cells in normal brain in humans. In further studies in mice, we found that aneuploid neurons can be integrated into the normal circuitry of the brain, indicating that the neurons are not simply dead or inert components but rather have the potential to modify properties of neural circuitry by virtue of their altered genomes. LYSOPHOSPHOLIPIDS Lysophospholipids such as LPA are simple phospholipids that act as extracellular signals that use cognate G protein–coupled receptors to bring about myriad effects. The 2 best studied lysophospholipids are LPA and sphingosine 1-phosphate (S1P). We continue to generate new lines of mice that lack the genes for single and multiple receptors and to characterize the mutant phenotypes. A null mutation in LPA3 resulted in a reduced-fertility phenotype that was attributed to alterations in embryo implantation (Fig. 1). We are elucidating the downstream Fig. 2. Nuclei isolated from the brains of different patients containing 1 (E), 2 (F), 3 (G), or 4 (H) copies of chromosome 21. The signaling effects of LPA3 in normal implantation. NORMAL NEURAL ANEUPLOIDY large, dark region indicates staining with DAPI (4′,6-diamidino-2- It is now clear that many cells in the brain have phenylindole), whole-chromosome paint appears in light gray, and the chromosome 21 point probes are indicated by arrows. A com- nonidentical genomes by virtue of being aneuploid, that plete overlap between the paint and the point probe occurs, as is, the cells have gained and/or lost chromosomes. The seen at higher magnification in the insets. Arrowheads indicate the initial research on aneuploidy was done in mice, raising numbers of chromosome 21 per cell. Scale bar, 5 µm. the question of whether this phenomenon also existed in In mice, we found that indeed, aneuploid neurons humans. Use of double labeling with point probes, which can have distant connections and physiologic activities, recognize a relatively discrete part of a chromosome, suggesting that these genomically distinct cells can and “paints,” which recognize much of a given chromosome, allowed the unambiguous identification of aneu- function in normal neural circuitry. Currently, we are ploid neurons and glia in normal human brain (Fig. 2). determining the extent, forms, and roles of aneuploid This finding led us to ask the additional question of neural cells in normal and diseased mammalian brains. whether such cells were capable of normal function. MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 233

PUBLICATIONS functions, including B cells, eosinophils, macrophages, Barbeito, L., Chun, J., Binder, L.I., Neto, V.M., Perry, G., Scazzochio, C., Violini, G. The end of a Chilean institute. Science 308:792, 2005. dendritic cells, and natural killer cells. Siglecs are a subfamily of the immunoglobulin superfamily that have Chun, J. Lysophospholipids in the nervous system. Prostaglandins Other Lipid Mediat. 77:46, 2005. in common a unique N-terminal Ig domain that confers

Gon, Y., Wood, M.R., Kiosses, W.B., Jo, E., Sanna, M.G., Chun, J., Rosen, H. the ability to bind to sialic acid–containing carbohydrate S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung groups (sialosides) of glycoproteins and glycolipids. barrier integrity and is potentiated by TNF. Proc. Natl. Acad. Sci. U. S. A. 102:9270, 2005. The cytoplasmic domains of most siglecs contain tyrosine-based inhibitory motifs characteristic of acces- Goparaju, S.K., Jolly, P.S., Watterson, K.R., Bektas, M., Alvarez, S., Sarkar, S., Mel, L., Ishii, I., Chun, J., Milstien, S., Spiegel, S. The S1P2 receptor negatively sory proteins that regulate transmembrane signaling regulates platelet-derived growth factor-induced motility and proliferation. Mol. Cell. Biol. 25:4237, 2005. and endocytosis of cell-surface receptor proteins. The diverse specificity for their sialoside ligands and their Kingsbury, M.A., Friedman, B., McConnell, M.J., Rehen, S.K., Yang, A.H., Kaushal, D., Chun, J. Aneuploid neurons are functionally active and integrated into variable cytoplasmic regulatory elements provide siglecs brain circuitry. Proc. Natl. Acad. Sci. U. S. A. 102:6143, 2005. with attributes for unique roles in the cell-surface biol- Li, H., Ye, X., Mahanivong, C., Bian, D., Chun, J., Huang, S. Signaling mecha- ogy of each cell that expresses them. nisms responsible for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells. J. Biol. Chem. 280:10564, 2005. The best understood siglec is CD22 (siglec-2), an accessory molecule of the B-cell receptor (BCR) complex Rehen, S.K., Yung, Y.C., McCreight, M.P., Kaushal, D., Yang, A.H., Almeida, B.S.V., Kingsbury, M.A., Cabral, K.M.S., McConnell, M.J., Anliker, B., Fontanoz, that has both positive and negative effects on receptor M., Chun, J. Constitutional aneuploidy in the normal human brain. J. Neurosci. signaling. The carbohydrate ligand recognized by CD22 is 25:2176, 2005. the sequence Siaα2-6Galbβ1-4GlcNAc found on glyco- Simon, M.F., Daviaud, D., Pradere, J.P., Grès, S., Guigné, C., Wabitsch, M., Chun, J., Valet, P., Saulnier-Blache, J.S. Lysophosphatidic acid inhibits adipocyte proteins of both B cells (cis ligands) and on cells that differentiation via lysophosphatidic acid 1 receptor-dependent down-regulation of interact with B cells (e.g., T cells, trans ligands). Interac- peroxisome proliferator-activated receptor γ2 J. Biol. Chem. 280:1456, 2005. tions of CD22 with cis or trans ligands regulate aspects Tölle, M., Levkau, B., Keul, P., Brinkmann, V., Giebing, G., Schönfelder, G., of B-cell activation, proliferation, and development. Schäfers, M., von Wnuck Lipinski, K., Jankowski, J., Jankowski, V., Chun, J., Zidek, W., Van der Giet, M. Immunomodulator FTY720 induces eNOS-dependent arterial We found that CD22 is predominately associated vasodilation via the lysophospholipid receptor S1P3. Circ. Res. 96:913, 2005. with clathrin-coated pits in resting B cells, whereas BCRs Ye, X., Hama, K., Contos, J.J., Anliker, B., Inoue, A., Skinner, M.K., Suzuki, H., are minimally associated with clathrin domains. Mice Amano, T., Kennedy, G., Arai, H., Aoki, J., Chun, J. LPA3 lysophosphatidic acid signalling in embryo implantation and spacing. Nature 435:104, 2005. deficient in the ligand for CD22 have greater colocalization of CD22 and the BCR in fused raft-clathrin domains than do mice that have the ligand, accounting for the Chemical Glycobiology immunosuppression in deficient mice. In wild-type mice, after antigen activation, the BCR is endocytosed via J.C. Paulson, O. Blixt, L.K. Allin, H. Andersson-Sand, raft-clathrin domains, a logical site for the dampening O.V. Bohorov, B.E. Collins, S. Han, J. Hoffman, D. Lebus, of B-cell signaling by CD22. In resting cells, CD22 L. Liao, X. Liu, B. Ma, M. O’Reilly, N. Razi, P. Sobieszczuk, undergoes constitutive endocytosis, which can result in L. Stewart, H. Tateno, H. Tian, D. Vasiliu, Y. Zeng internalization of high-affinity ligands of CD22 (Fig. 1). e investigate the roles of glycan-binding pro- We also study siglec-F (murine) and siglec-8 teins that mediate cellular processes central (human), which are predominately expressed on eosino- = W to immunoregulation and human disease. We phils and recognize the sialoside Siaα2-3(6-SO4 )Galβ1- work at the interface of biology and chemistry to under- 4GlcNAc and are targets for modulating eosinophil stand how the interaction of glycan-binding proteins activation. Another siglec being actively investigated is with their ligands mediates cell-cell interactions, endocy- myelin-associated glycoprotein (siglec-4). This siglec is tosis, and cell signaling. Our multidisciplinary approach expressed on glial cells and recognizes the sialoside is complemented by a diverse group of chemists, bio- Siaα2-3Galβ1-3(Siaα2-6)GalNAc-R found on O-linked chemists, cell biologists, and molecular biologists. glycans of glycoproteins and glycolipids. Functionally, BIOLOGICAL ROLES OF SIGLECS myelin-associated glycoprotein stabilizes interactions The siglecs are a family of 11 sialic acid–binding between glial cells and axons essential for normal orga- proteins that function as cell-signaling coreceptors. nization of myelin and inhibits axonal regeneration, They are expressed on glial cells and on a variety of which is currently a target for pharmaceutical inter- leukocytes that mediate acquired and innate immune vention to promote nerve regeneration. 234 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Fig. 1. Relationship between microdomain localization of the BCR and CD22, a regulator of BCR signaling that binds glycan ligands.

A major barrier to studying the ligand-binding properties of siglecs and their role in siglec biology is the difficulty in creating synthetic probes that compete with endogenous (cis) ligands. Even highly multivalent poly- Fig. 2. Sialoside analog glycan microarray reveals high-affinity mers containing the natural glycan sequence recognized ligands for CD22. A, Sialoside ligands of CD22 with amino-termi- by a siglec will not bind to cells unless cis ligands are nated linkers are printed on N-hydroxyl succinimide (NHS)–activat- first destroyed. However, we found that high-affinity ed glass slides, resulting in a covalent amide bond. B, The natural analogs of the natural sialoside ligand of CD22 bind to ligand (3) with various substituents (1, 2, 4, 6) and a nonligand native B cells and are carried into the cell by receptor- control (5) are printed in 10 replicates at 10 two-fold diluted printing concentrations. Overlay with a fluorescence-labeled CD22-Ig mediated endocytosis. Similar constructs with the ligand chimera reveals the increased binding to various substituents com- of siglec-F are also bound and endocytosed by eosino- pared with the natural ligand. phils, but by a different endocytic mechanism. We have also developed potent inhibitors of myelin-associated corresponding siglec by using our flexible chemoenzy- glycoprotein that reverse its ability to block axon growth, matic synthesis strategies. and in collaborative studies with R. Schnaar, Johns BIOENGINEERING OF CELL-SURFACE SIALOSIDES Hopkins University, Baltimore, Maryland, we are investi- Sialic acids with substituents at the C-9 and C-5 gating the potential of the inhibitors to promote nerve positions are readily taken up by cells and incorporated growth in vivo. into cell-surface glycans of glycoproteins and glycolipids With these successes, we have embarked on a major by the natural glycosylation pathways. Taking advantage effort to identify high-affinity analogs of each siglec to of this concept, we developed a novel method for in produce ligand-based tools to investigate the biological situ photoaffinity cross-linking of CD22 to its ligands on roles of the siglecs in innate and adaptive immunity. the same cell (cis) or adjacent cell (trans) by using a SIALOSIDE ANALOG GLYCAN ARRAYS 9-aryl-azide-sialic acid. When exposed to ultraviolet We have developed a robotically printed glycan array light, CD22 is rapidly cross-linked to its cis ligands that displays sialoside analogs to assess the affinity of through protein-glycan covalent bonds (Fig. 3). The siglecs for unnatural substituents at the C-9 and C-5 striking finding is that in addition to glycan structure, positions of sialic acids. Even in the initial experiments microdomain localization of CD22 strongly influences with 65 acyl substituents at the C-9 position of sialic the glycoprotein ligands that CD22 interacts with. In acid, the method was a powerful one for identifying fact, the predominant cis ligands of CD22 were gly- substituents that increase the affinity of siglecs by cans of neighboring CD22 molecules, showing homo- 100-fold or more (Fig. 2). In collaboration with K.B. multimeric complexes of CD22 mediated by CD22’s Sharpless, Department of Chemistry, we have created ligand-binding domain. another 80 analogs by using by click chemistry to cou- Another application is to incorporate sialic acid ple members of a library of alkynes to sialosides con- analogs that increase or decrease the affinity of a siglec taining 9-azido-N-acetyl-neuraminic acid. Results from for its natural ligand to perturb the dynamics of inter- the array can be rapidly assimilated into the synthesis actions of the siglec with its cis ligand. For example, a of high-affinity ligands and ligand-based probes of the 9-biphenylcarboxyl substituent (Fig. 3) increases the MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 235

influenza and related H1 avian influenza viruses and the more recent avian influenza virus (H5N1) to identify mutations required to switch specificity from avian receptors to human-type receptors.

PUBLICATIONS Bochner, B.S., Alvarez, R.A., Mehta, P., Bovin, N.V., Blixt, O., White, J.R., Schnaar, R.L. Glycan array screening reveals a candidate ligand for siglec-8. J. Biol. Chem. 280:4307, 2005.

Collins, B.E., Blixt, O., Han, S., Duong, B., Li, H., Nathan, J.K., Bovin, N., Paul- son, J.C. High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J. Immunol. 177:2994, 2006.

Fig. 3. Bioengineering of cell-surface glycoproteins to carry sub- Collins, B.E., Smith, B.A., Bengtson, P., Paulson, J.C. Ablation of CD22 in ligand- stituents at the 9-position of N-acetyl–neuraminic acid that deficient mice restores B cell receptor signaling. Nat. Immunol. 7:199, 2006. increase affinity (biphenylcarboxyl) or allow in situ photoaffinity Comelli, E.M., Head, S.R., Gilmartin, T., Whisenant, T., Haslam, S.M., North, cross-linking (9-aryl-azide) of CD22 to its ligands. S.J., Wong, N.K., Kudo, T., Narimatsu, H., Esko, J.D., Drickamer, K., Dell, A., Paulson, J.C. A focused microarray approach to functional glycomics: transcrip- affinity for CD22 by 100-fold, resulting in the strong tional regulation of the glycome. Glycobiology 16:117, 2006.

CD22-mediated aggregation of B cells. These basic Comelli, E.M., Sutton-Smtih, M., Yan, Q., Amado, M., Panico, M., Gilmartin, T., approaches will be of general value in elucidating the Whisenant, T., Lanigan, C.M., Head, S.R., Goldberg, D., Morris, H., Dell, A., Paulson, J.C. Activation of murine CD4+ and CD8+ T lymphocytes leads to dra- biology of other members of the siglec family. matic remodeling of N-linked glycans. J. Immunol. 177:2431, 2006. CONSORTIUM FOR FUNCTIONAL GLYCOMICS Han, S., Collins, B.E., Paulson, J.C. Synthesis of 9-substituted sialic acids as Members of our laboratory also staff 2 scientific probes for CD22-ligand interactions on B. Oxford University Press, New York, in press. ACS Symposium Series. cores for the Consortium for Functional Glycomics, organized to elucidate the mechanisms by which Leppanen, A., Stowell, S., Blixt, O., Cummings, R.D. Dimeric galectin-1 binds with high affinity to α2,3-sialylated and non-sialylated terminal N-acetyllactosamine glycan-binding proteins mediate cell communication units on surface-bound extended glycans. J. Biol. Chem. 280:5549, 2005. (http://www.functionalglycomics.org/). In the past Paulson, J.C., Blixt, O., Collins, B.E. Sweet spots in functional glycomics. Nat. year, scientists in the Mouse Transgenics Core, led by Chem. Biol. 2:238, 2006. Peter Sobieszczuk, created 6 novel mouse strains Raman, R., Raguram, S., Venkataraman, G., Paulson, J.C., Sasisekharan, R. from C57Bl/6 embryonic stem cells that are deficient Glycomics: an integrated systems approach to structure-function relationships of in genes for key glycan-binding proteins that affect glycans. Nat. Methods 2:817, 2005. immune function. Scientists in the Glycan Array Synthe- Singh, T., Wu, J.H., Peumans, W.J., Rouge, P., Van Damme, E.J., Alvarez, R.A., Blixt, O., Wu, A.M. Carbohydrate specificity of an insecticidal lectin isolated from sis Core, led by Ola Blixt, have produced a library of the leaves of Glechoma hederacea (ground ivy) towards mammalian glycoconju- synthetic glycans by chemoenzymatic synthesis for gates. Biochem. J. 393:331, 2005.

use in numerous applications. In addition, scientists Stevens, J., Blixt, O., Glaser, L., Taubenberger, J.K., Palese, P., Paulson, J.C., in the Scripps DNA Microarray Core, led by Steve Wilson, I.A. Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J. Mol. Biol. Head, designed and conduced investigator-initiated 355:1143, 2006.

analysis with a custom-based microarray with genes of Stevens, J., Blixt, O., Paulson, J.C., Wilson, I.A. Glycan microarray technologies: tools relevance for the consortium. to survey host specificity of influenza viruses. Nat. Rev. Microbiol. 4:857, 2006.

A major achievement by staff in the Glycan Array Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C., Wilson, Synthesis Core is the development of the world largest I.A. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312:404, 2006. glycan microarray, which currently has more than 300 unique structures, mostly synthetic glycans produced Taniguchi, N., Nakamura, K., Narimatsu, H., von der Lieth, C.W., Paulson, J.C. Human Disease Glycomics/Proteome Initiative workshop and the 4th HUPO Annual by chemoenzymatic synthesis. Now produced in col- Congress. Proteomics 6:12, 2006.

laboration with the DNA Microarray Core, the microar- Tateno, H., Crocker, P.R., Paulson, J.C. Mouse siglec-F and human siglec-8 are ray is widely used by investigators around the world functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6′-sulfo-sialyl Lewis X as a preferred glycan ligand. Glycobiology to assess the specificity of glycan-binding proteins that 15:1125, 2005. mediate a broad scope of biological interactions. In an van Vliet, S.J., van Liempt, E., Saeland, E., Aarnoudse, C.A., Appelmelk, B., exemplary collaboration with I.A. Wilson and J. Stevens, Irimura, T., Geijtenbeek, T.B., Blixt, O., Alvarez, R., van Die, I., van Kooyk, Y. Department of Molecular Biology, this array was used Carbohydrate profiling reveals a distinctive role for the C-type lectin MGL in the recognition of helminth parasites and tumor antigens by dendritic cells. Int. to investigate the specificity of the 1918 pandemic Immunol. 17:661, 2005. 236 MOLECULAR BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

Vasiliu, D., Razi, N., Zhang, Y., Jacobsen, N., Allin, K., Liu, X., Hoffmann, J., Bohorov, O., Blixt, O. Large-scale chemoenzymatic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens. Carbohydr. Res. 3451:1447, 2006.

Westerlind, U., Hagback, P., Tidback, B., Wiik, L., Blixt, O., Razi, N., Norberg, T. Synthesis of deoxy and acylamino derivatives of lactose and use of these for probing the active site of Neisseria meningitidis N-acetylglucosaminyltransferase. Car- bohydr. Res. 340:221, 2005. Molecular and Experimental Medicine

Within the “neurovascular unit” neuron activation controls microvascular vasore- activity via the astrocytes, which are a necessary part of the microvessel. During ischemic stroke, microvessel and neuron activation occur rapidly and simultaneously. Members of Gregory del Zoppo’s laboratory have demonstrated that loss of vascular basal lamina matrix with the rapid generation of pro-MMP-2, its activation systems, and other proteases accompany neuron injury. Furthermore, when the endothelial blood-brain barrier becomes permeable, fibrin is deposited in microvessels where thrombin is generated when plasma contacts perivascular tissue factor (TF). Recently, the laboratory has shown that tissue factor pathway inhibitor (TFPI) is also generated by the endothelium and by activated microglial cells in the ischemic regions. These alterations are part of the evolution of injury in the neurovascular unit. Illustration prepared by Janet Hightower. Eric F. Johnson, Ph.D. Professor Acting Head, Division of Biochemistry MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 239

DEPARTMENT OF Darryl D’Lima, M.D. Eric F. Johnson, Ph.D. Giuseppe Remuzzi, M.D. MOLECULAR AND Assistant Professor Professor Adjunct Professor Acting Head, Division of EXPERIMENTAL Darlene J. Elias, M.D. Biochemistry Michael W. Robertson, Ph.D. MEDICINE Adjunct Associate Professor Associate Professor Thomas J. Kipps, M.D., STAFF Brunehilde Felding- Ph.D. Zaverio M. Ruggeri, M.D.** Habermann, Ph.D. Adjunct Professor Professor Ernest Beutler, M.D.* Associate Professor Head, Division of Chairman and Professor Lawrence E. Kline, D.O. Experimental Hemostasis Head, Division of Hematology Mitchell H. Friedlaender, Adjunct Associate Professor and Thrombosis M.D. Masahiro Aoki, M.D., Ph.D. Adjunct Professor James A. Koziol, Ph.D. Enrique Saldivar, M.D., Ph.D. Adjunct Assistant Professor Professor Adjunct Assistant Professor Jeffrey S. Friedman, M.D., Head, Division of Hiroshi Asahara, M.D., Ph.D. Ph.D. Biomathematics Daniel R. Salomon, M.D. Assistant Professor Assistant Professor Associate Professor Daniel F. Kripke, M.D Bonno N. Bouma, Ph.D. Theodore Friedmann, M.D. Adjunct Professor Alessandro Sette, Ph.D. Adjunct Professor Adjunct Professor Adjunct Professor Thomas J. Kunicki, Ph.D.* Associate Professor Joel N. Buxbaum, M.D. Andrew J. Gale, Ph.D. Farhad F. Shadan, M.D., Professor Assistant Professor Ph.D. Pauline L. Lee, Ph.D. Head, Division of Research Adjunct Assistant Professor Associate Professor Rheumatology Roberta A. Gottlieb, M.D. Associate Professor Sanford J. Shattil, M.D. Stuart A. Lipton, M.D., Ph.D. Dennis A. Carson, M.D. Adjunct Professor Adjunct Professor Adjunct Professor John H. Griffin, Ph.D.** Professor Alexander R. Shikhman, Sergio D. Catz, Ph.D. Martin Lotz, M.D. M.D., Ph.D. Assistant Professor Professor Andras Gruber, M.D. Adjunct Assistant Professor Head, Division of Arthritis Adjunct Assistant Professor Francis V. Chisari, M.D. Research Inmaculada Silos-Santiago, Professor Luca G. Guidotti, D.V.M., M.D., Ph.D. Head, Division of Christopher Lee Marsh, M.D. Ph.D. Adjunct Associate Professor Experimental Pathology Adjunct Associate Professor Associate Professor Gregg J. Silverman, M.D. Clifford W. Colwell, Jr., M.D. Robert McMillan, M.D. Asa B. Gustafsson, Ph.D. Adjunct Professor Adjunct Professor Professor Emeritus Assistant Professor Ronald A. Simon, M.D. Laura M. Crisa, M.D. William E. Miller, M.D. Anne M. Hanneken, M.D. Assistant Professor Adjunct Assistant Professor Adjunct Professor Associate Professor Arthur D. Dawson, M.D. Kevin V. Morris, Ph.D. Peter J. Sims, M.D., Mary J. Heeb, Ph.D.** Adjunct Professor Assistant Professor Ph.D.*** Associate Professor Professor Albert B. Deisseroth, M.D., Jorge J. Nieva, M.D. University of Rochester Ph.D. James A. Hoch, Ph.D. Assistant Professor Rochester, New York Adjunct Professor Professor Head, Division of Cellular Marta Perego, Ph.D. Jack C. Sipe, M.D. Gregory J. del Zoppo, M.D.** Biology Associate Professor Associate Professor Associate Professor Frank M. Huennekens, Ph.D. Paul J. Pockros, M.D. Donald D. Stevenson, M.D. Thomas F. Deuel, M.D. Professor Emeritus Adjunct Assistant Professor Adjunct Professor Professor Head, Division of Molecular Shaun Phillip Jackson, Ph.D. K. Michael Pollard, Ph.D. Eng M. Tan, M.D. Oncology Adjunct Associate Professor Associate Professor Professor Emeritus 240 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

Bruce E. Torbett, Ph.D. Uzen Savas, Ph.D. Giulio Cattarossi, Ph.D. Sharookh B. Kapadia, Ph.D. Associate Professor Stefan Wieland, Ph.D. David M. Cauvi, Ph.D. Jung Hwan Kim, Ph.D. Susan L. Uprichard, Ph.D. Assistant Professor Akemi Yagi, Ph.D. Yunchao Chang, Ph.D. Joseph S. Krueger, Ph.D.

Kottayil I. Varughese, Ji Zhao, Ph.D.*** Emily I. Chen, Ph.D. Pablo G. Landart, Ph.D. Ph.D.** Department of Biomedical Associate Professor Sciences Guofeng Cheng, Ph.D. Alan Yueh-Luen Lee, Ph.D. Scripps Florida Peter K. Vogt, Ph.D. Stephanie Cherqui, Ph.D. Shi-Sheng Li, Ph.D. Professor Quansheng Zhou, Ph.D. Head, Division of Oncovirology Ian D. Dang, Ph.D.*** Enbo Liu, Ph.D.*** United States Patent and The Burnham Institute Matthias G. von Herrath, SENIOR RESEARCH Trademark Office La Jolla, California M.D. ASSOCIATES Rockville, Maryland Adjunct Associate Professor Miao-Chia Lo, Ph.D. Hiroshi Deguchi, M.D., Ph.D. Chinh T. Dao, Ph.D. Therese Wiedmer, Ph.D.*** Jiann-Kae Luo, Ph.D. Associate Professor Yuichi Kamikubo, Ph.D. Maria F. Del Papa, Ph.D. University of Rochester Holly N. Maier, Ph.D. Rochester, New York Richard D. Milner, M.D., Adam Denley, Ph.D. Ph.D. Mathieu Marella, Ph.D. Xiaohua Wu, Ph.D. Alejandra R. Diaz, Ph.D. Assistant Professor Laurent O. Mosnier, Ph.D. Florent M. Martin, Ph.D. Jonathon M. Flanagan, Ph.D. Takao Yagi, Ph.D. Deirdre M. O’Sullivan, Ph.D. Yuri Martina, Ph.D.*** Associate Professor Tatsuya Fukushima, Ph.D. Adaltis Keith Stephenson, Ph.D. Rome, Italy Dong-Er Zhang, Ph.D. Michael J. Giffin, Ph.D. Associate Professor Jill M. Waalen, M.D. Yasunori Mishima, M.D. Shawn Patrick Grogan, Ph.D. Subramanian Yegneswaran, Daniela Beatriz Munafo, Ph.D. STAFF SCIENTISTS Marco Gymnopoulos, Ph.D. Ph.D.

Andreas G. Bader, Ph.D. Wolf-Achim Hassenpflug, Eiko Nakamaru-Ogiso, Ph.D. RESEARCH ASSOCIATES M.D. Joseph R. Biggs, Ph.D. Eun-Young Ahn, Ph.D. Akiko Okumura, Ph.D. Dominik R. Haudenschild, Reha Celikel, Ph.D. Shinichi Asabe, Ph.D. Ph.D. Fumihiko Okumura, Ph.D.

Mei-Hui Hsu, Ph.D. Dong Bai, Ph.D. Gonzalo Herradon, Ph.D. Erin N. Olson, Ph.D.

Chengqun Huang, M.D., Jennifer L. Barber-Singh, Matteo Iannacone, M.D. Mee Young Park, Ph.D. Ph.D. Ph.D. Masanori Isogawa, M.D. Natalie M. Pecheniuk, Ph.D. Jennifer L. Johnson, Ph.D. Cristina Bongiorni, Ph.D. Tatsuo Ito, M.D. Luke F.Peterson, Ph.D. Klaus Kuhn, Ph.D. Kristen E. Bower, Ph.D. Hao Jiang, Ph.D. Pablo Perez Pinera, M.D. Sunil M. Kurian, Ph.D. Anita Y. Boyapati, Ph.D. Sohye Kang, Ph.D. Gian Marco Podda, M.D. Patrizia Marchese, Ph.D. Nathan R. Brady, Ph.D. Mou-Chieh Kao, Ph.D.*** Natalia Reixach, Ph.D. Tsaiwei Olee, Ph.D. Katia Maria Cabral, Ph.D. National Tsing Hua University Rosamund Leila Reynald, Brian Savage, Ph.D. Anna E. Cartier, Ph.D. Hsinchu, Taiwan Ph.D. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 241

Bruno Sainz, Jr., Ph.D. Jason K. Yano, Ph.D.*** Takeda San Diego, Inc. Stefaan J. Sansen, Ph.D. San Diego, California

Francesca Scaramozzino, Zhengyi Ye, Ph.D. Ph.D. Jinseong Yi, Ph.D. Jin Shi, Ph.D. Xiaoyan Yin, Ph.D. Misako Shibakura, Ph.D.*** Okayama University Medical Antonella Zampolli, Ph.D. School Okayama, Japan Wei Zhang, Ph.D.

Christina H. Swan, Ph.D. Li Zhao, Ph.D.

Hendrik Szurmant, Ph.D. Jin Zhong, Ph.D.

Noboru Taniguchi, M.D. Weiguo Zou, Ph.D.

Jesus Torres-Bacete, Ph.D. Masahiko Zuka, M.D., Ph.D.

Jaroslav Truksa, Ph.D. SCIENTIFIC ASSOCIATES Masanao Tsuda, Ph.D. Fanny E. Almus, Ph.D. Billyana C. Tsvetanova, Ph.D. Jose A. Fernandez, Ph.D. Ji Wang, Ph.D. Gabriele E. Foos, Ph.D. Yang Wang, Ph.D.*** Terri P. Gelbart, B.S., M.T. University of Texas MD Anderson Cancer Center Byoung Boo Seo, Ph.D. Houston,Texas

Zhuangzhi Wang, Ph.D. * Joint appointment in The Skaggs Martin R.Weber, Ph.D.*** Institute for Chemical Biology Bristol Myers-Squibb ** Joint appointment in Department Pharmaceutical Research of Cell Biology Institute *** Appointment completed, new loca- Wallingford, Connecticut tion shown

Andrea K. White, Ph.D.*** Chico State University Chico, California

Robert A. White, Ph.D.

Adam C. Wilson, Ph.D.

Tetsuo Yamashita, Ph.D.

Ming Yan, Ph.D.

Xia Yang, Ph.D. 242 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

John Griffin and his colleagues have been leaders in the area of thrombotic diseases (thrombophilia) for many years. Dr. Griffin’s discoveries 25 years ago led to the understanding of the physiologic function of protein C. A deficiency of this anticoagulant protein is associated with increased risk of venous thrombosis. As a result of some of these early studies, activated protein C is now used in the treatment of endotoxin shock. Recently, Dr. Griffin and his colleagues have shown that activated protein C can reduce bleeding induced by tissue plasminogen activator, one of the treatments for stroke. Members of the Griffin laboratory are currently also studying the role of lipoproteins in the risk of venous thrombosis; Dr. Griffin and his colleagues at the Green Hospital have found that low levels of high-density lipoprotein are an important risk factor. Bruce Torbett’s group is continuing to make progress toward the development of an innovative treatment for HIV infections. Their approach is based on the finding that CCR5 is 1 of the 2 main chemokine receptors

Ernest Beutler, M.D. for HIV entry into cells. Delivering a single-chain antibody against this receptor protects cells against entry of HIV. Torbett uses HIV-derived vectors for gene deliv- Chairman’s Overview ery, a technology that his laboratory helped to pioneer. Most of the work in Daniel Salomon’s laboratory is he faculty of the Department of Molecular and directed at improving surgical transplantation of organs Experimental Medicine comprises a group of 44 such as liver, heart, or kidney. One experimental approach T eclectic investigators whose focus is often in the to overcoming the shortage of human organs is to trans- area referred to as translational medicine. Despite the plant pig organs into human patients. Pigs are similar in “translational” nature of much of our research, only 18 size to humans, and if such xenotransplantation could be of our faculty members hold an M.D. degree, and only 3 achieved, there would be a virtually unlimited supply of of these are involved in direct patient care. This reflects organs. Although some of the formidable immunologic a longstanding national trend in which M.D.s bifurcate barriers to the transplantation of pig organs into humans their careers into either bench or bedside, and only rarely have been overcome, pigs harbor endogenous retro- both. Most, but not all, of the research in the department viruses. This has raised concerns not only about the is preclinical or even pre-preclinical. Indeed, only about safety of patients who are receiving these transplants but 5% of the research support in the department comes from conceivably about the safety of the human species as a industry; virtually all the rest comes from the National whole. Animal viruses can wreak havoc on humankind, as Institutes of Health, mostly to individual investigators in exemplified by bird influenza. Scientists in Dr. Salomon’s the form of R01 grants. laboratory have engineered a murine model consisting of The pages that follow this overview contain the sum- mice transgenic for the human porcine retrovirus receptor, maries of the year’s work by the investigators themselves. which his laboratory helped to identify and characterize. These summaries demonstrate the breadth of biomedical His recent finding that these animals are infected with problems currently under investigation in this department. retrovirus has very important implications for the use of But significant biomedical research rarely advances in porcine organs in human organ transplantation. 1-year segments. Some members of our faculty have Peter Vogt, head of the Division of Oncovirology, is played a significant role in developing whole areas of a preeminent pioneer in the field of viruses and cancer, knowledge. Here I try to encapsulate what some of our having discovered that viruses can “steal” genes from scientists are doing and, to some extent, what they have vertebrates and turn them into cancer-causing genes. done leading up to this work. Scientists in his laboratory have continued to expand our MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 243 understanding of how mutated genes cause cancer. In and we are striving to untangle the role of a complex series the past year, he and his colleagues demonstrated that of factors including inflammatory cytokines IL-6 and IL-1, the phosphatidylinosital-3′-kinase that is mutated in bone morphogenic proteins, and iron itself that serve to human tumors actually gains in enzymatic function and regulate the production of hepcidin. These studies may that it can cause malignant transformation both in cell lead to more efficient treatment of diseases in which body culture and in intact animals. iron content is increased (hemochromatosis) or deficient Dong-Er Zhang and her group have continued to and of the treatment of the anemia of chronic inflamma- expand our understanding of how the fusion gene AML1- tion, in which hepcidin is also involved. ETO causes acute myeloid leukemia in humans. In the Roberta Gottlieb and her colleagues demonstrated past year, Dr. Zhang and her group have been able to that the death of cardiomyocytes after a heart attack show that an alternately spliced form of this fusion gene was not necessarily due to necrosis but was largely a is a potent inducer of leukemia. Members of the Zhang result of apoptosis. This opened the door to a variety of laboratory have also pioneered the study of UBP43, an therapeutic strategies. Members of her laboratory are now enzyme that removes an ubiquitin-like protein from its focused on the study of autophagy in this process. targets. They have now found that it serves as a novel James Hoch and Marta Perego have pioneered phos- inhibitor to regulate interferon signaling. phorelay signal transduction pathways in sporulating The cytochrome P450s are a large group of enzymes bacteria and the regulation of phosphate flow in these that function to metabolize both endogenous and exoge- phosphorelays by a large number of phosphatases. They nous compounds that need to be degraded. Eric Johnson, have now turned their attention to unraveling the cellu- acting head of the Division of Biochemistry, has pioneered lar regulatory mechanisms that control transcription of our understanding of the structure of these compounds, the anthrax toxin genes. an important step in understanding of how they actually Pleotrophin is a cytokine identified and cloned by function. In the past year, Dr. Johnson has succeeded Thomas Deuel, head of the Division of Molecular Oncol- in determining the structure of the cytochrome P450 ogy. Pleotrophin has many different functions because that metabolizes nicotine in humans. This discovery it inactivates a phosphatase that has several different could aid in the design of drugs that may help smokers substrates, which have been identified by Dr. Deuel and kick the habit. his colleagues. Among its various functions are the dis- Brunhilde Felding-Haberman has developed a murine ruption of normal cytoskeletal architecture, inhibition of model of human breast cancer. Having isolated human neurite outgrowth in PC12 cells, and angiogenesis, par- antibodies directed at the activated confirmer of integrin ticularly in tumor growth. Further understanding of the

αVβ3 on human cells, she has used this model to study functioning of pleotrophin and developing methods for the effectiveness of the antibody both in preventing and enhancing or inhibiting its activities offer many possi- in treating metastases. She is also using it to determine bilities for treatment of several different disorders. whether neural stem cells may be effective in inhibiting Joel Buxbaum, head of the Division of Research brain metastases of breast cancer cells. Rheumatology, has a longstanding interest in the mis- My own laboratory has a long history of studying folding of proteins giving rise to deposits generically known single-gene diseases. The discovery of glucose-6-phos- as “amyloid.” A single amino acid substitution may greatly phate dehydrogenase deficiency was the first of these increase the propensity of a protein to misfold. One such and led to my origination of the X-inactivation hypothesis. substitution is very common among people of African Since that time my colleagues and I have investigated origin as a result of a single base-pair change in the gene many of the inherited red-cell enzyme deficiencies, the encoding transthyretin. In the case of this particular mis- glycolipid storage disorders, particularly Gaucher disease, folding protein, deposits occur in the heart, and scien- and inborn errors of iron metabolism. It is the latter area tists in the Buxbaum laboratory have recently shown that that is currently receiving most attention from my group. 10% of African Americans over the age of 65 with con- The pathways that regulate the amount of iron absorbed gestive heart failure carry this mutation. This is quite a from the intestine and therefore maintain total body iron remarkable finding, with obvious health implications for within normal limits have proved to be much more com- this ethnic group. plex than anyone had imagined. The 25 amino acid pep- Francis Chisari, head of the Division of Experimental tide hepcidin seems to play a particularly important role, Pathology, is an internationally recognized expert in the 244 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE field of hepatitis. More than 20 years ago, using then effort—is the Manhattan project of World War II, the very new transgenic mouse technology, he produced the project that engineered and built the first atomic bombs. first small animal model of hepatitis B infection—actually Those leading the National Institutes of Health seem to the first transgenic mouse model of any human patho- have become enamored of this approach to bioscience, gen. In more recent years, scientists in his laboratory with the idea that large networks, interdisciplinary have also been studying hepatitis C. The agent for this approaches, and extensive collaboration are needed to important infection was discovered in 1989, and the better understand Nature. Indeed, some problems require development of a tissue culture model of this infection this sort of approach. They include the genome project, has eluded investigators in this field until last year, when multicenter clinical trials, and large epidemiologic stud- Chisari’s group and two other laboratories simultane- ies. But what must not be forgotten is that truly great, ously developed the first tissue culture model of infec- innovative ideas do not usually arise in committees, and tion. Dr. Chisari and his group are now using this model that such large efforts are generally based on fundamen- to identify entry and egress mechanisms in the virus, to tal ideas and techniques developed by single investiga- define metabolic and signaling pathways that regulate tors heading small research grants. Most of the research the infection, and ultimately to develop antiviral drugs in the Department of Molecular and Experimental Medi- to treat chronic infection. cine is of the latter ilk. Members of our faculty have Osteoarthritis, essentially the wearing out of joints, contributed some of the fundamental ideas on which affects most people sooner or later. One of the major today’s science is based, and this is what we continue underlying problems is that as cartilage cells are destroyed to try to achieve. or die, they are not replaced. Scientists in the Division of Arthritis Research, headed by Martin Lotz, are studying animal models to develop means of preventing cartilage loss, and they have recently found that inhibitors of caspase injected into the joint in animals in these models of osteoarthritis prevent the destruction of cartilage. This opens up the possibility of a new treatment for this common, debilitating disorder, which Dr. Lotz and his group will study in animals with the hope that a human therapeutic may emerge. Zaverio Ruggeri, head of the Division of Experimental Hemostasis and Thrombosis, is internationally known for his insights into the mechanism of thrombosis. He has shown that the process of platelets adherence to endothelium is very different in the static systems that were once used than in the dynamic flow systems that he has pioneered. Working together with Tom Kunicki in his division, Dr. Ruggeri is mapping new genes that are important in the regulation of clot formation at the sites of blood vessel injury. In collaboration with Luca Giodotti, in the Division of Experimental Pathology, members of Dr. Ruggeri’s group are studying an unexpected role of platelets in immune-mediated processes, and particularly in viral clearance. To some extent, one may dichotomize biomedical research into big research projects in which many principal investigators, often at several different institutions, are addressing a problem of some significance, and smaller projects headed by a single principal investigator. The prototype of big science—a large-scale scientific MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 245

INVESTIGATORS’REPORTS that mechanical dynamic compression of chondrocytes led to rearrangement of the actin cytoskeleton. Currently we are investigating the role of a signaling pathway that DIVISION OF ARTHRITIS RESEARCH involves Rho kinase and the actin-regulating protein cofilin. These studies may reveal novel pathways and Martin Lotz, M.D., Division Head therapeutic targets for stimulating formation of the extracellular matrix of cartilage. Joint Injury and Osteoarthritis

D. D’Lima, C.W. Colwell, Jr., M. Lotz Physiology of Facilitated Glucose cute or chronic mechanical injury is a risk factor Transporter GLUT1 in Cartilage for the development of osteoarthritis. We are Homeostasis and Osteoarthritis A investigating mechanisms that mediate the effects of mechanical injury on joint integrity and potential therapeutic approaches that target these mechanisms. We A.R. Shikhman, D.C. Brinson, J. Valbracht, M. Lotz have established in vitro models to apply mechanical rticular cartilage is an avascular tissue that func- stress to cartilage explants and cells in 3-dimensional tions under nearly anaerobic conditions and there- cultures. Application of high-intensity mechanical stress A fore depends on glucose supply for the generation causes cell death that is mediated in part by apoptotic of energy. Glucose is the main precursor for UDP-hex- mechanisms. Cell death after mechanical injury can be osamines and UDP-uronic acids, which are used by prevented by pharmacologic inhibitors of caspases; this chondrocytes in the synthesis of glycosaminoglycans. treatment results in the maintenance of biosynthetically Transmembranous glucose transport facilitated by a active cartilage cells. group of glucose transporter proteins termed GLUTs is In an animal model of posttraumatic osteoarthritis the first rate-limiting step in glucose metabolism. Human induced by transection of the anterior cruciate ligament, articular chondrocytes express several specific GLUTs, intraarticular injection of caspase inhibitors reduced the including GLUT1, GLUT3, GLUT6, GLUT8, and GLUT10. severity of cartilage lesions. We plan to examine new GLUT1 is the most abundant glucose transporter in chemical classes of caspase inhibitors in this model. Our human articular chondrocytes. Expression of GLUT1 in long-term goal is to use the inhibitors to treat patients chondrocytes is regulated by proinflammatory cytokines with posttraumatic arthritis. via signaling pathways that depend on protein kinase C and p38 MAP kinase. Overexpression of GLUT1 occurs in osteoarthritic cartilage. Small interfering RNA inhibits Mechanotransduction in expression of GLUT1 in unstimulated and IL-1β–stimulated human articular chondrocytes. Inhibition of this Chondrocytes expression does not significantly change basal facilitated glucose transport, but it abrogates surplus glu- D. D’Lima, D. Haudenschild, M. Lotz cose transport induced by cytokines or growth factors, hysiologic levels of mechanical load are impor- indicating that GLUT1 is required to provide glucose tant in stimulating cartilage cells to maintain supply in activated cells but not in resting cells. P the composition of the extracellular matrix. We Inhibition of GLUT1 expression in chondrocytes is are elucidating the signaling mechanisms that trans- associated with suppression of glycolysis and lactate duce mechanical stimuli into biochemical responses production. GLUT1-regulated lactate production is more in cartilage cells. Dynamic compression of cartilage sensitive to the inhibition of GLUT1 expression than is in vitro exerts anabolic effects and stimulates the pro- transmembranous glucose transport, indicating that one duction of proteins that make up the cartilage extra- of the potential functions of GLUT1 is regulation of intracellular matrix. Mechanical deformation of cells can cellular glucose flow through the glucose-consuming directly or indirectly through receptor-mediated events metabolic pathways (glycolysis, pentose-phosphate affect the organization of the cytoskeleton. We found shunt, and hexosamine pathway). 246 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

Inhibition of GLUT1 expression suppresses basal mediated acetylation requires acetyl coenzyme A as a and growth factor–stimulated thymidine transport and substrate, we performed in vitro transcription assays chondrocyte proliferation and depends on phosphoryla- in the presence and absence of this coenzyme. We tion of AMP-activated protein kinase. Chondrocytes with found that p300 did not stimulate Sox9-dependent suppressed GLUT1 are also characterized by an increase transcription in the absence of the coenzyme. In his- in basal and growth factor–stimulated production of tone acetyltransferase assays, both p300 and Sox9 hyaluronan and expression of hyaluronan synthase 2 synergistically acetylated histones, which were assem- that does not depend on activation of the kinase. Fur- bled on a chromatin template. In reporter assays, the thermore, inhibition of GLUT1 expression by small inter- addition of p300 increased the relative luciferase activity fering RNA also reduces IL-1β–induced production of in a Sox9-dependent manner but not in assays in which nitric oxide. These data indicate that GLUT1 is a novel we used a mutant p300 deficient in histone acetyl- regulator of chondrocyte activation and that its func- transferase. These results suggest that the altered tions extend beyond its glucose-transporting activity. chromatin structure caused by the histone acetyltransferase activity of p300 may have an important role in PUBLICATIONS Cecil, D.L., Johnson, K., Rediske, J., Lotz, M., Schmidt, A.M., Terkeltaub, R. Sox9-dependent transcription. Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced In chondrocytes, histone hyperacetylation activates glycation end products. J. Immunol. 175:8296, 2005. the expression of genes that encode the extracellular D’Lima, D., Hermida, J., Hashimoto, S., Colwell, C., Lotz, M. Caspase inhibitors matrix of cartilage. To assess the relationship between reduce severity of cartilage lesions in experimental osteoarthritis. Arthritis Rheum. 54:1814, 2006. expression of COL2A1 and histone acetylation on the COL2A1 enhancer region, we used trichostatin A, a Hiraoka, K., Grogan, S., Olee, T., Lotz, M. Mesenchymal progenitor cells in adult human articular cartilage. Biorheology, in press. histone deacetylase inhibitor, to induce histone hyper-

Shikhman, A.R. Glucosamine and osteoarthritis. Future Rheumatol. 1:67, 2006. acetylation in human chondrocytes. We found that treatment with trichostatin A stimulated COL2A1 expression in chondrocytes. Transcriptional Regulation of Taken together, these findings indicate that p300 stimulates Sox9-dependent transcription by modifying Chondrogenesis via Chromatin histone acetylation. These results suggest that chromatin regulation, such as regulation via histone deacetylase-1 Modification inhibitors in chondrocytes, could be a new therapeutic strategy for treatment of arthritis. H. Asahara, T. Ito, K. Yoshida, N. Taniguchi, M. Tsuda

PUBLICATIONS hondrogenesis is a multistep pathway in which Furumatsu, T., Tsuda, M., Yoshida, K., Taniguchi, N., Ito, T., Hashimoto, M., Ito, T., multipotential mesenchymal stem cells differenti- Asahara, H. Sox9 and p300 cooperatively regulate chromatin-mediated transcription. J. Biol. Chem. 280:35203, 2005. C ate into chondrocytes. The transcription factor Sox9 regulates chondrocyte differentiation and cartilage- specific expression of genes, such as COL2A1, which encodes collagen type II α1. During the past year, we DIVISION OF BIOCHEMISTRY used an in vitro chromatin assembly model to investigate the function of p300 in Sox9-dependent transcription. Eric F. Johnson, Ph.D., Acting Division Head Using chromatin templates, we determined whether Sox9 transcriptional activity requires p300. We found that addition of p300 upregulated the transcriptional Cytochrome P450: Regulation, activation by recombinant Sox9, showing that both p300 and Sox9 are necessary for activation of chro- Structure, and Function matin-mediated transcription. E.F. Johnson, K.J. Griffin, M.-H. Hsu, R.L. Reynald, S. Sox9-dependent transcription is regulated by p300- Sansen, Ü. Savas, J.K. Yano mediated histone acetylation on chromatin. Using naked or chromatinized DNA, we explored the relationship nzymes in the cytochrome P450 superfamily pri- between p300-induced histone acetylation and Sox9- marily serve 2 purposes in human physiology. dependent transcriptional activation. Because p300- E Some P450s catalyze specific biotransforma- MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 247 tions in autocrine, paracrine, and endocrine signal drug-drug interactions that can arise from inhibition of transduction pathways. A second, relatively large group P450s if multidrug therapies are used. of P450 monooxygenases play defensive roles by con- Mammalian P450s are tethered to the endoplasmic verting toxic compounds to less toxic forms that are reticulum by a transmembrane segment at the amino more soluble and more easily excreted than are the par- terminus and by additional interactions of the catalytic ent compounds. Each xenobiotic-metabolizing P450 domain with the cytoplasmic side of the membrane. generally oxidizes structurally diverse substrates, lead- Although membrane proteins are difficult to crystallize, ing to a wide-ranging protective capacity for elimina- we developed methods to express, purify, and crystallize tion of toxic chemicals. Often the expression levels of genetically modified mammalian P450s that retain a these enzymes are increased in response to exposure native catalytic domain. Using this approach, we have to xenobiotics or altered physiologic states. We wish determined the atomic structures of several of the most to understand how the structural diversity and genetic important drug-metabolizing P450s: 1A2, 2A6, 2C8, regulation of P450s that metabolize xenobiotics con- 2C9, and 3A4. Through these studies, we discovered tribute to a person’s ability to avoid the adverse effects how the flexibility of the P450s and the diversity of their of environmental chemicals and alter the clearance and amino acid sequences can shape catalytic specificity. bioavailability of therapeutic drugs. The P450 2A6 is also the principal nicotine-oxidiz- Although extensive information on the conditional ing enzyme. Although 2A6 plays a prominent role in expression of P450 genes in experimental species is detoxification of nicotine, it also can activate the tobacco available, in humans the transcriptional responses of smoke–specific carcinogen nitrosamine 4-(methylnitro- P450 genes to environmental stimuli and to physiologic samino)-1-(3-pyridyl)-1-butanone to its carcinogenic changes are poorly understood. To address this problem, form. Several reports indicate that because of the we use human cell lines, primary cultures of human increased side effects of nicotine, persons who are cells, and transgenic mice to study mechanisms that genetically deficient in 2A6 activity are less likely to regulate human family 4 P450 genes. These genes smoke than are persons not genetically deficient in this encode enzymes that are involved in both signal trans- activity. In collaboration with J. Cashman, Human Bio- duction and the metabolism of endogenous lipids and molecular Research Institute, La Jolla, California, we xenobiotics. Studies with cell lines are providing new are developing inhibitors of 2A6 that could reduce smok- information about endocrine and autocrine signal trans- ing behavior and diminish the likelihood of tobacco- duction pathways that govern the conditional expression related lung cancers. of these genes in response to nutritional, hormonal, and xenobiotic signals. Research is in progress to test PUBLICATIONS Johnson, E.F., Stout, C.D. Structural diversity of human xenobiotic-metabolizing cyto- whether more complex physiologic conditions such as chrome P450 monooxygenases. Biochem. Biophys. Res. Commun. 338:331, 2005. pregnancy or energy (caloric) restriction alter the expres- Yano, J.K., Hsu, M.-H., Griffin, K.J., Stout, C.D., Johnson, E.F. Structures of sion of the human enzymes in transgenic mice. human microsomal cytochrome P450 2A6 complexed with coumarin and Recently, we discovered that the human long chain methoxsalen. Nat. Struct. Mol. Biol. 12:822, 2005. fatty acid ω-hydroxylase, CYP4F2, is induced in primary cultures of human hepatocytes and in cell lines by several drugs, termed statins, that are used to lower Neutrophil Dysfunction and serum levels of cholesterol. The induction of CYP4F2 could contribute to the reported reduction by statins Human Immunodeficiencies of long chain fatty acids that accumulate in X-linked adrenoleukodystrophy. S.D. Catz, B.A. Ellis, D.B. Munafo, M. Park, S. Pacquelet, In collaboration with C.D. Stout, Department of J.L. Johnson Molecular Biology, we are defining the atomic structures RAB27A AND THE SECRETORY MACHINERY of individual human P450s to understand the struc- IN GRANULOCYTES tural basis for the broad yet unique catalytic selectiv- eutrophils kill microorganisms via microbicidal ity of each enzyme. This information can be used to products released to the phagosome or to the better understand the adverse affects of the oxidation N extracellular space. In resting neutrophils, these of drugs and toxins and the potential for metabolic microbicidal molecules are segregated in secretory 248 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE organelles, thus protecting the host from uncontrolled p47phox activated NADPH oxidase in a cell-free system activation. The secretory machinery used by these organ- and IRAK-4 overexpression increased NADPH oxidase elles is poorly characterized. activity in response to lipopolysaccharide. We discovered that the small GTPase Rab27a, which We concluded that IRAK-4 is responsible for the is absent in patients with the immunodeficiency Griscelli phosphorylation of p47phox and NADPH oxidase activa- syndrome, is a main component of the secretory machin- tion after lipopolysaccharide stimulation. This finding may ery in granulocytes. We also found that Rab27a is pre- have physiologic connotations in the human immunodefi- sent in a large proportion of granules that contain matrix ciency triggered by IRAK-4 deficiency that is character- metalloproteinase 9 and in a minor subpopulation of ized by susceptibility to pyogenic bacterial infections. granules that contain myeloperoxidase. Interference with the Rab27a secretory machinery impaired secre- PUBLICATIONS Johnson, J.L., Ellis, B.A., Noack, D., Seabra, M.C., Catz, S.D. The Rab27a-bind- tion of the metalloproteinase and myeloperoxidase in ing protein, JFC1, regulates androgen-dependent secretion of prostate-specific anti- permeabilized neutrophils. In HL-60 promyelocytic cells, gen and prostatic-specific acid phosphatase. Biochem. J. 391(Pt. 3):699, 2005. the expression of Rab27a was dramatically increased when the cells differentiated to granulocytes but not when they differentiated to monocytes, supporting a Preserving Vision in Glaucoma role for this small GTPase in the secretory machinery of granulocytes. and Macular Degeneration We developed a RNA interference approach to A. Hanneken, J. Johnson downregulate Rab27a in HL-60 cells and showed that Rab27a-deficient cells have impaired myeloperoxidase etinal nerve cell damage is the primary cause secretion. Similarly, we discovered that Rab27a-deficient of visual loss in patients with glaucoma and mac- mice have a marked decreased in myeloperoxidase secre- R ular degeneration. Recent evidence suggests tion in response to lipopolysaccharide. We concluded that under certain circumstances, retinal cells can be that Rab27a is a main component of the secretory protected from dying and nerve cells can be rescued machinery of the secretory organelles in neutrophils. from death by specific dietary flavonoids found in plant CROSS TALK BETWEEN IL-1 RECEPTOR–ASSOCIATED extracts. We have identified a group of flavonoids that KINASE-4 AND NADPH OXIDASE are particularly effective in protecting eye-derived cells Exposure of neutrophils to lipopolysaccharide ampli- from the type of injury associated with macular degen- fies their oxidative response to formylated peptides in eration and glaucoma. The ability of flavonoids to a process referred to as “priming.” The relationship restore the health of injured retinal cells and induce between the signaling downstream of Toll-like receptor the outgrowth of neurites gives these compounds a 4 after lipopolysaccharide stimulation and the activation unique set of advantages. of NADPH oxidase remains elusive. Phosphorylation of Macular degeneration leads to the death of the the NADPH oxidase cytosolic factor p47phox is essen- retinal pigment epithelial cells, whereas glaucoma leads tial for activation of the NADPH oxidase. We examined to the death of retinal ganglion cells. We have screened the hypothesis that IL-1 receptor–associated kinase-4 multiple different flavonoids for their ability to protect (IRAK-4), the main regulatory kinase downstream of retinal pigment epithelial cells and retinal ganglion Toll-like receptor 4 activation, regulates NADPH oxidase cells from cell death induced by oxidative stress (Fig. 1). through phosphorylation of p47phox. Figure 2 shows the protective effect of luteolin on cul- We discovered that p47phox is a substrate for IRAK-4 tures of retinal pigment epithelial cells exposed to oxi- and that IRAK-4–phosphorylated p47phox can be subse- dative stress, the type of injury associated with macular quently phosphorylated by protein kinase C. We iden- degeneration. Luteolin also prevents cell death induced tified, by mass spectrometry, a novel threonine-rich by oxidative stress in retinal ganglion cells. regulatory domain in p47phox. Lipopolysaccharide-depen- Recently, we found that the effective flavonoids have dent phosphorylation of p47phox was enhanced by the specific mechanisms of action. Some enhance the pro- inhibition of p38 MAP kinase, confirming that the kinase duction of glutathione, the cell’s primary defense against responsible for p47phox phosphorylation operates oxidative injury. Others block the production of reac- upstream of p38 MAP kinase. IRAK-4–phosphorylated tive oxygen species, which cause cellular injury and MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 249

Fig. 2. Luteolin protects retinal pigment epithelial cells from oxi-

dative stress–induced cell death. H2O2 = hydrogen peroxide; t-BOOH = tert-butyl hydroperoxide. epithelial cells and retinal ganglion cells from oxidative stress–induced death (Table 2). We are validating Fig. 1. Chemical structures of the dietary flavonoids. EGCG = and expanding these results; we hope to identify addi- (–)-epigallocatechin gallate. tional compounds and combinations that have greater death. Additionally, some flavonoids can activate the potency and efficacy. antioxidant response element in cells and so induce the This research is the result of a partnership formed expression of genes that increase resistance to oxidative between the Scripps Mericos Eye Institute and Scripps injury (Table 1). We are investigating these flavonoids Table 2. Dietary flavonoids that protect retinal cells from injury in more detail to determine whether these compounds and death in macular degeneration. are also capable of protecting cells in long-term pro- Flavonoid Dietary source tection assays. At this point, we have compiled a list of specific Luteolin Spinach, wild greens, hot peppers, celery, thyme, parsley, mint dietary flavonoids that protect both retinal pigment

Table 1. Protective mechanisms of different flavonoids. Quercetin Onions (especially yellow), cranberries, cocoa, wild greens, capers, fennel, Flavonoid Involved in Scavenges Activates spinach, chives, celery, cherries, glutathione reactive antioxidant metabolism oxygen species response blueberries, apples, kale, red wine element Eriodictyol Peppermint, citrus juices (lemon, lime, Luteolin No Yes No sour orange) Fisetin Yes Yes Yes Fisetin Strawberries, tomatoes, onion, oranges, Quercetin Yes Yes Yes apples, peaches, grapes, kiwifruit, Eriodictyol Yes Yes Yes persimmons 250 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

Research aimed at bringing together the promise of biomedical research and the practice of medicine.

PUBLICATIONS Hanneken, A., Lin, F.-F., Johnson, J., Maher, P. Flavonoids protect human retinal pigment epithelial cells from oxidative stress-induced cell death. Invest. Ophthal- mol. Vis. Sci., in press.

Maher, P., Hanneken, A. Flavonoids protect retinal ganglion cells from oxidative stress-induced cell death. Invest. Ophthalmol. Vis. Sci. 46:4796, 2005.

NADH Dehydrogenases Fig. 1. Essential amino acid residues of the membrane domain subunit NuoK of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli.E indicates glutamic acid; R, arginine. T. Yagi, A. Matsuno-Yagi, B.B. Seo, E. Nakamaru-Ogiso, M.-C. Kao, T. Yamashita, M. Marella, J. Barber-Singh, cysteine residues: at positions 180, 351, 354, 357, J. Torres-Bacete and 398. We mutated the individual cysteines to alanine. STRUCTURE AND FUNCTION OF PROTON- The mutated NuoF subunits were isolated and sub- TRANSLOCATING NADH-QUINONE OXIDOREDUCTASE jected to various physicochemical analyses including he proton-translocating NADH dehydrogenase of electron paramagnetic resonance spectroscopy. The mitochondria (complex I) is responsible for energy data indicate that the cysteines at positions 351, 354, T coupling in the respiratory chain. Complex I is 357, and 398, but not the cysteine at position 180, composed of 46 unlike subunits and contains 1 FMN are involved in the ligation of cluster N3. and 8 iron-sulfur clusters as cofactors. The proton- MOLECULAR REMEDY OF COMPLEX I DEFECTS translocating NADH dehydrogenase of bacteria, NDH-1, Studies suggest that defects in mitochondrial com- is similar to complex I in terms of electron carriers and plex I are involved in many human diseases, such as inhibitor specificity. However, in contrast to complex I, Leigh syndrome and sporadic Parkinson’s disease. How- NDH-1 is composed of 14 unlike subunits (NuoA–NuoN). ever, no effective remedies for complex I deficiencies Both NDH-1 and complex I consist of 2 major have been established. We have adopted a gene ther- domains: the peripheral segment and the membrane apy approach in which we use the gene NDI1, which segment. The peripheral domain of NDH-1 contains encodes Ndi1, the single polypeptide NADH dehydro- 7 subunits (NuoB–NuoG and NuoI) and bears all the genase of Saccharomyces cerevisiae. cofactors, so this domain participates in electron trans- Our earlier experiments indicated that Ndi1 can fer. The membrane domain of NDH-1 appears to be replace or supplement the functionality of complex I in composed of 7 subunits (NuoA, NuoH, and NuoJ–NuoN), various cultured cells. In addition, by using NDI1–recom- which are homologs of mitochondrial DNA-encoded binant adeno-associated virus particles, we found that subunits (ND1–ND6 and ND4L) of complex I. This Ndi1 can be expressed in mitochondria in skeletal mus- domain catalyzes proton translocation. cles and brains of rats and mice. The expressed Ndi1 In one of our current projects, we are identifying was functionally active. amino acid residues in the membrane subunits essen- For this approach to be useful, the mature protein tial for proton translocation.For these studies, we use must have protective effects against complex I defects a chromosomal DNA mutagenesis approach. We found in vivo. Currently, well-established animal models of that in the NuoK subunit, mutations in glutamic acid complex I diseases are not available. However, the at positions 36 and 72 and in arginine at positions 25 parkinsonian signs in mice treated with 1-methyl-4- and 26 (Fig. 1) suppress the energy-transducing activ- phenyl-1,2,3,6-tetrahydropyridine (MPTP) might be ity of NDH-1, suggesting that these residues may be due to inhibition of complex I by MPTP. We determined directly involved in proton translocation. whether the expressed Ndi1 enzyme has protective In another project, we are characterizing the cofac- effects against the parkinsonian signs in MPTP-treated tors of NDH-1. As described earlier, NDH-1 contains mice. As shown in Figure 2, Ndi1 expressed in mouse 8–9 iron-sulfur clusters. We attempted to identify the substantia nigra suppressed dopaminergic neuronal amino acid residues that coordinate cluster N3 in the deficits induced by MPTP such as decreases in tyro- NuoF subunit. The NuoF subunit contains 5 conserved sine hydroxylase in the substantia nigra and the stria- MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 251 Asymmetries in the Spatial Distributions of Enhancing Lesions and Hypointense Lesions in Relapsing-Remitting Multiple Sclerosis

J.A. Koziol, S. Wagner,* D.F. Sobel,** A.C. Feng, H.P. Adams

* Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany

** Scripps Clinic, La Jolla, California

e examined the spatial distributions of enhancing lesions and hypointense lesions (“black W holes”) in 24 patients with relapsing-remitting multiple sclerosis enrolled in a clinical study at Scripps Clinic. We tested the hypotheses that lesions occur randomly throughout the brain and that the spatial patterns of lesions are homogeneous among patients. Fig. 2. Protective effects of Ndi1 expressed in mouse striatum against decreases in tyrosine hydroxylase induced by treatment with We investigated within patients whether enhancing MPTP. Upper panel, control; middle panel, MPTP treatment; lower lesions and black holes have bilateral symmetry about panel, MPTP treatment after injection of NDI1–recombinant adeno- the following planes: midtransaxial, midcoronal, and associated virus particles into the left substantia nigra in mouse brain. midsagittal. Using patients’ monthly magnetic resonance tum and decreases in striatal levels of dopamine. The images, we counted lesions in 10 locations: brain stem, data indicate that NDI1 will be a promising therapeu- cerebellum, periventricular region (frontal, temporal, tic tool in the treatment of diseases caused by impair- parietal, and occipital), and other white matter (frontal, ments in complex I. temporal, parietal, and occipital). We found a pronounced lack of midtransaxial sym- PUBLICATIONS metry in the locations of the lesions; most of the lesions Betarbet, R., Canet-Aviles, R.M., Sherer, T.B., Mastroberardino, P.G., McLendon, C., Kim, J.-H., Lund, S., Na, H.-M., Taylor, G., Bence, N.F., Kopito, R., Seo, were supratentorial. We also noted lack of symmetry B.B., Yagi, T., Matsuno-Yagi, A., Klinefelter, G., Cookson, M.R., Greenamyre, J.T. about the coronal and sagittal axes, although it was Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, α-synuclein, and the ubiquitin-proteasome system. more subtle than the lack of symmetry about the mid- Neurobiol. Dis. 22:404, 2006. transaxial plane. In addition, lesions preferentially

Kao, M.-C., Nakamaru-Ogiso, E., Matsuno-Yagi, A., Yagi, T. Characterization of the appeared in the anterior rather than the posterior part membrane domain subunit NuoK (ND4L) of the NADH-quinone oxidoreductase of the brain. We found no consistent pattern of left- or from Escherichia coli. Biochemistry 44:9545, 2005. right-sided predominance in the locations of lesions, Seo, B.B., Nakamaru-Ogiso, E., Flotte, T.R., Matsuno-Yagi, A., Yagi, T. In vivo even within periventricular or other white matter, across complementation of complex I by the yeast Ndi1 enzyme: possible application for treatment of Parkinson disease. J. Biol. Chem. 281:14250, 2006. all patients. But, we found a distinct lack of symmetry

Velazquez, I., Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., Yagi, T. Amino acid residues about the midcoronal plane (anterior vs posterior) and associated with cluster N3 in the NuoF subunit of the proton-translocating NADH- the midsagittal plane. quinone oxidoreductase from Escherichia coli. FEBS Lett. 579:3164, 2005. We next investigated whether patients have similar Yagi, T., Seo, B.B., Nakamaru-Ogiso, E., Marella, M., Barber-Singh, J., Yamashita, patterns of locations of lesions. We used cluster analy- T., Kao, M.-C., Matsuno-Yagi, A. Can a single subunit yeast NADH dehydrogenase (Ndi1) remedy diseases caused by respiratory complex I defects? Rejuvenation Res. ses to detect subsets of patients that might have similar 9:191, 2006. patterns of lesion locations. Figure 1 shows the loca-

Yagi, T., Seo, B.B., Nakamaru-Ogiso, E., Marella, M., Barber-Singh, J., Yamashita, tion patterns for the 4 tightest nondegenerate clusters T., Matsuno-Yagi, A. Possibility of transkingdom gene therapy for complex I dis- of patients on the basis of enhancing lesions and for eases. Biochim. Biophys. Acta, in press. the 3 tightest nondegenerate clusters on the basis of black holes. 252 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

metries. We found an extreme lack of bilateral symmetry about the midtransaxial, midcoronal, and midsagittal planes in terms of frequencies of both enhancing lesions and black holes in our cohort. These asymmetries suggest that in individual patients, particular lobes or regions might be more vulnerable than other regions to pathologic changes associated with the formation of lesions, even if the processes leading to lesions are assumed to be ubiquitous. Perhaps asymmetries in the locations of lesions reflect various asymmetries within the CNS white matter and consequent heterogeneities in the histopathologic features of lesions. An immediate implication is that potential therapeutic agents could have differential effects on these processes, so that the treatment of choice for an individual patient with multiple sclerosis may depend on identifying the underlying mechanisms of disease.

PUBLICATIONS Koziol, J.A., Wagner, S., Sobel, D.F., Feng, A.C., Adams, H.P. Asymmetries in the spatial distributions of enhancing lesions and black holes in relapsing-remitting MS. J. Clin. Neurosci. 2:895, 2005.

DIVISION OF CELLULAR BIOLOGY

James A. Hoch, Ph.D., Division Head

Fig. 1. Distinct clusters of patients with relapsing-remitting multiple sclerosis have similar patterns of locations of lesions on mag- Sensor Kinases That Regulate netic resonance images. A, Total numbers of enhancing lesions. B, Total numbers of black holes. Abbreviations: BS, brain stem; CE, Sporulation and the Synthesis cerebellum; FP, frontal periventricular region; TP, temporal periventricular region; PP, parietal periventricular region; OP, occipital periven- of Toxins tricular region; FW, frontal white matter; TW, temporal white matter; PW, parietal white matter; OW, occipital white matter. J.A. Hoch, M. Perego, T. Fukushima, F. Scaramozzino, H. Szurmant, B. Tsvetanova, A. Wilson The clusters are quite distinctive. In all of the clusters of enhancing lesions, parietal lesions dominate; in ormation of endospores in Bacillus subtilis is a particular, both periventricular and other white matter model for understanding the mechanism of devel- lesions in clusters 1 and 2, but solely other white matter F opmentally programmed gene expression. Sev- lesions in clusters 3 and 4. Occipital lesions are also eral dozen genetically dispersed sporulation operons are present in clusters 2 and 4 and frontal white matter regulated coordinately as temporal classes during the lesions in cluster 3. Cluster 1 of black holes constitutes time required to complete the formation of spores. This solely parietal white matter lesions. Cluster 3 is slightly complex developmental program is under the control of expanded relative to cluster 1, encompassing both the spo0 genes, which control entry of the cell into frontal and parietal white matter lesions, whereas clus- sporulation and the production of toxins and virulence ter 2 is rather dispersed throughout the white matter. factors in pathogens such as Bacillus anthracis. We developed statistical techniques to characterize The transcription factor Spo0A is the key master the asymmetries of the locations, and we investigated regulator of the initiation of developmental transcrip- statistical measures of individual and subgroup asym- tion. The activity of the protein is controlled by a rever- MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 253 sible phosphorylation-dephosphorylation mechanism. Computational Analysis of The pathway to Spo0A activation is a series of phosphorylation reactions involving sequentially the Spo0F Molecular Specificity in and Spo0B proteins, for which we coined the term phosphorelay. The probability that sporulation will be 2-Component Signaling initiated depends on the competition between kinases and phosphatases. J.A. Hoch, R.A. White, H. Szurmant, T. Hwa* Previously, we identified 5 sensor kinases involved * University of California, San Diego, California in signaling the initiation of sporulation in B subtilis. n both prokaryotes and eukaryotes, a large number Our studies in B anthracis revealed 9 sensor kinases of pathways with proteins with identical structural for sporulation that differ from those of B subtilis in the I folds are used to interpret and propagate vastly dif- signal-sensing domains. Deletion studies indicated that ferent signals specific for unique targets. A central ques- sporulation in B anthracis is a consequence of the con- tion in understanding signal transduction is how does certed activities of several of these sensor kinases. The a signaling protein distinguish its true partner from the sporulation pathway regulates the production of the much larger number of similar partners present in the genes for anthrax toxin through at least one Spo0A- cell. Without this specificity, unintended cross talk controlled regulator. Studies on the transcription of the among the pathways will greatly reduce the fidelity of gene that encodes the protective antigen component signal transduction. On the other hand, designed cross of the toxin revealed the extent of the promoter region talk at specific stages between specific pathways pro- responsible for gene activation and the relationship of vides a means of combinatorial signal integration that the region to the positive transcription activator AtxA. may greatly increase the signal-processing capability The goal of these studies is to understand the specific of the cell. In collaboration with T. Hwa, University of and global controls that regulate virulence in B anthracis. California, San Diego, we are examining the molecular We have initiated a program to study the regulation interactions that underlie partner recognition; the focus of sporulation in clostridia, which are increasingly preva- of these studies is the 2-component system, the prevalent as causes of nosocomial infections and as producers lent signaling system used in bacteria. of extremely lethal toxins. Genomic data on these anaero- We have developed a sequence-based method, inde- bic bacteria indicated that the sporulation phosphorelay pendent of structural considerations, for identifying spec- pathway of aerobic Bacillus species is partially absent in ificity-determining interactions between proteins for a species-specific manner in clostridia. Some species, for which genomic data indicate a large number of func- example, Clostridium tetani, have the spo0B gene but no tionally coupled pairs. This method was applied to the spo0F gene, whereas others, including Clostridium dif- phosphotransfer domains of 2-component signaling pro- ficile and Clostridium botulinum, lack both genes. In teins, primarily in the OmpR/EnvZ family. Using the studies of C botulinum, we identified a sensor kinase method, we identified a network of residue-residue inter- for sporulation that phosphorylates Spo0A directly. The actions and generated a 3-dimensional structure con- Spo0B protein of C tetani is fully functional in B subtilis, sistent with the exemplary cocrystal structure obtained but the pathway to the phosphorylation of this protein in for the Spo0B-Spo0F complex and mutation studies of C tetani is not understood. The potential for therapeutic this pair of proteins. We also identified an interaction applications based on these studies is excellent, because network that links long-distance interactions with pair the regulation of sporulation also is involved in the specificity of 2-component signaling proteins. expression of genes that encode clostridial toxins and The method provides a simple scoring procedure perhaps genes for other virulence factors. that can be used to identify potential cross-phosphorylation between functional pairs and to assign orphan 2-component signaling proteins with no known mate to their signaling partners. Applied to the interconnected triad of 2-component signaling protein pairs in Bacillus subtilis, the procedure produced results consistent with recent discoveries of phosphotransfer cross talk, indicating that higher order signaling networks can be elu- 254 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE cidated. Although currently we are applying the method kinases and aspartyl phosphate phosphatases. Phos- to 2-component signal transduction systems for which phatases belong to 2 families: Rap and Spo0E. structural and mutational data allow proof of principle, Rap proteins are characterized by a structural orga- the method may generate interaction structures for less nization in tetratricopeptide repeats. Tetratricopeptide characterized protein pairs if sufficient functional pairs repeat domains are thought to be ancient modules that exist in genomic data. promote protein-protein interactions. The Rap proteins act as negative regulators of the initiation of sporulation by dephosphorylating the Spo0F response regulator inter- Molecular Dynamics of mediate of the phosphorelay. The activity of Rap proteins is generally inhibited by specific Phr pentapeptides Response Regulators encoded within precursor proteins that follow an export- import processing pathway that generates the active J.A. Hoch, J. Cavanagh* inhibitor. The Spo0E proteins act as negative regula- * North Carolina State University, Raleigh, North Carolina tors of the phosphorelay by dephosphorylating the n the basis of studies of the backbone dynamics Spo0A response regulator and master transcription fac- of the response regulator Spo0F, we proposed tor for the initiation of sporulation. O a model in which communication of informa- We have identified the Rap and Spo0E proteins that tion through the core of the Spo0F protein, between control the phosphorelay in Bacillus subtilis and Bacillus buried and surface-bound residues, is responsible for anthracis. Bacillus anthracis has 6 genes that encode the dissociation of sensor kinases from response regu- Rap proteins; each of these genes is followed by a gene lators after phosphorylation. We defined a region on that encodes a Phr pentapeptide. Five Rap-Phr genes Spo0F that moves in a dynamically concerted fashion, are chromosomally located; the sixth gene is present on driven by the motion of the imidazole ring of histidine the pXO1 plasmid, which also carries the genes that at position 101. encode toxin protein components of B anthracis. The imidazole ring moves in response to a conforma- Through genetic and biochemical analysis, we deter- tional change in the aspartic acid binding pocket upon mined that 1 chromosomally encoded system and the phosphorylation. Movement of the ring disrupts packing plasmid-encoded Rap-Phr system regulate initiation of interactions, a condition that alters the topology of the sporulation in B anthracis and thus may influence kinase recognition site, thereby causing the kinase to toxin production and virulence. We also identified the dissociate. Low concentrations of copper ions are potent products of 4 chromosomally located genes as mem- inhibitors of sporulation. Using nuclear magnetic reso- bers of the Spo0E family that dephosphorylate the nance and micro electrospray ionization–mass spectrom- Spo0A protein. We are now focusing on the molecular etry, we found multiple metal-bound species of Spo0F mechanism of interaction between Rap proteins and in the presence of copper. One of the copper-binding their specific Phr peptide inhibitors or their target sites was histidine at position 101, where bound copper Spo0F. We are also examining the surfaces used by alters the dynamics and conformation of the active site Spo0E and Spo0A to interact with each other. residues of Spo0F, making Spo0F nonfunctional. Signal Transduction in Negative Regulation of Enterococcus faecalis Development in Bacilli M. Perego, F. Del Papa M. Perego, C. Bongiorni, A. Diaz nterococci are commensal bacteria within the nitiation of sporulation in gram-positive bacilli is intestinal tract in mammals but also can cause regulated by a multicomponent signal transduction E disease in compromised hosts. The acquisition I system called a phosphorelay. Multiple positive and of resistance to multiple antibiotics by enterococci negative signals are integrated by the phosphorelay makes infections caused by these microorganisms through the opposing activities of histidine protein clinically challenging. The ability of the bacteria to MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 255 adapt and respond to different environmental stimuli, lution of molecular specificity in protein-protein inter- including the host environment, led us to investigate actions. In the past year, we switched our attention to the role of 2-component signal transduction in the proteins that regulate essential and important signal physiology and pathogenesis of Enterococcus faecalis. transduction systems. Using a bioinformatic approach, we identified 17 The YycFG 2-component signal transduction system 2-component systems consisting of a sensory histidine is essential for the growth of gram-positive bacteria, kinase and a cognate response regulator and an addi- including pathogenic staphylococci and streptococci. tional orphan response regulator. We inactivated each This system regulates and coordinates the synthesis of response regulator with the exception of the ortholog the constituents of cell walls and membranes with growth of the YycF essential protein of gram-positive organ- and division. The activity of this system is regulated by isms. We tested the effect of the deletions on a num- at least 2 proteins located on the outer surface of the ber of physiologic conditions and detected defects in cellular membrane, and one of these, YycH, was purified growth, antibiotic resistance, stress response, and for- and crystallized. The crystal structure of YycH was solved mation of biofilms. We are using these mutant strains by using 2-wavelength selenium anomalous dispersion to analyze the role of signal transduction in pathogen- data and was refined by using 2.3-Å data to an R-factor esis in vivo and to determine the extent of the regulon of 25.2%. The molecule consists of 3 domains with a controlled by each 2-component system. unique 3-dimensional structure (Fig. 1). Although a cal- Analysis of the 2-component system encoded by the gene fsr revealed that this system is the only one that affects growth of enterococci as a biofilm on solid surfaces. The role of the fsr system in biofilms is to activate transcription of the gene that encodes gelatinase, a zinc-metallo protease. Gelatinase is required for the formation of biofilms by E faecalis.We also found that full activation of gelatinase activity depends on a self- cleavage process at the C-terminal end of the gelatinase protein. Because growth of bacteria as biofilm is strongly associated with the development of human infections, such as infective endocarditis and urinary tract infections, our findings suggest that gelatinase may present a unique target for therapeutic intervention against biofilm-based enterococcal infection.

Regulatory Proteins: Structure, Molecular Recognition, and Phosphosignaling

K.I. Varughese, H. Szurmant, J.A. Hoch

n recent studies, we have focused on the crystal Fig. 1. YycH is a membrane-bound periplasmic protein that reg- structures of the phosphorelay proteins Spo0F and ulates the activity of YycG kinase, perhaps by interacting with the sensing domain of the kinase. The YycH molecule is made up of 3 Spo0B, Spo0A bound to its target DNA, and the com- I domains and has a novel 3-dimensional structure. plex consisting of Spo0F and Spo0B. The structure of the Spo0F-Spo0B complex showed for the first time cium-binding site was also discovered in this structure, how response regulator proteins bind to histidine phos- the orthologs in other bacterial species do not contain photransfer domains of sensor kinases and was the this motif. This structure has been extremely useful in key initial finding that opened up the study of the evo- studies to determine the function of YycH. 256 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

The regulation of toxin production by Bacillus DIVISION OF EXPERIMENTAL anthracis is critical for the ability of this organism to invade and grow in the body. The regulatory circuits HEMOSTASIS AND THROMBOSIS for the expression of the genes for anthrax toxin and the secretion of the toxin are complex, but a key protein Zaverio M. Ruggeri, M.D., Division Head is the product of the gene pagR. We were able to express and crystallize the 99 amino acid PagR transcription factor encoded by this gene. Crystals of the selenome- Regulation of Allogeneic thionine protein diffracted to 1.75 Å, and the structure was determined by using the multiple anomalous Immune Responses to Cell diffraction technique. We found that the PagR protein consists of 5 Transplants α-helices and a 2-stranded β-sheet. Two monomers form an elongated dimer. This structure is highly simi- L. Crisa, R. Prinsen, V. Cirulli,* B.E. Torbett lar to that of the metalloregulatory proteins of the * Whittier Institute, La Jolla, California Smt/ArsR family. However, PagR lacks the critical resi- lass I and class II MHC antigens are the primary dues required for metal binding, making it unlikely that barrier to acceptance of allografts. However, cer- metals are involved in its regulatory properties. The C tain class I MHC antigens may also trigger regu- structure provides a platform for mutagenesis experi- latory immune responses. Thus, in humans, HLA-G, a ments to uncover the role of this protein in the com- nonpolymorphic class Ib HLA molecule, may mediate plex regulation of anthrax toxin. immunologic tolerance at sites of immune privilege, such as the anterior chamber of the eye, the testis, the thy- PUBLICATIONS Bongiorni, C., Stoessel, R., Shoemaker, D., Perego, M. Rap phosphatase of virulence mus, and the cytotrophoblast. plasmid pXO1 inhibits Bacillus anthracis sporulation. J. Bacteriol. 188:487, 2006. Several explanations for the immunoregulatory

Brunsing, R.L., La Clair, C., Tang, S., Chiang, C., Hancock, L.E., Perego, M., functions of HLA-G have been considered. The lim- Hoch, J.A. Characterization of sporulation histidine kinases of Bacillus anthracis. ited polymorphism of HLA-G in humans may allow J. Bacteriol. 187:6972, 2005. the recognition of tissues expressing high levels of this Kojetin, D.J., Thompson, R.J., Benson, L.M., Naylor, S., Waterman, J., Davies, molecule as “self,” thereby preventing the activation K.G., Opperman, C.H., Stephenson, K., Hoch, J.A., Cavanagh, J. Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the of autoreactive or alloreactive T cells and natural killer possibility for in vitro metalloregulation in the initiation of sporulation. Biometals cells. Alternatively, HLA-G may foster the development 18:449, 2005. of specific immunoregulatory lymphocytes capable of Low, L.Y., Yang, C., Perego, M., Osterman, A., Liddington, R.C. Structure and lytic activity of a Bacillus anthracis prophage endolysin. J. Biol. Chem. downregulating alloreactivity. Our previous finding that 280:35433, 2005. HLA-G is expressed in the thymic medullary epithelium

Musumeci, L., Bongiorni, C., Tautz, L., Edwards, R.A., Osterman, A., Perego, M., in humans strongly supports both possibilities. Thus, Mustelin, T., Bottini, N. Low-molecular-weight protein tyrosine phosphatases of the purpose of HLA-G expression in the thymic medulla Bacillus subtilis. J. Bacteriol. 187:4945, 2005. may be to both educate developing T cells to recognize Szurmant, H., Nelson, K., Kim, E.J., Perego, M., Hoch, J.A. YycH regulates the HLA-G as self and induce the selection of HLA-G–spe- activity of the essential YycFG two-component system in Bacillus subtilis. J. Bac- teriol. 187:5419, 2005. cific immunoregulatory T-cell populations. We are investigating the immune responses elicited Szurmant, H., Zhao, H., Mohan, M.A., Hoch, J.A., Varughese, K.I. The crystal structure of YycH involved in the regulation of the essential YycFG two-component system by HLA-G in human thymocytes and peripheral T cells. in Bacillus subtilis reveals a novel tertiary structure. Protein Sci. 15:929, 2006. Our goals are to dissect the molecular mechanisms of White, A.K., Hoch, J.A., Grynberg, M., Godzik, A., Perego, M. Sensor domains HLA-G immune functions and then use this information encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase. J. Bacteriol., in press. to bioengineer HLA-G expression in tissues suitable for transplantation. Particular emphasis is given to models Worner, K., Szurmant, H., Chiang, C., Hoch, J.A. Phosphorylation and functional analysis of the sporulation initiation factor Spo0A from Clostridium botulinum. of pancreatic islet transplantation for the treatment of Mol. Microbiol. 59:1000, 2006. diabetes. For this purpose, we have generated lines of human pancreatic cells that express either low or high levels of membrane-bound or soluble recombinant HLA- G. These HLA-Glow and HLA-Ghigh cell lines are useful MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 257 tools for studies of HLA-G functions both in vitro and Stroke is a vascular disorder that causes neuronal injury. in vivo in models of cell transplantation. The social impact of the neurologic and behavioral con- Another promising line of research for the bioengi- sequences of this injury is enormous. We hypothesized neering of cells for transplantation was provided by our that alterations in the intercellular matrix of cerebral work on the identification of endothelial cell progeni- microvessels and their cellular adhesion receptors by tors in human cord blood. While studying human thy- active proteases are reflected by injury to neighboring mopoiesis in a chimeric mice model in which mice are neurons and the extracellular matrix of the neurons. The reconstituted with human cord blood, we discovered results of our studies have supported the concept of that cord blood hematopoietic stem cells engrafted in the neurovascular unit. these mice not only reconstituted the bone marrow and We have extended our studies to 3 related areas: repopulated the human thymic grafts but also contrib- (1) the distribution of expression of adhesion receptors uted to the formation of new blood vessels at sites of in the matrix of the neurovascular unit, (2) the impact wound healing. of hypoxia with glucose deprivation on cellular compo- We are characterizing this population of putative nents of the microvascular permeability barrier, and endothelial progenitors to be used as another target (3) the responses of intrinsic CNS inflammatory cells cell type for transplantation. Specifically, we have defined (microglia and oligodendroglia) to focal ischemia and some of the growth and differentiation signals required other types of injury and the impact of the responses for the expansion ex vivo of human bone marrow–derived on the neurovascular unit. endothelial progenitors. Currently, using a mouse model Our results have provided cell models that clearly of bone marrow–derived vasculogenesis, we are char- indicate the relationship between the neurovascular acterizing the immunologic and angiogenic properties of unit and ischemic stroke. The cellular models help us bone marrow–derived endothelium. Ultimately, cotrans- understand how the microvasculature and the related planting HLA-G–transduced allogeneic tissue along with neurons within the neurovascular unit interact. HLA-G–bioengineered endothelial cell progenitors and/or The integrity of microvessels depends on adhesion enhancing recruitment of bone marrow–derived endo- of endothelial cells and astrocytes to the basal lamina. thelium with intrinsic immunomodulatory properties We have shown that in addition to β1 integrins, αβ-dys- may endow tissue grafts with an additional level of troglycan is expressed predominantly on the luminal side immunoprotection. This approach may be useful in of astrocyte end-feet in cerebral microvessels. In vivo, developing novel strategies for the induction of immu- dystroglycan is found on the entire microvasculature. nologic tolerance and/or the avoidance of rejection Both integrin α6β4 and dystroglycan appear through- after transplantation. out the microvasculature of the striatal gray matter,

but only α6β4 is found on large penetrating vessels of PUBLICATIONS Cirulli, V., Zalatan, J., McMaster, M., Prinsen, R., Salomon, D.R., Ricordi, C., the cortical gray matter. Dystroglycan is a functional Torbett, B.E., Meda, P., Crisa, L. The class I HLA repertoire of pancreatic islets laminin receptor for astrocytes in vitro. The reasons comprises the nonclassical class Ib antigen HLA-G. Diabetes 55:1214, 2006. for the significantly different distributions of these 2 receptors are being studied. The effects of focal ischemia on brain microvessels Cerebral Microvessel-Neuron can be studied in vitro by depriving endothelial cells Responses to Ischemia and astrocytes of oxygen and glucose. The expression of β1 integrins on endothelial cells is relatively resis- G.J. del Zoppo, R. Milner, J. Hallenbeck,* E. Lo** tant to oxygen and glucose deprivation and actually * National Institutes of Health, Bethesda, Maryland increases in response to this treatment. But dystrogly- ** Massachusetts General Hospital, Boston, Massachusetts can expression decreases significantly after oxygen and glucose deprivation. These changes recapitulate the nderstanding the interactions between neurons loss of dystroglycan expression within the brain micro- and their supply microvessels can provide insight vasculature that occurs in focal ischemia. The disap- U into communication and control of neuronal pearance of dystroglycan from astrocytes during oxygen activation and the coordinate responses of neurons and and glucose deprivation can be blocked by some pro- microvessels to local injury, as during ischemic stroke. tease inhibitors. 258 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

A common early event in many brain injuries is the grin adhesion receptors in a constitutively activated, breakdown of the blood-brain barrier, a situation that high-affinity format. Activation-inducing mutant inte- leads to deposition of the serum proteins fibronectin grin subunits endowed human breast cancer cells with and vitronectin. We have shown that fibronectin and a highly aggressive, metastatic phenotype when tested vitronectin stimulate microglial activation and expres- in immunodeficient mice, establishing a cause-and-effect sion of the proteolytic enzymes matrix metallopro- relationship between integrin activation and metastatic teinase 9 and matrix metalloproteinase 12 and that activity in breast cancer. A clinical relevance of this this activation is mediated via integrins α5β1 and finding was evident from a screening of malignant tumor αvβ5, respectively. cells that we isolated from blood samples and malig- Other experiments have revealed that interference nant effusions of patients with advanced breast cancer. with cellular adhesion in the neurovascular unit signif- Functional analyses of tumor cell migration and the icantly disrupts tight junctions within the blood-brain ability of the cells to interact with components of the barrier. These studies suggest several novel therapeu- vessel wall under blood flow conditions such as those tic avenues to reduce the pathologic changes that that occur in the venous circulation revealed that inte- occur after ischemic injury in the brain. grins are expressed in a constitutively activated form exclusively in metastatic and invasive cells. Analyses PUBLICATIONS Adams, H., Adams, R., del Zoppo, G., Goldstein, L.B., Stroke Council of the of the development of metastasis in the animal model American Heart Association, American Stroke Association. Guidelines for the early revealed that activated integrin αvβ3 promotes tumor management of patients with ischemic stroke: 2005 guidelines update a scientific statement from the Stroke Council of the American Heart Association/American cell survival in the absence of an adhesive matrix and Stroke Association [published corrections appear in Stroke 36:1352 and 1626, supports the initial steps of tumor cell colonization of 2005]. Stroke 36:916, 2005. target organs distant from the bloodstream. del Zoppo, G.J. Stroke and neurovascular protection. N. Engl. J. Med. 354:553, INHIBITION OF BREAST CANCER METASTASIS BY 2006. ANTIBODIES FROM CANCER PA TIENTS Mabuchi, T., Lucero, J., Koziol, J.A., del Zoppo, G.J. Focal cerebral ischemia pref- After we discovered that the activated conformer erentially affects neurons distant from their neighboring microvessels. J. Cereb. Blood Flow Metab. 25:257, 2005. of integrin αvβ3 was a functional target on metastatic

NINDS ICH Workshop Participants. Priorities for clinical research in intracerebral breast cancer cells, we used our breast cancer cell hemorrhage: report from a National Institute of Neurological Disorders and Stroke model to isolate human antibodies directed against workshop. Stroke 36:e23, 2005. this target. We isolated 2 antibodies that specifically

recognize the activated conformer of integrin αvβ3. These antibodies mimic natural ligands of the receptor; Harnessing Host Defense and they express an arginine–glycine–aspartic acid integrin Regeneration to Target Breast recognition motif in the third complementarity-determining region of the heavy chain. The antibodies can Cancer Metastasis be used to detect metastatic human tumor cells and inhibit critical tumor cell functions that depend on B.F. Felding-Habermann, J.S. Krueger, D. O’Sullivan, activation of integrin αvβ3. Importantly, treatment of W. Hassenpflug, J.S. Forsyth, B.M. Maruszak, E.I. Chen, experimental mice with the antibodies prevented for- J.R.Yates, K.D. Janda, R.A. Lerner, J.F. Kroener,* mation of metastases and reduced existing metastatic E.Y. Snyder** tumor burden (Fig. 1). Our current goal is to optimize * Scripps Clinic, La Jolla, California these antibodies and, by using chemical modification, ** Burnham Institute, La Jolla, California harness them for treatment of metastatic breast cancer. FUNCTIONAL TARGETS IN BREAST CANCER HARNESSING NEURAL STEM CELLS FOR THERAPY METASTASIS OF BREAST CANCER BRAIN METASTASES ur goals are to understand mechanisms of tumor Improved diagnosis and treatment prolong the lives metastasis and to develop novel approaches for of patients with breast cancer, but brain metastases O early detection and therapeutic inhibition of eventually develop in nearly 30% of patients who have metastatic disease. Our studies on adhesive, migratory, advanced disease. No current therapy can prevent or and invasive properties of human tumor cells revealed effectively eliminate brain metastases. Therefore, we that a metastatic subset of cancer cells expresses inte- seek to develop a novel treatment approach that is MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 259

to reach metastatic brain lesions and locally convert a harmless prodrug to highly toxic 5-fluorouracil at the site of tumor growth. With this interdisciplinary team effort, we hope to provide a new opportunity for treatment of brain metastases in patients who have breast cancer.

PUBLICATIONS Chen, E.I., Hewel, J., Felding-Habermann, B., Yates, J.R. III. Large scale protein profiling by combination of protein fractionation and multidimensional protein iden- Fig. 1. Treatment with human antibodies against activated integrin tification technology (MudPIT). Mol. Cell. Proteomics 5:53, 2006.

αvβ3 reduces the growth rate of lung metastases in advanced breast Lillo, A.M., Kim, Y., Liu, Y., Ballatore, C., Anichini, A., Mortarini, R., Zhou, B., cancer. Human breast cancer cells isolated from a blood sample from Felding-Habermann, B., Janda, K.D. Targeting heat-shock proteins on cancer cells: a patient with breast cancer were labeled with a bioluminescent selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry, in press. marker and then injected into mice. The growth rate of lung metastases (tumor burden) in the animals was monotired by using weekly noninvasive bioluminescence imaging. On day 56, treatment groups were selected so that each group contained mice with light, interme- The Antithrombotic, diate, or heavy tumor burdens. The groups were treated with daily injections of wild-type (WT) phage, phage displaying antibody 1 (Ab1), Anti-inflammatory, and or phage displaying antibody 5 (Ab5). The growth rate of the tumor burden was determined for each animal at the beginning and the end Antiapoptotic Protein C Pathway of treatment. The graph shows data points for changes in metastatic lung signal reflecting the growth rate of the tumor burden in each J.H. Griffin, B.N. Bouma, M. Chopp,* H. Deguchi, D.J. Elias, animal; a horizontal line indicates the median measurement. Overall, S. Eichinger,** F. Espana,*** J.A. Fernández, P. Kyrle,** the 3 groups differed significantly (F = 5.50, P = .04). Pairwise S. Li, Y.M. Lee, L. Mosnier, S. Navarro, N. Pecheniuk, X. Xu, comparison via a multiple comparison procedure indicated that the X. Yang, S. Yegneswaran, B.V. Zlokovic**** Ab1 group differed significantly from the WT group (.05 α level). * Henry Ford Hospital, Detroit Michigan based on the body’s own mechanisms for healing and ** Universität Wien, Vienna, Austria regeneration. We have established unique, trackable *** Universitat de València, València, Spain human breast cancer cell models in which noninvasive **** University of Rochester, Rochester, New York bioluminescence imaging is used to detect cancer spread- arious host defense systems act in concert in nor- ing, onset and development of brain metastases, and mal physiology. Coagulation pathways, fibrinoly- response to treatment (Fig. 2). V sis pathways, and anticoagulant mechanisms prevent bleeding while avoiding harmful blood clots. The protein C pathway provides antithrombotic, anti- inflammatory, and antiapoptotic activities and is a focus of our research. ANTIAPOPTOTIC AND CYTOPROTECTIVE EFFECTS OF ACTIVATED PROTEIN C Fig. 2. Cell and animal model for brain metastasis of human breast cancer. Noninvasive bioluminescence imaging is used to The antiapoptotic activity of activated protein C monitor widespread metastatic colonization of breast cancer target (APC), first described in 2001, may provide cytopro- organs in immunodeficient mice injected with human breast cancer tective activity that reduces cell death after a variety cells labeled with a bioluminescent marker. Breast cancer cells iso- of cellular injuries. Recombinant APC, a well-defined lated from a brain lesion home to various regions of the brain (A) anticoagulant enzyme, reduced mortality in patients and maintain expression of epithelial antigens (B). with severe sepsis in a phase 3 clinical trial. However, In collaboration with E.Y. Snyder of the Burnham 2 potent anticoagulants, antithrombin III and recombi- Institute, La Jolla, California, a pioneer and expert in the nant tissue factor pathway inhibitor, did not, suggesting biology of neural stem cells, we are testing the hypothe- the physiologic relevance of APC’s less well-defined sis that brain metastases can be inhibited with neural anti-inflammatory and antiapoptotic activities. Therapy stem cells because the stem cells have a proven propen- with recombinant APC is associated with an increased sity to seek out diseased areas in the brain. We are risk for serious bleeding complications because of the using neural stem cells armed with cytosine deaminase anticoagulant activity of the enzyme. 260 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

To generate recombinant APC variants that have strates can be assembled to express their activities. reduced anticoagulant activity and thus are less likely Although dyslipoproteinemia is associated with arterial to cause bleeding, we dissected APC’s anticoagulant atherothrombosis, little is known about plasma lipopro- activity from its cytoprotective activity by using site- teins in patients with venous thrombosis. We used directed mutagenesis. Using staurosporine-induced nuclear magnetic resonance spectroscopy and antigenic endothelial cell apoptosis assays, we showed that muta- levels of apolipoproteins AI and B to determine the tions to alanine in 2 APC surface loops that severely concentrations of subclasses of lipoproteins in blood reduced anticoagulant activity resulted in two APC samples from 49 men less than 45 years old who had variants that retained normal antiapoptotic activity. venous thrombosis and from matched control subjects. Like wild-type APC, these 2 mutants require protease Patients with venous thrombosis had significantly lower activated receptor-1 and endothelial cell protein C levels of high-density lipoprotein (HDL) particles, large receptor for cytoprotective activity. HDL particles, HDL-cholesterol, and apolipoprotein AI Thus, it is possible to reduce anticoagulant activity and significantly higher levels of low-density lipopro- while preserving antiapoptotic activity of recombinant tein (LDL) particles and small LDL particles. The quar- APC variants. We are using animal models of injury to tile-based odds ratios for decreased levels of HDL determine if therapy with such APC variants can reduce particles and apolipoprotein AI were 6.5 for patients serious risks for bleeding while providing the benefi- and 6.0 for control subjects. cial effects of APC acting directly on cells. In collaboration with S. Eichinger and P. Kyrle, Uni- NEUROPROTECTIVE ACTIVITIES OF ACTIVATED versität Wien, we did similar new studies to determine PROTEIN C if dyslipoproteinemia exists in patients who have recur- Stroke is a major cause of morbidity and mortality. rent venous thrombosis. The results provided strong evi- In collaboration with B. Zlokovic and colleagues, Uni- dence supporting the hypothesis that HDL, notably large versity of Rochester, we used human brain endothe- HDL particles, protects against recurrence of venous lium in vitro and murine in vivo stroke models to study thrombosis. These clinical findings plus the results of the neuroprotective activities of the protein C pathway. other in vitro experiments support the emerging concept Previously we showed that intravenous infusions of that procoagulant and anticoagulant lipids and lipopro- recombinant APC reduced the size of brain infarctions teins may contribute to a yin-yang balance that influ- and brain edema induced by ischemia. Although throm- ences the upregulation and downregulation of thrombin bolytic effects of tissue plasminogen activator (tPA) generation and that alterations of this lipoprotein balance are beneficial, its neurotoxic effects are a problem. We may alter the hemostatic balance in patients who have found that APC reduces the neurotoxic effects of tPA life-threatening thrombotic events. and blunts tPA-induced apoptosis of both endothelial cells and neurons. Remarkably, new studies in murine and rat models Antithrombotic Mechanisms of ischemic stroke indicate that recombinant murine

APC reduces bleeding induced by the thrombolytic agent M.J. Heeb, B.N. Bouma, K.M.S. Cabral, M.O. Hall,* L. Tonnu tPA. APC stabilizes the blood-brain barrier against bleed- * University of California, Los Angeles, California ing because it blunts the tPA-induced increases in mRNA and protein levels of matrix metalloprotease-9, which e study plasma proteins that regulate blood causes breakdown of the blood-brain barrier. coagulation and can prevent thrombosis, a Thus, we think that APC merits clinical trials as W factor in half of all deaths in the United a neuroprotective agent in patients with ischemic States. Knowledge of the mechanisms of action of stroke. Furthermore, we speculate that APC may add these anticoagulant proteins may lead to improved substantially to the effectiveness of tPA therapy for antithrombotic therapies and preventive measures. stroke in humans. PROTEIN S INFLUENCE OF LIPIDS ON BLOOD COAGULATION Protein S, a vitamin K–dependent protein, is a cofac- Lipid-containing surfaces, including cell membranes tor for activated protein C during inactivation of proco- and lipoproteins, provide sites where both procoagu- agulant factors Va and VIIIa. We showed that protein lant and anticoagulant enzymes, cofactors, and sub- S also exerts direct anticoagulant activity by inhibiting MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 261 factors Va, VIIIa, and Xa and by competing with proco- ble phospholipids or phospholipid vesicles. Formation agulant factors for limiting phospholipids. of complexes of Xa and ZPI was promoted by either Because the finding that protein S has direct anti- phospholipid species. Thus, phospholipids most likely coagulant effects was questioned, we determined the are required to promote a necessary conformation of molecular forms of protein S in plasma and the activity ZPI or factor Xa, and not to colocalize ZPI with factor of the forms. Multimers of protein S in plasma were Xa on a surface. Using surface plasmon resonance bind- detected by using several methods, including an enzyme- ing techniques and activity measurements, we found linked immunosorbent assay that could in principle be that calcium ions, but not protein Z or phospholipids, used to detect multimers of any protein. We developed were required for ZPI inhibition of factor IXa and for a novel assay for detecting direct anticoagulant activity association of factor IXa with ZPI. of protein S and showed that plasma protein S had Although factor Va, the cofactor for factor Xa, pro- strong activity and that monomers, multimers, and tected factor Xa from inhibition by ZPI or protein Z, the complexes consisting of protein S and C4b-binding same was not true for factor VIIIa, the cofactor for factor protein all had similar direct anticoagulant activity that IXa. Rather, low concentrations of factor VIIIa promoted matched the activity of affinity-purified protein S mono- ZPI binding to and inhibition of factor IXa. Surface plas- mers and multimers. mon resonance studies indicted that ZPI binds to fac- Protein S purified by methods used by most other tor VIIIa with nanomolar affinity. Possibly, factor VIIIa laboratories had weak direct anticoagulant activity that replaces the function of protein Z during ZPI inhibition did not match the activity of plasma protein S, explain- of factor IXa. ing a conflicting report in the literature. That report We had reported that low levels of protein Z were maintained that all plasma protein S is monomeric but associated with the risk for ischemic stroke in men but that only artifactual multimers of protein S, not mono- not in women. Surprisingly, further analysis of data for mers, have good direct anticoagulant effects. We are women revealed that low levels of the protein were investigating why some purification methods lead to associated with the risk for stroke in the younger half low direct inhibition by protein S; the findings should of women subjects (24–57 years old) but not in the reveal structural features of protein S that are impor- older half.We will determine whether lipid profiles that tant in its direct anticoagulant effects. change with estrogen levels can explain this finding. Protein S inhibition of prothrombinase (factor Xa–factor Va) in plasma or in a purified system did not depend on tissue factor pathway inhibitor. We are Structure and Function of examining whether other modes of protein S activity depend on this inhibitor, as recently reported. In col- Coagulation Cofactors laborative studies, protein S served as a ligand for receptor tyrosine kinase Mer, promoting necessary A.J. Gale, T. Cramer, J. Riceberg, D. Rozenshteyn, phagocytosis of shed outer rod segments of rat retinal J.-L. Pellequer* pigment epithelium. We are continuing structure-function *CEA/DSV/DIEP, Bagnols ser Cèze, France studies of protein S and studies of protein S in animal oagulation factors Va and VIIIa are highly homol- models of thrombosis. ogous cofactors of the serine proteases factor Xa PROTEIN Z–DEPENDENT PROTEASE INHIBITOR C and factor IXa, respectively. These cofactors are The unusual serpin protein Z–dependent protease the primary targets of activated protein C (APC) in its inhibitor (ZPI) requires protein Z, negatively charged downregulation of the procoagulant pathway. In collabo- phospholipids, and calcium ions to inhibit procoagu- ration with J.-L. Pellequer in France, we used homol- lant factor Xa. We found that ZPI also inhibited factor ogy modeling techniques to model the 3-dimensional IXa, a close homolog of factor Xa, independently of structures of these multidomain proteins. We are using protein Z. We sought to understand the requirement these models and other structures to develop models of for phospholipids for inhibition of factor Xa and to learn various coagulation complexes. We use the models as the requirements for inhibition of factor IXa by ZPI and guides to create mutants of these cofactors and acti- protein Z. ZPI and protein Z inhibited factor Xa with vated protein C in order to investigate mechanisms of equal efficiency in the presence of either small, solu- cofactor function and downregulation. For example, we 262 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE engineered disulfide bonds between domains in both Gruber, A., Fernández, J.A., Bush, L., Marzec, U., Griffin, J.H., Hanson, S.R., DeCera, E. Limited generation of activated protein C during infusion of the protein factor Va and factor VIIIa. In factor Va, the disulfide C activator thrombin analog W215A/E217A in primates. J. Thromb. Haemost. bond facilitated investigation of the mechanisms of 4:392, 2006. inactivation of factor Va by APC cleavage. Hall, M.O., Obin, M.S., Heeb, M.J., Burgess, B.L., Abrams, T.A. Both protein S and Gas6 stimulate outer segment phagocytosis by cultured rat retinal pigment epi- Factor VIIIa however, is inactivated by 2 mecha- thelial cells. Exp. Eye Res. 81:581, 2005. nisms. Thrombin activation of factor VIII results in a Heeb, M.J., Schuck, P., Xu, X. Protein S multimers and monomers each have heterotrimer that consists of the A1 subunit, the A2 direct anticoagulant activity. J. Thromb. Haemost. 4:385, 2006. subunit, and the light chain. Both spontaneous disso- Ilmakunnas, M., Pesonen, E.J., Hockerstedt, K., Makisalo, H., Fernández, J.A., ciation of the A2 subunit and proteolytic cleavage of Griffin, J.G., Repo, H., Siitonen, S., Petaja, J. Graft protein C entrapment is associated with reduced phagocyte activation during reperfusion in human liver trans- factor VIIIa by APC inactivate factor VIIIa. Hemophilia plantation. Crit. Care Med. 34:426, 2006. A, a deficiency of factor VIII, is treated by infusions of Lindstrom, O., Kylanpaa, L., Mentula, P., Puolakkainen, P., Kemppainen, E., purified recombinant factor VIII. But the usefulness of Haapiainen, R., Fernández, J.A., Griffin, J.H., Repo, H., Petaja, J. Upregulated factor VIII is limited because it is unstable after acti- but insufficient generation of activated protein C is associated with development of multiorgan failure in severe acute pancreatitis. Crit. Care 10:R16, 2006. vation by thrombin as a result of the spontaneous dis- Mosnier, L.O., Griffin, J.H. Protein C anticoagulant activity in relation to anti- sociation of the A2 subunit. inflammatory and anti-apoptotic activities. Front. Biosci. 11:2381, 2006. We generated 2 mutants of factor VIII in which 2 Seligsohn, U., Griffin, J.H. Hereditary thrombophilia. In: William’s Hematology, newly introduced cysteine residues form a de novo disul- 7th ed. Beutler, E., et al. (Eds.). McGraw-Hill, New York, 2005, p. 1981. fide bridge that cross-links the A2 and A3 domains. Tanaka, K.A., Fernández, J.A., Marzec, U.M., Kelly, A.B., Mohri, M., Griffin, J.H., These interdomain disulfides prevent the spontaneous Hanson, S.R., Gruber, A. Soluble thrombomodulin is antithrombotic in the pres- dissociation of the A2 subunit. We are using the mutants ence of neutralising antibodies to protein C and reduces circulating activated protein C levels in primates. Br. J. Haemotol. 132:107, 2006. as tools to investigate mechanisms of factor VIIIa inacti- Turunen, A.J., Fernández, J.A., Lindgren, L., Salmela, K.T., Kyllonen, L.E., Mak- vation alone and in combination with mutants of APC isalo, H., Griffin, J.H., Siitonen, S.M., Petaja, J., Pesonen, E.J. Activated protein cleavage sites. Additionally, the mutants may provide a C reduces graft neutrophil activation in clinical renal transplantation. Am. J. Trans- plant. 5:2204, 2005. new, improved therapy for hemophilia A. Therefore, we are investigating the function of these stabilized variants von dem Borne, P.A.K., Cox, L.M., Bouma, B.N. Factor XI enhances fibrin generation and inhibits fibrinolysis in a coagulation model initiated by surface-coated tis- in mice to determine if the variants have improved func- sue factor. Blood Coagul. Fibrinolysis 17:251, 2006. tional properties in vivo. Zlokovic, B.V., Zhang, C., Liu, D., Fernández, J., Griffin, J.H., Chopp, M. Functional We are also characterizing the interactions of APC recovery after embolic stroke in rodents by activated protein C. Ann. Neurol. 58:474, 2005. with factor VIIIa by using mutants of residues on the surface of APC that may be involved in factor VIIIa binding. Conversely we will make mutants of factor VIIIa residues that may be involved in APC binding. Molecular Insights Into Analysis of these mutants will allow us to delineate the Platelet Adhesion binding interaction between APC and factor VIIIa. In other studies, we are investigating how the neutrophil T.J. Kunicki, Y. Cheli, L. Baronciani, M.T. Canciani, proteases cathepsin G and elastase modulate the func- F. Gianniello, S.T. Head, T.S. Mondala, D.R. Salomon, tions of factor VIII and factor VIIIa, and we are char- A.B. Federici, O. Inoue,* K. Suzuki-Inoue,* O.J. McCarty, acterizing the APC cofactor activity of factor V. M. Moroi,* Z.M. Ruggeri, Y. Ozaki,* S.P. Watson, K. Furihata *Yamanashi Medical University, Yamanashi, Japan PUBLICATIONS Bouma, B.N., van Mourik, J.A. Unraveling the mystery of von Willebrand factor. J. REGULATION BY HETEROGENEOUS NUCLEAR Thromb. Haemost. 4:489, 2006. RIBONUCLEOPROTEIN L OF DIFFERENCES IN THE

Bouma, B.N., von dem Borne, P.A.K., Meijers, J.C.M. Discovery of thrombin acti- EXPRESSION OF MOUSE INTEGRIN α 2 β 1 vatable fibrinolysis inhibitor. J. Thromb. Haemost. 4:257, 2006. n mice, heritable variations οf the integrin subunit Gale, A.F., Radtke, K.P., Cunningham, M.A., Chamberlain, D., Pellequer, J.-L., gene ITGA2 control cellular expression of the integrin Griffin, J.H. Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants. J. Thromb. Haemost. 4:1315, 2006. I α2β1 and in blood platelets, significantly influence Griffin, J.H. Control of coagulation reactions. In: William’s Hematology, 7th ed. adhesion to collagens in vitro. We found that a 2-fold Beutler, E., et al. (Eds.). McGraw-Hill, New York, 2005, p. 1695. difference in platelet levels of α2β1 between 11 inbred Griffin, J.H., Fernández, J.A., Mosnier, L.O., Liu, D., Cheng, T., Guo, H., Zlokovic, mouse strains is controlled by the length of a cytosine- B.V. The promise of protein C. Blood Cells Mol. Dis. 36:211, 2006. adenine (CA) dinucleotide repeat at a single site within MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 263 intron 1. A total of 7 strains with a 21 CA repeat stimulates spreading of human and mouse platelets sequence expressed twice the level of platelet α2β1 through a pathway that depends not only on α6β1 but as did 4 strains with a 6 CA repeat sequence and had also on GPVI. Studies with GP6–/– mouse platelets indian equivalent gain of platelet adhesive function. These cated that GPVI is not required for platelet adhesion to differences in expression and function coincided with laminin. However, formation of lamellipodia on laminin an increase in the affinity of heterogeneous nuclear is completely inhibited in the absence of GPVI, whereas ribonucleoprotein L for the 21 CA repeat sequence formation of filopodia remains normal. Direct binding relative to the 6 CA repeat sequence. and surface plasmon resonance spectroscopy confirmed Using cell-free in vitro mRNA splicing, we found that GPVI is a receptor for laminin. In our model, α6β1 that increased binding of the ribonucleoprotein results brings laminin in proximity to GPVI, which then binds in increased splicing efficiency and fidelity. Treatment to laminin and mediates formation of lamellipodia and with short interfering RNA specific for the ribonucleo- platelet spreading. protein abolished the splicing enhancer activity of the INFLUENCE OF N -LINKED GLYCOSYLATION ON THE 21 CA repeat sequence in vivo and attenuated the FUNCTION OF PLATELET GPVI The binding sites on GPVI for collagens and the expression of endogenous α2β1 by established murine cell lines. Our findings indicate that the increase in snake venom C-type lectin convulxin have not been determined precisely. We used recombinant human surface α2β1 on mouse platelets is a consequence of increased efficiency of ITGA2 pre-mRNA splicing through GPVI to evaluate the effect of N-linked glycosylation of a mechanism that is regulated by heterogeneous nuclear asparagine 92–glycine–serine 94 on binding to type I collagen, collagen-related peptide, and convulxin. Degly- ribonucleoprotein L and that depends on the length of cosylation of GPVI with peptide-N-glycosidase F (spe- the CA repeat sequences at this specific site in intron 1. cific for complex N-linked glycans) or tunicamycin but ASSOCIATION OF ITGA2 HAPLOTYPES AND BLEED- not endoglycosidase H (specific for N-linked glycans ING SEVERITY IN VON WILLEBRAND DISEASE rich in mannose) decreased the molecular weight of the Diagnosis of von Willebrand disease is difficult glycoprotein and attenuated cell binding to both colla- because of low heritability and the influence of modifier gen-related peptide and convulxin. Replacing asparagine genes. Using a qualitative trait locus method, we ana- or serine with alanine significantly decreased cell adhe- lyzed the association of bleeding severity with 8 can- sion to collagen-related peptide and to a lesser degree didate gene haplotypes within pedigrees of 11 index to type I collagen and convulxin, but neither amino acid cases of von Willebrand disease type 2: 2 type 2A, 3 change altered surface expression of GPVI. Our results type 2B, and 6 type 2M. These pedigrees included 47 clearly implicate N-linked glycosylation at asparagine affected and 49 unaffected relatives. As expected, the 92 as an important factor contributing to maximal GPVI bleeding score was most strongly influenced by the adhesion to type I collagen, collagen-related peptide, serum level of von Willebrand factor. After Bonferroni and convulxin, in that order. correction for multiple testing, only a single candidate gene haplotype was associated with a significant increase PUBLICATIONS Cheli, Y., Kunicki, T.J. hnRNP L regulates differences in expression of mouse inte- in bleeding severity: the ITGA2 promoter haplotype –52T. grin α2β1. Blood 107:4391, 2006.

Our findings support the hypothesis that genetic Inoue, O., Suzuki-Inoue, K., McCarty, O.J., Moroi, M., Ruggeri, Z.M., Kunicki, differences in the expression of the integrin subunit α T.J., Ozaki, Y., Watson, S.P. Laminin stimulates spreading of platelets through inte- 2 grin α β -dependent activation of GPVI. Blood 107:1405, 2006. influence the bleeding phenotype of von Willebrand 6 1 Kunicki, T.J. Platelet immunology. In: Hemostasis and Thrombosis: Basic Princi- disease. Thus, attenuation of the expression of recep- ples and Clinical Practice, 5th ed. Colman, R.W., Marder, V.J., Clowes, A.W., tors for collagen on platelets can influence risk for George, J.N., Goldhaber, S.Z. (Eds.). Lippincott Williams & Wilkins, Philadelphia, 2006, p. 461. morbidity in patients, such as those with von Wille- brand disease, who have compromised hemostasis. Kunicki, T.J., Baronciani, L., Canciani, M.T., Gianniello, F., Head, S.R., Mondala, T.S., Salomon, D.R., Federici, A.B. An association of candidate gene haplotypes LAMININ-STIMULATED SPREADING OF PLATELETS and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees. J. Thromb. Haemost. 4:137, 2006. THROUGH INTEGRIN α 6 β 1 -DEPENDENT ACTIVATION OF GLYCOPROTEIN VI Kunicki, T.J., Cheli, Y., Moroi, M., Furihata, K. The influence of N-linked glycosylation on the function of platelet glycoprotein VI. Blood 106:2744, 2005. Glycoprotein VI (GPVI) is an important platelet receptor for collagens. Laminin also supports platelet adhe- Kunicki, T.J., Nugent, D.J. Human platelet antigens. In: Blood Banking and Transfu- sion Medicine: Basic Principles and Practice, 2nd ed. Hillyer, C.D., Silberstein, L.E., sion through the integrin α6β1. We observed that laminin Ness, P.M., Anderson K.N., Robach, J.D. (Eds.). Saunders, Philadelphia, 2006 p. 63. 264 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE Functional Genomics in Organ ics based on single nucleotide polymorphisms to establish profiles to diagnose acute and chronic transplant Transplantation and Islet Cell rejection. These studies include patients with both kidney and liver transplants. A major objective of these efforts Xenotransplantation is to identify new pathways that drive the immune response and cell biology of organ transplants that D.R. Salomon, S.M. Kurian, S. Cherqui, Y. Martina, might be used as the next generation of targets for ther- K. Marcucci, D. Valente, H. Ospina, D. Campbell, C. Marsh, apy. For example, with all current drug therapies, the S. Head,* C. Lanigan,* J.R. Yates,** J. Hewel,** target is the patient’s immune response; none target the P.Y. Kwok,*** J. Warrington,**** S. Horvath,***** transplant itself, even though the function of the trans- † †† †† J. Papp,***** C.A. Wilson, R. Bartel, J. Gavin plant is the ultimate determinant of success or failure. * DNA Microarray Core, Scripps Research We would like to test the hypothesis that gene ** Department of Cell Biology, Scripps Research expression profiles can be used to create a metric or *** University of California, San Francisco, California simple diagnostic test for adequate immunosuppression. **** Affymetrix, Santa Clara, California Physicians could then adjust a patient’s drugs on the ***** University of California, Los Angeles, California basis of an objective measure. Our long-term goal is to † Food and Drug Administration, Bethesda, Maryland identify genes, proteins, and genetic polymorphisms †† MicroIslets, La Jolla, California that determine the outcome of a transplant to create a uccessful transplantation requires the orchestra- systems biology–based understanding of clinical trans- tion of complex mechanisms set in motion by plantation at the molecular level. surgical implantation of cells or organs into a S PIG ISLET XENOTRANSPLANTATION AND THE RISK patient. Regulation of the immune response with FOR INFECTIOUS DISEASE immunosuppressive drugs has received the most atten- We are using novel technology to (1) create a pro- tion. But equally important is the unique cell biology tective alginate capsule around pig islets to prevent of the transplanted tissue that evolves under stress rejection and (2) modify the capsule with therapeutic after transplantation and ultimately determines the molecules to enhance islet survival and function after function of the transplant. transplantation. Pig insulin works well in humans with One challenge, called functional genomics, is to diabetes, and pigs can be genetically engineered and understand the expression and function of genes and can be available in great numbers. We are also using proteins after transplantation. How do immunosuppressive drugs work at this fundamental level? What is the our genomics tools to study how endothelial progenitors, difference between a successful and an unsuccessful the progenitor cells for blood vessels, can be included transplant? Another challenge is to develop an unlimited to further advance the success of cell transplantation, supply of healthy tissue for transplantation, for exam- a proof of concept for composite tissue engineering. ple, pancreatic islet cells to cure diabetes. Animals could Although xenotransplantation is a logical strategy be used as donors, called xenotransplantation, although to address current shortages of human donor organs, the potential risks for infectious disease inherent in using a critical concern is the potential of moving infections animal donors need to be better understood so that from the animals to humans. We established a new this method can be used safely. One strategy would be mouse model for pig islet xenotransplantation, showed to create technologies to protect cell transplants from that multiple tissues become infected with pig endoge- rejection and optimize the function of the cells. Deliv- nous retrovirus, identified the human receptors for this ering therapeutic molecules to the transplanted tissue retrovirus, identified functional defects in nonhuman could enhance the success of engraftment and function. primate cells for viral entry and assembly, and contin- Finally, progenitor cells could be used to enhance the ued to refine our understanding of the viral biology and formation of new blood vessels, called angiogenesis. potential risks. We think these studies are a necessary Revascularization of cell transplants is a critical step complement to our work in safely advancing clinical in successful engraftment and function. islet xenotransplantation. FUNCTIONAL GENOMICS IN ORGAN TRANSPLANTATION PUBLICATIONS Cherqui, S., Kurian, S.M., Schussler, O., Hewel, J.A., Yates, J.R. III, Salomon, We are using high-density gene chip arrays, tandem D.R. Isolation and angiogenesis by endothelial progenitors in the fetal liver. Stem mass spectrometry proteomics, and complex trait genet- Cells 24:44, 2006. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 265

Cirulli, V., Zalatan, J., McMaster, M., Prinsen, R., Salomon, D.R., Ricordi, C., to inhibitors to a form that is broadly resistant resulted Torbett, B.E., Meda, P., Crisa, L. The class I HLA repertoire of pancreatic islets comprises the nonclassical class Ib antigen HLA-G. Diabetes 55:1214, 2006. in profound changes in the protease structure. Struc- tural changes in the resistant proteases included alter- Gemeniano, M., Mpanju, O., Salomon, D.R., Eiden, M.V., Wilson, C.A. The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is ations in the flap and basal regions and alteration from modulated by residues in the C-terminal region of its surface envelope protein. Virology 346:108, 2006. a symmetric to an asymmetric protease. These structural changes provide insight into the biochemical basis Kunicki, T.J., Baronciani, L., Canciani, M.T., Gianniello, F., Head, S.R., Mondala, T.S., Salomon, D.R., Federici, A.B. An association of candidate gene haplotypes for the loss of activity of protease inhibitors. To better and bleeding severity in von Willebrand disease type 2A, 2B, and 2M pedigrees. J. understand how structure contributes to the biochemi- Thromb. Haemost. 4:137, 2006. cal basis of resistance, we are continuing investigations Kurian, S.M., Flechner, S.M., Kaouk, J., Modlin, C., Goldfarb, D., Cook, D.J., Head, S., Salomon, D.R. Laparoscopic donor nephrectomy gene expression profil- on the relationship between structure and function in ing reveals upregulation of stress and ischemia associated genes when compared wild-type proteases and in mutant proteases that are to control kidneys. Transplantation 80:1067, 2005. broadly resistant to inhibitors. Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., HIV-1 VECTOR DELIVERY OF CCR5-INTRABODY Wilson, C.A., Patience, C., Salomon, D.R. Mice transgenic for a human porcine endogenous retroviral receptor are susceptible to productive viral infection [pub- GENES TO HUMAN HEMATOPOIETIC CELLS lished correction appears in J. Virol. 80:5100, 2006]. J. Virol. 80:3135, 2006. CXCR4 and CCR5 are the main chemokine receptors for HIV-1 entry into cells, and blocking these receptors Control of HIV Type 1, Gene limits entry of the virus. Naturally occurring polymorphisms of the gene for CCR5 indicate that disruption Delivery, and Regulation of of the gene provides protection from viruses that use CCR5 to gain entry. Because polymorphisms are pre- Hematopoietic Development sent in healthy persons, the use of genetic intervention strategies that prevent or limit expression of CCR5 B.E. Torbett, K.S. Barnett, L. Crisa, K.M. Fischer, G.E. Foos, may provide protection from initial infection and limit M.J. Giffin, V.D. Kutilek, P.A. McClintock, R.C. Prinsen, the spread of virus. With C.F. Barbas, Department of J.H. Savage, C.H. Swan, M.P. Tschan, J.A. Witkowski, Molecular Biology, we showed that intracellular expres- S. De Rozieres,* J.H. Elder,* H. Heaslet,* Y.-C. Lin,* sion of a CCR5-specific single-chain antibody (intra- C.D. Stout* body) efficiently disrupted expression of CCR5 on the * Department of Molecular Biology, Scripps Research T-cell surface and protected cells from HIV-1 infection. ur research interests include the structural and Moreover, we found that human stem cells expressing biochemical evolution of the resistance of HIV the CCR5-intrabody develop into T cells and that the O type 1 (HIV-1) proteases, gene delivery strate- decreased expression of CCR5 protected cells against gies to disrupt cellular entry of HIV-1, and normal and HIV-1 challenge and imparted a survival advantage in abnormal regulation of myeloid development by the the presence of HIV-1 infection. transcription factors PU.1 and cyclin D–interacting In current studies, we are disrupting the function of Myb-like protein (DMP1). viruses that use either the CXCR4 or the CCR5 recep- HIV-1 PROTEASE RESISTANCE tor for entry, the so-called R5X4 viruses. To accomplish In patients infected with HIV-1, treatment with inhib- our goals, we are using combination vectors that geneti- itors of HIV reverse transcriptase and protease sup- cally target chemokine receptors and viral and cellular presses replication of the virus. However, HIV-1 variants pathways critical for viral entry and replication. evolve that escape the approved drug treatments by REGULATION OF MYELOID DEVELOPMENT BY PU.1 developing a broad-based resistance to the protease AND DMP1 inhibitors. A molecular understanding of the resistance PU.1, a member of the Ets family of transcription to protease inhibitors is needed so that new inhibitors factors, is expressed solely in hematopoietic cells and can be developed that target drug-resistant viruses is necessary for directing myeloid development and for and, importantly, are less likely to induce inhibitor- regulating genes required for monocyte/macrophage and resistant viruses. neutrophil function. PU.1 has 3 major domains: the In collaboration with J.H. Elder, C.D. Stout, and transactivation, PEST, and Ets/DNA-binding domains. H. Heaslet, Department of Molecular Biology, we showed PU.1 interacts with other transcription factors, and that evolution of HIV protease from a form susceptible domains of PU.1 have been implicated in its function. 266 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

Myeloid development is controlled by temporal gene DIVISION OF EXPERIMENTAL expression of PU.1 and interactions among specific transcription factors. We are addressing which PU.1 PATHOLOGY domains regulate myeloid lineage–specific commitment, differentiation, and function. To determine which tran- Francis V. Chisari, M.D., Division Head scription factors interact with PU.1 and direct myeloid development, we use a strategy in which the gene for PU.1 is expressed only under certain conditions and a Molecular Biology of Hepatitis B proteomics approach. These studies are enabling us to and C Viruses and the Immune identify gene programs regulated by PU.1. Cancer often originates from inactivation and/or Response to Their Antigens deregulation of the control of gene expression. The transcription factor DMP1 positively regulates expression of epatitis B and C viruses are noncytopathic human p14ARF and CD13/aminopeptidase N, thus play- DNA and RNA viruses that cause acute and ing a role in cell-cycle control, differentiation, and func- H chronic hepatitis and hepatocellular carci- tion of hematopoietic and nonhematopoietic cells. The noma. More than 500 million persons worldwide are tumor suppressor ARF is critical for positive regulation chronically infected, and more than 2 million persons of p53, which in turn controls cellular proliferation and die of these infections every year. The focus of our modulates apoptosis. We have identified 2 novel and research is to unravel the life cycle of these viruses, developmentally expressed human DMP1 splice vari- discover the roles played by the innate and adaptive ants, β and γ. We found that the β variant functions as immune responses in the control of the infections, and a dominant-negative regulator of the originally reported elucidate the mechanisms responsible for viral clear- DMP1 protein. Currently, we are investigating the molec- ance and disease pathogenesis. Our goal is to devise ular and biological roles of the various isoforms in the novel strategies to prevent and cure these infections. development of normal and leukemic cells.

PUBLICATIONS Britschgi, C., Rizzi, M., Grob, T.J., Tschan, M.P., Hügli, B., Reddy, V.A., Andres, Suppression of Cytotoxic A.-C., Torbett, B.E., Tobler, A., Fey, M.F. Identification of the p53 family-responsive element in the promoter region of the tumor suppressor gene hypermethylated in cancer 1. Oncogene 25:2030, 2006. T Lymphocyte Function by Cirulli, V., Zalatan, J., McMaster, M., Prinsen, R., Salomon, D.R., Ricordi, C., PD-1–PD-L1 Interactions Torbett, B.E., Meda, P., Crisa, L. The class I HLA repertoire of pancreatic islets comprises the nonclassical class 1b antigen HLA-G. Diabetes 55:1214, 2006. After Antigen Recognition of Heaslet, H., Kutilek, V.D., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout, D.C. Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3. J. Mol. Biol. 4:967, 2006. Hepatitis B Virus in the Liver

Swan, C.H., Bühler, B., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection H. Maier, M. Isogawa, F.V. Chisari and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther., in press.

Swan, C.H., Torbett, B.E. Can gene delivery close the door to HIV-1 entry after roduction of IFN-γ by cytotoxic T lymphocytes escape? J. Med. Primatol., in press. (CTLs) specific for hepatitis B virus (HBV) is P rapidly induced when the lymphocytes enter the liver of mice transgenic for HBV and then is rapidly suppressed, despite the continued presence of antigen. Suppression of IFN-γ production by the CTLs coincides with the upregulation of PD-1, a cell-surface signaling molecule known to inhibit T-cell function. To determine whether PD-1 plays a role in the functional suppression of IFN-γ secretion by CTLs, we treated HBV transgenic mice with blocking antibodies specific for PD-L1, the most widely expressed PD-1 ligand, and adoptively transferred HBV-specific CTLs to the treated MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 267 mice. Treatment with antibodies to PD-L1 resulted in totropic virus infections in vivo, the events could explain an increase in the frequency of intrahepatic HBV-spe- the propensity of hepatotropic viruses to persist. cific CTLs producing IFN-γ and enhanced clearance of HBV from the liver. These results indicate that interactions between Effect of the Size of the PD-1 and PD-L1 contribute to the suppression of IFN-γ secretion after antigen recognition in the liver. Block- Viral Inoculum on the Course ade of inhibitory pathways such as PD-1–PD-L1 may prevent viral persistence and chronic infection in and Outcome of Hepatitis B instances in which the CTL response is suppressed Virus Infection by this mechanism.

S.F. Wieland, S. Asabe, K. Purton, R. Purcell,* F.V. Chisari Impact of Intrahepatic Antigen * National Institute of Allergy and Infectious Diseases, Bethesda, Maryland o monitor the influence of viral dose on the kinet- Recognition on Priming of the ics of viral spread and the course of infection, + T we infected 6 chimpanzees with serial 1000- CD8 T-Cell Response in T-Cell fold dilutions of a monoclonal preparation of hepatitis B virus (HBV). We observed a strict dose response in Receptor Transgenic Mice the kinetics of viral spread in 4 HBV-naive animals infected with 1010, 107, 104, 101 and 100 genome M. Isogawa, F.V. Chisari equivalents of HBV. Unexpectedly, the course of infec- ecause our findings that interactions between tion in animals receiving 101 and 100 genome equiva- PD-1 and PD-L1 suppress the function of cyto- lents was greatly prolonged compared with the course B toxic T lymphocytes specific for hepatitis B virus in recipients of larger doses, and peak viral titers were (HBV) after antigen recognition in the liver reflect the comparable to the peak titer in the animal that received impact of antigen recognition by effector/memory cells, 1010 genome equivalents. the findings may not reflect what occurs in an immuno- These counterintuitive results suggest that a low- logically naive host during HBV infection. To define the dose HBV inoculum favors the development of persistent immunologic events that occur during priming of HBV- infection, presumably because the low level of viral specific T cells, we generated T-cell receptor (TCR) trans- antigens present in the inoculum does not prime an genic mice that have CD8+ T cells specific for the HBV adequate adaptive immune response. Consequently, core and envelope proteins. naive T cells make initial contact with HBV antigen in Preliminary results indicated that naive T cells the liver, a situation that results in partial activation of respond to HBV quite differently than do antigen-expe- the cells and failure to produce antiviral cytokines that rienced effector/memory T cells. When naive T cells control the infection. Experiments designed to test this from TCR transgenic mice were transferred into HBV hypothesis are in progress. transgenic mice, the transferred cells proliferated in the liver and caused liver disease but did not inhibit HBV replication because they could not produce IFN-γ upon Coevolution of Virus and Host antigen recognition. In contrast, the naive HBV-specific CD8+ T cells produced IFN-γ after they were transferred During Persistent Hepatitis C into HBV transgenic mice when the mice were system- ically infected with recombinant vaccinia virus expressing Virus Infection the corresponding HBV antigens. J. Zhong, P. Gastaminza, J. Chung, G. Cheng, M. Isogawa, These results suggest that when naive CD8+ T cells F.V. Chisari recognize antigen exclusively in the liver, their proliferative and cytolytic programs are induced without initiating he virologic and cellular consequences of persis- cytokine production and, therefore, without suppress- tent hepatitis C virus (HCV) infection have been ing viral replication. If similar events occur during hepa- T elusive because of the lack of the requisite experi- 268 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE mental systems to study HCV infections. We recently Cooperativity and Cholesterol established the conditions required to persistently infect Huh-7 cells derived from a human hepatoma in vitro Dependence of CD81 and with an HCV genotype 2a infectious molecular clone. Persistent in vitro infection is characterized by the selec- Scavenger Receptor B Type I in tion of viral variants that have accelerated expansion the Initiation of Hepatitis C kinetics, higher peak viral titers, and greater buoyant densities than do wild-type virus. Virus Infection Sequencing analysis revealed the evolution of a single adaptive mutation in the HCV E2 envelope protein S.B. Kapadia, T. Baumert,* J. McKeating,** F.V. Chisari that was largely responsible for the variant phenotype. * University of Freiburg, Freiburg, Germany Furthermore, as the virus became more aggressive, cells ** University of Birmingham, Birmingham, England emerged that were resistant to infection, with escape sing surrogate models of hepatitis C virus (HCV) mechanisms operative at the levels of viral entry, HCV infection, we identified several cellular proteins RNA replication, or both. Collectively, these results U as possible receptors for entry of HCV into cells. reveal the existence of coevolutionary events during Among the proteins, the tetraspanin CD81 and scavenger persistent HCV infection that favor survival of both receptor B type I, both of which localize to specialized virus and host. plasma membrane domains enriched in cholesterol, may be key players in HCV entry. Using our in vitro HCV infection system, we showed that CD81 and scavenger Antiviral Peptides That Prevent receptor B type I are both required for authentic HCV infection in vitro, that the 2 proteins function coopera- Hepatitis C Virus Infection tively to initiate HCV entry, and that CD81-mediated HCV entry depends, in part, on membrane cholesterol. G. Cheng, P. Gastaminza, A. Montero,* M.R. Ghadiri,* F.V. Chisari * Department of Chemistry, Scripps Research Establishment of Hepatitis C sing a hepatitis C virus (HCV) in vitro infection system, we identified several HCV-derived syn- Virus Infection in Highly U thetic peptides that inhibit HCV infection by at least 90% at nontoxic concentrations. The most potent Differentiated, Growth-Arrested peptide, an 18-mer that contains the sequence of the putative membrane anchor domain of the HCV NS5A Hepatocyte Cell Lines In Vitro protein, and the peptide’s D-amino acid congener com- B. Sainz, F.V. Chisari pletely and permanently blocked HCV infection with no cellular toxic effects. The peptide appears to inactivate hen actively dividing, poorly differentiated HCV extracellularly; it has no effect on HCV replicon cells derived from a human hepatoma (Huh- RNA replication and it must be added to cells together W 7 cells) are cultured in the presence of 1% with virus to prevent the infection. dimethyl sulfoxide, which induces the differentiation Consistent with this hypothesis, the peptide rapidly of primary hepatocytes in vitro, the cells become cyto- increases the permeability of cholesterol-phospholipid logically differentiated and transition into a nondivid- liposomes in a dye-release assay, suggesting that it could ing state and the induction of hepatocyte-specific genes. destabilize or otherwise compromise the integrity of the We recently showed that these cells are highly permis- viral membrane. N- and C-terminal truncation of the sive for hepatitis C virus infection and that these cultures peptide indicates that full antiviral and liposome dye- can be persistently infected. Because hepatitis C virus release activities are retained when 4 amino acids are naturally replicates in highly differentiated nondividing removed from its C terminus and that both activities human hepatocytes, this system may more accurately are lost when the peptide is shortened any further.Fur- mimic the cellular conditions under which the virus ther analysis of the antiviral activity of this peptide and replicates in vivo than do models based on poorly dif- the remaining 12 antiviral peptides is under way. ferentiated, rapidly dividing cell lines. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 269

PUBLICATIONS injected different isolates of lymphocytic choriomenin- Bui, H.H., Sidney, J., Peters, B., Sathiamurthy, M., Sinichi, A., Purton, K.A., Mothé, B.R., Chisari, F.V., Watkins, D.I., Sette, A. Automated generation and gitis virus (LCMV) into adult immunocompetent mice evaluation of specific MHC binding predictive tools: ARB matrix applications. that had or had not been depleted of platelets. Immunogenetics 57:304, 2005. In the mice not depleted of platelets, despite a Cheng, G., Zhong, J., Chisari, F.V. Inhibition of dsRNA-induced signaling in hepati- marked thrombocytopenia (80% reduction in normal tis C virus-infected cells by NS3 protease-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. U. S. A. 103:8499, 2006. platelet counts) associated with platelet dysfunction,

Dryden, K.A., Wieland, S.F., Whitten-Bauer, C., Gerin, J.L., Chisari, F.V., Yeager, acute infection with LCMV resulted in a mild hemorrhage M. Native hepatitis B virions and capsids visualized by electron cryo-microscopy. (20% decrease in hematocrit and presence of fecal Mol. Cell, in press. blood) and rapid clearance of the infection. Depletion Gilbert, R.J.C., Beales, L., Blond, D., Simon, M.N., Lin, B.Y., Chisari, F.V., Stu- of platelets (98% reduction of normal platelet counts), art, D.I., Rowlands, D.J. Hepatitis B small surface antigen particles are octahe- dral. Proc. Natl. Acad. Sci. U. S. A. 102:14783, 2005. but not treatment with an anticoagulant, resulted in a severe hemorrhage (80% decrease in hematocrit) that Guidotti, L.G., Chisari, F.V. Immunobiology and pathogenesis of viral hepatitis. In: Mechanisms of Disease. Abbas, A.K., Downing, J.R., Kumar, V. (Eds.). Annual was often lethal and involved mostly the skin. Lethal Reviews, Palo Alto, CA, 2006, p. 23. Annual Review of Pathology; Vol. 1. hemorrhage required IFN-α/β–dependent signaling but Iannacone, M., Sitia, G., Isogawa, M., Marchese, P., Castro, M.G., Lowenstein, was independent of CTL-induced immunopathologic P.R., Chisari, F.V., Ruggeri, Z.M., Guidotti, L.G. Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat. Med. 11:1167, 2005. changes or TNF-α–mediated responses. Platelet-depleted mice that survived for up to 5–6 days after LCMV Isogawa, M., Robek, M.D., Furuichi, Y., Chisari, F.V. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo. J. Virol. 79:7269, 2005. infection had reduced CTL numbers in the infected organs and did not clear the virus. Platelet transfusion Kimura, K., Moriwaki, H., Nagaki, M., Saio, M., Nakamoto, Y., Naito, M., Kuwata, K., Chisari, F.V. Pathogenic role of B Cells in anti-CD40-induced necroin- prevented death, ameliorated hemorrhage, restored CTL flammatory liver disease. Am. J. Pathol. 168:786, 2006. responses, and cleared LCMV, indicating that platelets

Murray, J., Wieland, S.F., Purcell, R.H., Chisari, F.V. Dynamics of hepatitis B virus protect mice from lethal hemorrhagic LCMV infection clearance in chimpanzees. Proc. Natl. Acad. Sci. U. S. A. 102:17780, 2005. and mediate viral clearance. The results also suggest Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D.R., that similar events may happen in patients infected Wieland, S.F., Uprichard, S.L., Wakita, T., Chisari, F.V. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. U. S. A. 102:9294, 2005. with arenaviruses such as Lassa virus or Junin virus, in which thrombocytopenia, platelet dysfunction, and exceptionally high levels of circulating IFN-α/β have Role of Platelets in Hemorrhagic been associated with hemorrhage, impaired cellular immunity, lack of viral clearance, and death. Lymphocytic Choriomeningitis PUBLICATIONS Guidotti, L.G., Chisari, F.V. Immunobiology and pathogenesis of viral hepatitis. Virus Infection in Mice Annu. Rev. Pathol. Mech. Dis. 1:23, 2006.

Iannacone, M., Sitia, G., Guidotti, L.G. Pathogenetic and antiviral immune L.G. Guidotti, M. Iannacone, G. Sitia, J.K. Whitmire,* responses against hepatitis B virus. Future Virol. 1:189, 2006. P. Marchese, F.V. Chisari, Z.M. Ruggeri Iannacone, M., Sitia, G., Isogawa, M., Marchese, P., Castro, M.G., Lowenstein, * Molecular and Integrative Neurosciences Department, Scripps Research P.R., Chisari, F.V., Ruggeri, Z.M., Guidotti, L.G. Platelets mediate cytotoxic T lymphocyte-induced liver damage. Nat. Med. 11:1167, 2005. e recently showed that an initial immune- Sitia, G., De Bona, A., Bagaglio, S., Paties, C., Uberti-Foppa, C., Guidotti, L.G., induced inflammatory response to viral anti- Lazzarin, A., Morsica, G. HIV/HCV co-infected patients naive to antiretroviral ther- gens expressed within the liver results in apy show higher intrahepatic levels of CD3, IFN-γ and TNF-α mRNAs when com- W pared to either co-infected patients treated with ART or HCV mono-infected changes of the vessel wall that promote adhesion and patients. Antivir. Ther., in press. activation of platelets. In turn, platelet adhesion and activation favor the exit of virus-specific cytotoxic T lymphocytes (CTLs) from the bloodstream and accumulation of the lymphocytes within the infected organ. These studies indicate that platelets play an unexpected and important role in the pathogenesis of CTL-mediated antiviral activity and liver damage. To investigate the contribution of platelets in the control of an acute viral infection that involves multiple tissues and organs, we 270 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

DIVISION OF HEMATOLOGY One of these is the measurement of iron absorption by mice; we feed them radioactive iron and estimate the amount of iron retained by using total body counting. Ernest Beutler, M.D., Division Head One attractive candidate regulator that we have studied is soluble transferrin receptor. Using the technique of hydrodynamic transfection, we were able to greatly Studies in Human increase the levels of this putative regulator in the Genetic Disease plasma of mice. Measuring iron absorption in these animals, we showed that soluble transferrin receptor had no influence on iron absorption. E. Beutler, K. Crain, J. Flanagan, T. Gelbart, P. Lee, H. Peng, The central regulator of iron absorption is hepcidin, J. Truksa, J. Waalen, L. Wang, C. West and how this 25 amino acid peptide is regulated by iron ur epidemiologic studies of hemochromatosis remains a mystery, particularly since the iron regulation have greatly expanded our understanding of the can only be observed in whole animals but not in iso- O phenotype of homozygotes for the common lated hepatocytes. We have used the hydrodynamic C282Y mutation in HFE, the gene responsible for the transfection method to introduce hepcidin promoter most common form of primary hemochromatosis. Our luciferase constructs into whole mice to identify the finding that only about 1% of C282Y homozygotes man- region of the hepcidin promoter that is regulated by iron. ifested the disease was so counter to common belief Hepcidin is also regulated by inflammation. Using DNase that it was regarded with great skepticism—even hos- footprinting and electrophoretic mobility shift assays, we tility. This reaction was not surprising, because it had have examined the regions of the hepcidin promoter that been suggested, on the basis of clinical impressions, are essential for regulation by inflammation. that the penetrance of the disease phenotype was as Several proteins without any obvious connection to high as 95% among men. However, our results have iron metabolism apparently play major roles in iron home- now been amply confirmed by many other studies, and ostasis. For example, hemojuvelin, which is closely this finding has had a major impact on attitudes regard- related to retinal guidance molecules and most highly ing the appropriateness of general population screen- expressed in skeletal muscle, is essential for regulating for this disorder. ing hepcidin expression. Bone morphogenic proteins Moreover, longitudinal studies of our HFE C282Y have proved to be potent stimulators of hepcidin tran- homozygous patients have shown a remarkable lack of scription; again, their relationship to iron homeostasis clinical progression, even among patients with markedly was entirely unexpected. We are attempting to unravel elevated serum ferritin levels and abnormal liver func- these relationships. tion tests. The DNA samples we collected in the course In collaboration with Jeff Kelly, Department of Chem- of these studies also provided us and others with a valu- istry, we have established a transgenic mouse model to able resource. The 30,000 plus DNA samples have made express a mutant human glucocerebrosidase that is the it possible to perform large studies examining the rela- most common cause of Gaucher disease. Our purpose tionship between genetic polymorphisms and a variety is to use this animal as a model to study the effective- of diseases. The samples have enabled studies of genetic ness, in vivo, of chaperone compounds that have been shown by researchers in Dr. Kelly’s laboratory to increase factors, including those that may affect aging, hyper- the amount of enzyme made by cultured cells producing tension, drug addiction, obesity, arthritis, atherosclero- this mutant enzyme. These studies could lead to a better sis, amyloidosis, and lung infection. In addition, the treatment for this inherited glycolipid storage disorder. laboratory data that we gathered made it possible for us to establish more robust standards for the diagno- PUBLICATIONS sis of anemia than had been previously available and Barton, J.C., Lee, P.L. Disparate phenotypic expression of ALAS2 R452H (nt 1407 G→A) in two brothers, one with severe sideroblastic anemia and iron over- have shown that the prevalence of unexplained ane- load, hepatic cirrhosis, and hepatocellular carcinoma. Blood Cells Mol. Dis. mia among the elderly is not nearly as high as had 36:342, 2006. been suggested. Barton, J.C., Lee, P.L., Bertoli, L.F., Beutler, E. Iron overload in an African Ameri- We continue to construct model systems to allow us can woman with SS hemoglobinopathy and a promoter mutation in the X-linked erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. Blood Cells Mol. Dis. to better understand how body iron content is regulated. 34:226, 2005. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 271

Barton, J.C., Lee, P.L., West, C., Bottomley, S.S. Iron overload and prolonged Waalen, J., Beutler, E. Hereditary hemochromatosis: screening and management. ingestion of iron supplements: clinical features and mutation analysis of hemochro- Curr. Hematol. Rep. 5:34, 2006. matosis-associated genes in four cases. Am. J. Hematol., in press. Zimran, A., Elstein, D., Beutler, E. Low-dose therapy trumps high-dose therapy Beutler, E. Gaucher disease: multiple lessons from a single gene disorder. Acta again in the treatment of Gaucher disease. Blood, in press. Paediatr. Suppl. 95:103, 2006.

Beutler, E. Hemochromatosis. In: Encyclopedic Reference of Genomics and Pro- teomics in Molecular Medicine. Ganten, D., Ruckpaul, K. (Eds.). Springer, New York, in press. Cell Death in the Heart

Beutler, E. Hemochromatosis: genetics and pathophysiology. Annu. Rev. Med. 57:331, 2006. R.A. Gottlieb, N. Brady, A. Cartier, Å.B. Gustafsson,

Beutler, E. Lysosomal storage diseases: natural history and ethical and economic A. Hamacher-Brady, C. Huang, D. Kubli, M.R. Sayen, aspects. Mol. Genet. Metab., in press. P. Wentworth, Jr.,* M. Yeager,** H. Rosen***

Beutler, E. Red blood cell enzymopathies. In: Clinical Hematology. Young, N.S., * Department of Chemistry, Scripps Research Gerson, S.L., High, K.A. (Eds.). Mosby, St. Louis, 2006, p. 308. ** Department of Cell Biology, Scripps Research Beutler, E. Screening for hemochromatosis. Bloodmed Exclusives. September 28, *** Department of Immunology, Scripps Research 2005. Available at: http://www.BloodMed.com. REGULATION OF CELL DEATH PATHWAYS IN Beutler, E. The treatment of Gaucher disease in countries with limited health care resources. Ind. J. Hum. Genet. 11:121, 2005. MYOCARDIAL ISCHEMIA AND REPERFUSION

Beutler, E., Gelbart, T., Scott, C.R. Hematologically important mutations: Gaucher yocardial infarctions result in the death of half disease. Blood Cells Mol. Dis. 35:355, 2005. a million persons in the United States each Beutler, E., Waalen, J. The definition of anemia: what is the lower limit of normal M year. We are interested in understanding the of the blood hemoglobin concentration? Blood 107:1747, 2006.\ molecular events that commit cells to a death program Beutler, E., Waalen, J., Gelbart, T. Chronic inflammation does not appear to mod- after ischemia and reperfusion in the heart. Although ify the homozygous hereditary hemochromatosis phenotype. Blood Cells Mol. Dis. 35:326, 2005. ischemia itself is deleterious because of energy depletion, further damage ensues upon reperfusion, when a Lee, P., Promrat, K., Mallette, C., Flynn, M., Beutler, E. A juvenile hemochromatosis patient homozygous for a novel deletion of cDNA nucleotide 81 of hemoju- burst of reactive oxygen species is produced and when velin. Acta Haematol. 115:123, 2006. apoptosis, or programmed cell death, is activated in Lee, P.L., Barton, J.C. Hemochromatosis and severe iron overload associated with vulnerable cells. Because apoptosis is a tightly regulated compound heterozygosity for TFR2 R455Q and two novel mutations TFR2 R396X and G792R. Acta Haematol. 115:102, 2006. program, it may be possible to interfere with the process and salvage cardiac cells. In addition to apoptosis, cells Lee, P.L., Barton, J.C., Rao, S.V., Acton, R.T., Adler, B.K., Beutler, E. Three kin- ships with ALAS2 P520L (c. 1559 C→T) mutation, two in association with severe can die via necrosis, which has generally been regarded iron overload, and one with sideroblastic anemia and severe iron overload. Blood as an unregulated process that can occur after expo- Cells Mol. Dis. 36:292, 2006. sure to high levels of oxidants. However, recent evi- Lee, P.L., Beutler, E. Hemochromatosis. In: Handbook of Molecular Diagnostics. Grody, W.W., Nakamura, R.M., Strom, C. (Eds.). Elsevier, San Diego, in press. dence suggests that so-called necrotic cell death may also be subject to biochemical regulation. We are Lee, P.L., West, C., Crain, K., Wang, L. Genetic polymorphisms and susceptibility to lung disease. J. Negat. Results BioMed. 5:5, 2006. defining the biochemical events of cell death in the heart, both apoptosis and necrosis, to identify poten- Noel, N., Flanagan, J., Bajo, M.J.R., Kalko, S.G., del Mar Mañú, M., Fuster, J.L.G., Perez de Ossa, P., Carreras, J., Beutler, E., Vives-Corrons, J.-L. Two new phospho- tial therapeutic targets to mitigate reperfusion injury. glycerate kinase mutations associated with chronic haemolytic anaemia and neurological dysfunction in two patients from Spain. Br. J. Haematol. 132:523, 2006. Using isolated perfused rat hearts subjected to global ischemia and reperfusion, we found that calpain Sawkar, A.R., Adamski-Werner, S.L., Cheng, W.C., Wong, C.-H., Beutler, E., Zim- mer, K.P., Kelly, J.W. Gaucher disease-associated glucocerebrosidases show muta- is activated during reperfusion, leading to cleavage of tion-dependent chemical chaperoning profiles. Chem. Biol. 12:1235, 2005. Bid, a proapoptotic member of the Bcl-2 family of anti- Sipe, J.C., Arbour, N., Gerber, A., Beutler, E. Reduced endocannabinoid immune apoptotic proteins. Bid targets the mitochondria, result- modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders. J. Leukoc. Biol. 78:231, 2005. ing in energetic failure and release of proapoptotic factors. The protein apoptosis repressor with caspase recruit- Waalen, J., Beutler, E. Beware of multiple comparisons: a study of symptoms associated with mutations of the HFE hemochromatosis gene. Clin. Chim. Acta ment domain is expressed at high levels in cardiac and 361:128, 2005. skeletal muscle and is strongly protective against cell Waalen, J., Beutler, E. Effect of correcting transferrin saturation for body mass death mediated by oxidative stress. We have shown index in HFE C282Y homozygotes. J. Hepatol. 44:433, 2006. that this protein interacts with Bax, another proapop- Waalen, J., Beutler, E. Gaucher disease as a model for an orphan disease. In: totic protein, to prevent apoptosis through the mito- Gaucher Disease. Futerman, T., Zimran, A. (Eds.). CRC Press, Boca Raton, FL, in press. chondrial pathway. 272 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

Cyclophilin D, a protein found in the inner membrane of mitochondria, regulates the mitochondrial permeability transition pore. In collaboration with J. Molkentin, Children’s Hospital Medical Center, Cin- cinnati, Ohio, we are examining the mitochondrial alterations mediated by deletion and overexpression of cyclophilin D. REGULATION AND SIGNIFICANCE OF AUTOPHAGY IN THE MYOCARDIUM Autophagy is a cell-autonomous mechanism to remove damaged or unwanted organelles (Fig. 1). Using high-resolution fluorescence microscopy with 3-dimensional deconvolution to image live cells expressing the autophagy marker LC3-GFP (Fig. 2), we evaluated the upregulation of autophagy in HL-1 cardiomyocytes subjected to starvation or simulated ischemia and reper- Fig. 2. HL-1 cells were transfected with LC3-GFP and imaged after simulated ischemia and 90 minutes of reperfusion (sI/R) or incubation in normoxic (but nutrient-limited) buffer (KH) for the same amount of time. The punctate structures indicate the formation of autophagosomal vesicles (AVs). The level of autophagic activity (flux) was revealed by treating cells with inhibitors (+ i) to prevent lysosomal fusion, resulting in the accumulation of AVs. The bar graph quantifies the upregulation of autophagy by starvation and sI/R and the impairment of flux by sI/R.

fusion. We found that autophagy is upregulated in nutrient-limited conditions, but this response is completely suppressed during simulated ischemia and only partially recovers during reperfusion. Nevertheless, the induction of autophagy is part of a cytoprotective response to ischemia-reperfusion injury. We hypothesize that autophagy removes damaged proapoptotic mitochondria, thereby averting apoptosis. Mitochon- dria must fragment into small spherules before they can be engulfed by an autophagosome. We are now

Fig. 1. Autophagy in simulated ischemia and reperfusion. Induc- exploring the control of autophagy in the heart and tion of autophagy requires activity of Beclin1 and its interacting examining the conditions that lead to fragmentation partner, class III phosphatidylinositol-3′-kinase (PI3-K; hVps34), and removal of mitochondria via autophagy. resulting in the generation of phosphatidylinositol-3′-phosphate, and it CHARACTERIZATION OF THERAPEUTIC AGENTS is negatively regulated by class I PI3-K through mTOR. Formation of FOR ISCHEMIA-REPERFUSION INJURY the phagophore requires conjugation of Atg12 to lysine 130 of Atg5 We made the serendipitous discovery that chloram- as a prerequisite for recruiting LC3-II. Sequestration of cytoplasmic material can be nonspecific or selective; mechanisms that may gov- phenicol and other inhibitors of cytochrome P450 ern selectivity are incompletely understood. In order to accomplish monooxygenases reduce ischemia-reperfusion injury degradation of the autophagosome and its cargo, the autophagosome in the heart. These drugs are protective even when is then transported to and fuses with the acidic lysosome, generating administered after ischemia, suggesting that they may the autophagolysosome. Within the autophagolysosome, lysosomal have therapeutic potential in the treatment of myocar- proteases degrade the inner autophagosomal membrane and cargo. dial infarction. Cytochrome P450 monooxygenases in During ischemia, autophagy is inhibited at the level of autophagosome formation. Upon reperfusion, autophagy partially recovers, with the heart metabolize arachidonic acid to eicosanoids submaximal induction and impaired degradation. Enhancing autophag- that regulate contractility and vasomotor tone. Some ic flux is protective against simulated ischemia-reperfusion injury. P450 enzymes are also potent sources of superoxide, MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 273 which may contribute to reperfusion injury. We are PUBLICATIONS Brady, N.R., Hamacher-Brady, A., Gottlieb, R.A. Proapoptotic BCL-2 family mem- investigating the basis for the protective effect of P450 bers and mitochondrial dysfunction during ischemia/reperfusion injury, a study inhibition. We are focusing on the downstream signal employing cardiac HL-1 cells and GFP biosensors. Biochim. Biophys. Acta, in press. transduction events such as activation of calpain and Brady, N.R., Hamacher-Brady, A., Westerhoff, H.V., Gottlieb, R.A. A wave of reactive oxygen species (ROS)-induced ROS release in a sea of excitable mitochondria. p38 MAP kinase. Antioxid. Redox Signal., in press. Analogs of sphingosine-1-phosphate (S1P) are being Gustafsson, Å.B., Gottlieb, R.A., Granville, D.J. TAT-mediated protein transduc- developed as immunomodulators. We have studied the tion: delivering biologically active proteins to the heart. Methods Mol. Med. effects on the heart of S1P and synthetic receptor-selec- 112:81, 2005. tive agonists. We found that an agonist selective for S1P1 Hamacher-Brady, A., Brady, N.R., Logue, S.E., Sayen, M.R., Jinno, M., Kirshen- baum, L.A., Gottlieb, R.A., Gustafsson, Å.B. Response to myocardial ischemia/ greatly exacerbated reperfusion arrhythmias. Recep- reperfusion injury involves Bnip3 and autophagy. Cell Death Differ., in press. tor-selective agonists will require further evaluation for safety in clinical trials in humans. ROLE OF BNIP3 IN THE MYOCARDIUM Genetics of the Endogenous Bnip3 is a member of the “BH3-only” subfamily of proapoptotic Bcl-2 proteins and is localized primarily Cannabinoid System to the mitochondria in cardiomyocytes. We found that Bnip3 contributes to ischemia-reperfusion injury and J.C. Sipe, A. Gerber that overexpression of Bnip3 leads to mitochondrial e focus on the genetics of the endogenous dysfunction and cell death in cardiac myocytes. We also cannabinoid system and the role of genetic found that Bnip3 induces extensive fragmentation of the W abnormalities in this system in human dis- mitochondrial network (Fig. 3), which coincides with eases. Several common human neurobehavioral disorders, such as drug addiction, obesity, anxiety, and chronic pain, most likely are related to malfunction of endocannabinoids, chemicals known as the brain’s own marijuana. We recently found a link between autoimmune diseases such as multiple sclerosis and abnormalities of endocannabinoid function in the immune system. Since our discovery in 2002 of a naturally occurring human mutation, P129T, in fatty acid amide hydrolase, the main enzyme that controls levels of endocannabinoid signaling in the nervous system, we have focused on disorders in humans that appear to be associated with genetic variations. In collaboration with B.F. Cravatt, Fig. 3. Overexpression of Bnip3 causes fragmentation of the Department of Cell Biology, we found that the P129T mitochondrial network. HL-1 cardiac myocytes were cotransfected with Mito-dsRed-2 (to label mitochondria) and pcDNA3.1 or mutation results in significantly reduced cellular activity Bnip3, and fluorescent images were obtained 48 hours later. and expression of fatty acid amide hydrolase. Previ- ously, we were the first investigators to show a signifi- a dramatic upregulation of autophagy. 3-Dimensional cant association between the P129T mutation and deconvolution rendering of fluorescent images revealed overweight and obesity, suggesting that this variation fragmented mitochondria inside autophagosomes. Inhi- may be an important risk factor for these major public bition of the formation of autophagosomes resulted in health problems. increased Bnip3-mediated cell death, supporting the More recently, we collected new information on the notion that autophagy might serve as a protective influence of P129T in human disorders of reward and response by sequestering damaged mitochondria. Cur- craving, such as drug abuse and addiction. Collaborative rently, we are elucidating the molecular mechanism by studies with several drug abuse centers resulted in more which Bnip3 mediates mitochondrial dysfunction and detailed data on the P129T variation. The mutation was cell death. We are also interested in identifying proteins not a risk factor for marijuana dependence in marijuana that interact with Bnip3 in the heart and in determin- users, but it was linked to abuse of sedative drugs. In ing the functional significance of this interaction. a study of heroin addicts done in collaboration with 274 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE colleagues in New York City, we found no increased risk with pleiotrophin. Substrates of RPTPβ/ζ that we have for heroin addiction in patients with the P129T muta- discovered include β-catenin, β-adducin, Fyn, anaplas- tion. However, in a study of subjects with abuse of tic lymphoma kinase (ALK), and TrkA, the receptor of several different types of drugs, we identified several nerve growth factor. Increased tyrosine phosphorylation mutations linked to P129T. We calculated that this of these substrates in pleiotrophin-stimulated cells leads mutation in humans is an ancient one that arose more to disruption of adherent junction complexes and loss than 100,000 years ago in Africa and was carried into of homophilic cell-cell adhesion, suggesting the impor- all racial groups after the African Diaspora. In collabo- tance of pleiotrophin signaling in the regulation of these ration with colleagues at the National Institute on Drug vital cell functions. Abuse, Baltimore, Maryland, we confirmed our earlier We recently discovered that pleiotrophin signaling findings that the P129T mutation is associated with critically regulates the renin-angiotensin biosynthesis multiple drugs of addiction. pathway and the catecholamine biosynthesis pathway and that pleiotrophin stimulates synthesis of a specific PUBLICATIONS Flanagan, J., Gerber, A.L., Cadet, J.L., Beutler, E., Sipe, J.C. The fatty acid amide cohort of collagens and elastin. Pleiotrophin also is a hydrolase 385 A/A (P129T) variant: haplotype analysis of an ancient missense potent angiogenic factor that stimulates growth of new mutation and confirmation of risk for drug abuse. Hum. Genet., in press. blood vessels when injected into ischemic myocardium. Proudnikov, D., Sipe, J.C., Barral, S., Ott, J., LaForge, S., Kreek, M.J. The 385 A Ptn, the gene for pleiotrophin, is also a proto-onco- allele coding the low activity fatty acid amide hydrolase may be associated with reduced vulnerability to heroin addiction in African-American males. Neurosci. gene and is constitutively expressed in many human Lett., in press. malignant cancers. In all cases studied, pleiotrophin

Tyndale, R.F., Payne, J.I., Gerber, A.L., Sipe, J.C. The fatty acid amide hydrolase signaling was essential to maintain the transformed C385A (P129T) missense mutation in THC users: studies of drug use and depen- phenotype of the cancers. Inappropriate pleiotrophin dence in Caucasians. Pharmacogenet. Genomics, in press. signaling thus is a potent promoter of tumor progression. We found that constitutive pleiotrophin signaling DIVISION OF MOLECULAR ONCOLOGY in malignant cells stimulates an epithelial-mesenchmal transition, loss of cell-cell adhesion, gain of a motile phenotype, degradation of cadherins, and expression Thomas F. Deuel, M.D., Division Head of different integrins at the cell surface. In vivo, pleiotrophin stimulates tumor angiogenesis, extensive remodeling Pleiotrophin: A Cytokine With of the microenvironment and induction of carcinoma- associated fibroblasts, morphologic changes of the car- Critical Roles in Growth and in cinoma cell itself to a more aggressive phenotype, and a more rapid and aggressive growth of the tumors. Development and Progression Our long-range goals are to expand knowledge of this unique pleiotrophin–RPTPβ/ζ signaling pathway, of Human Neoplasms to use pleiotrophin as a therapeutic agent to stimulate angiogenesis, and to target the pleiotrophin signaling T.F. Deuel, Y. Chang, L. Ezquerra-Ruiz, G. Herradon, pathway as a tool for treating progression of human P. Perez-Pinera, W. Zhang neoplasms with constitutive expression of Ptn. e recently identified and cloned pleiotrophin, SIGNALING an 18-kD cytokine with diverse roles in nor- In the past year, we identified ALK as a substrate W mal growth and in the development, differenti- of RPTP β/ζ. ALK is a receptor-type transmembrane ation, and progression of malignant tumors. Pleiotrophin tyrosine kinase with a unique mechanism of activation. signals through a unique mechanism; it inactivates the Inactivation of the tyrosine phosphatase activity of receptor protein tyrosine phosphatase (RPTP)β/ζ. Through RPTP β/ζ is responsible for ALK activation; the kinase inactivation of RPTPβ/ζ, pleiotrophin increases levels is not activated directly by a cytokine acting through a of tyrosine phosphorylation of the substrates of RPTPβ/ζ cell-surface receptor. due to the continued activities of unknown tyrosine We showed that pleiotrophin activates the tyrosine kinases that phosphorylate the same sites that normally kinase activity of ALK in pleiotrophin-stimulated cells are dephosphorylated by RPTPβ/ζ in cells not stimulated and that the activated ALK kinase phosphorylates MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 275

β-catenin. In addition, we found that the site phos- growth. We are now studying the mechanisms and path- phorylated in β-catenin by ALK is a site recognized ways of pleiotrophin signaling that lead to angiogene- and dephosphorylated by RPTP β/ζ. This tyrosine sis in both in vitro and in vivo models. phosphorylation site in β-catenin is potentially impor- BREAST CANCER tant, because when it is phosphorylated in pleiotro- To extend our studies on pleiotrophin in neoplasia, phin-stimulated cells, it disrupts the association of we used a dominant-negative Ptn and found that it β-catenin with N-cadherin needed for cells to adhere reversed the malignant phenotype of human breast can- to each other. Because disruption of homophilic cell- cer cells in vitro and in vivo. Currently, we are deter- cell adhesion is characteristic of highly malignant cells mining the mechanisms by which pleiotrophin signaling that express Ptn, our data suggest that one mechanism stimulates a malignant state in human breast cancer through which pleiotrophin stimulates a more aggres- cells. In vitro, we identified reciprocal signaling between sive phenotype in malignant cells is disruption of nor- breast cancer cells that express an activated Ptn and mal cytoskeletal architecture. In collaboration with activated stromal fibroblasts. We have now shown that J.R. Yates, Department of Cell Biology, we are using through reciprocal cross talk pleiotrophin secreted from mass spectrometry to identify the sites of tyrosine phos- human breast cancer cells cocultured with NIH 3T3 phorylation in β-catenin and in ALK that are phosphory- cells sharply upregulates protein kinase C δ and matrix lated in pleiotrophin-stimulated cells. metalloproteinase 9 in both the NIH 3T3 cells and the In PC12 cells, stimulation with pleiotrophin acti- human breast cancer cells. Furthermore, the upregula- vates TrkA. The cessation of growth and progression of tion of both protein kinase C δ and matrix metallopro- neurite outgrowth in PC12 cells stimulated with pleio- teinase 9 in both cells depends entirely on secretion of trophin are same as the cessation and progression of pleiotrophin from the breast cancer cell. neurite outgrowth that occur in PC12 cells stimulated To further test the relevance of pleiotrophin in pro- with nerve growth factor. We found that PC12 cells moting the growth of malignant breast cancers in vivo, require pleiotrophin to survive and that pleiotrophin we used bitransgenic mice predisposed to breast cancer. We found that constitutive pleiotrophin signaling acts through an autocrine mechanism. The activation driven by the mouse mammary tumor virus promoter, of TrkA requires phosphorylation of the same tyrosine which directs genes to mammary gland cells for expres- that is phosphorylated in pleiotrophin-stimulated cells. sion, does not induce breast cancer in mice; inappro- Thus, the mechanism of activation of TrkA is the same priate expression of pleiotrophin alone is insufficient to as the mechanism of activation of ALK, suggesting that induce breast cancer. However, inappropriate expres- the regulation of different tyrosine kinase receptors by sion of pleiotrophin cooperates with signals driven by pleiotrophin may be a unique mechanism of maintain- polyoma middle T antigen to accelerate the growth of ing the trophism of cells. mouse breast cancers and initiate formation of new ANGIOGENESIS blood vessels in the tumors. We found that expression of Ptn is upregulated in In ongoing collaborative studies with Z.-Y. Wang, developing microvasculature, macrophages, and astro- Creighton University, Omaha, Nebraska, we identified and cytes after acute ischemic brain injury and that pleiotro- partially characterized a novel form of the estrogen recep- phin directly injected into ischemic myocardium induces tor. The significance of this finding is under investigation. formation of functional neovasculature in vivo, including stimulating growth of new capillaries and arterioles that PUBLICATIONS functionally interconnect with existent coronary vascular Ezquerra, L., Herradon, G., Nguyen, T., Silos-Santiago, I., Deuel, T.F. Midkine is a potent regulator of the catecholamine biosynthesis pathway in mouse aorta. Life systems. Furthermore, in other studies, we showed that Sci., in press. reversal of endogenous pleiotrophin signaling in human Wang, Z.Y., Zhang, X.T., Shen, P., Loggie, B.W., Chang, Y.C., Deuel, T.F. A novel glioblastoma cells, via introduction of a dominant-negative variant of estrogen receptor α, hER-α36: tranduction of estrogen- and antiestrogen- dependent membrane-initiated mitogenic signaling. Proc. Natl. Acad. Sci. U. S. A., Ptn gene, reverses both the malignant and the angio- in press. genic phenotypes of these cells in vivo. Zhang, N., Zhong, R., Perez-Pinera, P., Herradon, G., Ezquerra, L., Wang, Z.Y., These findings indicate that pleiotrophin is an angio- Deuel, T.F. Identification of the angiogenesis signaling domain in pleiotrophin genic factor in vivo and that constitutive signaling of defines a mechanism of the angiogenic switch. Biochem. Biophys. Res. Commun. 343:653, 2006. the endogenous pleiotrophin in cancer cells is sufficient to initiate tumor angiogenesis and aggressive tumor 276 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE The S-Phase Checkpoint in prereplication complexes at replication origins. Consis- tent with this finding, overexpression of the replication Mammalian Cells licensing factor Cdt1 induces rereplication in certain tumor cell lines. We found that when the licensing X. Wu, E. Olson, E. Liu, A. Lee control is impaired by Cdt1 overexpression, the S-phase checkpoint is activated and rereplication is inhibited enome instability is a hallmark of the malignant through the ataxia telangiectasia–mutated and Rad3- phenotype and a driving force for tumorigene- related pathway. Our findings suggest that when the sis. S phase is genetically the most vulnerably G licensing control is compromised in mammalian cells, period of the cell cycle. In this phase, DNA must be the S-phase checkpoint provides another protection replicated faithfully in a timely fashion, and the entire mechanism to prevent DNA rereplication. genome must be duplicated exactly once per cell cycle. Errors or lesions generated spontaneously or in response to damaging environmental injuries must be repaired to DIVISION OF ONCOVIROLOGY maintain genome stability. The S-phase checkpoint monitors S-phase progression. It inhibits ongoing repli- Peter K. Vogt, Ph.D., Division Head cation as soon as DNA damage is detected, allowing time for DNA repair. In one area of our research, we focus on a disease- Molecular Genetics of Cancer linked protein complex termed Mre11/Rad50/Nbs1 (MRN). Mutations in the genes NBS1 and Mre11 lead P.K. Vogt, A. Bader, D. Bai, K. Bower, I. Dang, A. Denley, to the Nijmegen breakage syndrome and ataxia telang- A. Galkin, M. Gymnopoulos, H. Jiang, S. Kang, U. Karst, iectasia–like disorder, respectively. Cells derived from M. Scheerer, J. Shi, L. Zhao patients with Nijmegen breakage syndrome or ataxia he focus of our research is molecular mechanisms telangiectasia–like disorder undergo radioresistant DNA of carcinogenesis. We study viral and cellular synthesis, failing to suppress DNA replication in response T oncoproteins and tumor suppressors, defining to ionizing radiation. How MRN affects DNA replica- their functions in oncogenesis and identifying molecular tion to control the S-phase checkpoint, however, is targets for therapeutic intervention. In high-throughput not clear. screens, we look for small molecules that can interact We observed that MRN directly interacts with repli- with these targets and inhibit or reverse oncogenic cation protein A (RPA) and that this interaction is needed cellular transformation. for MRN to correctly localize to replication centers. Abol- ONCOGENIC TRANSFORMATION ishing the interaction of Mre11 with RPA leads to pro- Oncogenic transformation of cells requires changes nounced radioresistant DNA synthesis. We also found in gene activities, regulated at the level of transcription, that the interaction of MRN and RPA is required for sup- translation, or posttranslational modification. These pressing the initiation of replication upon DNA damage. changes result in a gain of function for specific growth- This suppression depends on the recruitment of MRN to promoting genes and a loss of function for growth- sites near the origins of chromosomal replication in S attenuating genes. phase by a direct interaction with RPA. These studies PHOSPHATIDYLINOSITOL-3′ -KINASE AS AN ONCO- indicate that in response to DNA damage MRN acts GENE AND CANCER TARGET directly at sites proximal to the origins of chromosomal Phosphatidylinositol-3′-kinase (PI3K) is a lipid replication to inhibit initiation of DNA replication, thereby kinase. It generates phosphatidylinositol 3,4,5-trisphos- providing an important mechanism underlying the intra- phate, an important second messenger molecule that S-phase checkpoint in mammalian cells. sets in motion complex growth-promoting signaling The second focus of our research is understanding chains in the cell. Gain of function in PI3K-dependent how DNA replication is controlled so that DNA is repli- signaling is common in cancer and has 3 principal cated once and only once per cell cycle. Rereplication of causes: amplification of the gene PIK3CA, which codes the genome, or even a segment of it, could lead to for the catalytic subunit p110α of class I PI3K; point genome instability. One key to the control of initiation mutations in PIK3CA; and loss of function in PTEN, of DNA replication is the tightly regulated assembly of MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 277 the antagonist of PI3K that functions as an important activated translation. A correlation exists between the tumor suppressor. Increased PI3K activity is a critical gain of function in Myc, tumor grade, and poor progno- determinant of the oncogenic cellular phenotype. When sis, suggesting that Myc plays an important role in the fused to a sequence that mediates membrane localiza- causation and progression of cancer. tion, p110α functions as a retroviral oncoprotein, induc- To function as a transcription factor, Myc must form ing transformation in cell culture and tumors in animals. a dimer with another protein, Max. The structure of Frequently occurring human cancers, such as breast the Myc-Max dimerization interface is known; single and colorectal cancers, have a high incidence of muta- amino acid substitutions at critical sites can break or tions in PIK3CA. The mutations are not randomly dis- stabilize dimerization. In collaboration with D.L. Boger tributed over the gene but are concentrated in 3 major and K.D. Janda, Department of Chemistry, we have hot spots along the coding sequence. This finding sug- isolated several small molecules that interfere with the gests that they are selected for and that they confer a dimerization of Myc and Max. As a consequence, these growth advantage to the cell.* The mutations are somatic molecules also prevent Myc DNA binding, Myc-depen- and cancer specific. We have shown that these muta- dent transcriptional activation, and Myc-induced onco- tions induce a gain of enzymatic function and that they genic transformation. activate the PI3K signals in the cell. The Myc-Max dimer belongs to a complex network The mutant PI3K proteins can transform cells in cul- that includes activators as well as repressors of tran- ture and induce tumors in animals. These tumors and the scription. All of the activators and repressors function cells transformed in culture are highly sensitive to the as dimers with the Max protein, making Max the com- TOR inhibitor rapamycin. TOR is a protein kinase and a mon denominator of the network.* Max is also the only component of the PI3K signal chain. Mutated PI3K is a component of the network that can form homodimers, highly attractive cancer target. The mutated protein is albeit weak and transcriptionally inactive homodimers. essential for the oncogenic phenotype of the tumor cell. Small molecules that specifically stabilize the Max The mutations do not occur in normal tissue. Because homodimer would make this essential partner unavail- PI3K is an enzyme, it can be readily manipulated with able for heterodimerization and for transcriptional reg- small molecules, and a gain of function is more easily ulatory activities. Such compounds would downregulate corrected than a loss of function. We have started a pro- the entire network. gram to identify small-molecule inhibitors that are spe- We have used the software program Autodock to cific for cancer-derived mutants of the enzyme. identify small molecules that bind preferentially to the Class I PI3K occurs in 4 isoforms encoded by dif- Max homodimer and enhance its stability. Two of these ferent genes. Cancer-specific mutations have been found compounds also effectively inhibit Myc-induced onco- only in the α isoform. We investigated the oncogenic genic transformation in cell culture. The principle of potential of the β, γ, and δ isoforms and discovered that downregulating the Myc network by stabilizing the Max these isoforms are oncogenic as wild-type proteins, homodimer was also validated by genetic experiments. inducing transformation in cell cultures. In contrast, the A mutant of Max was generated that formed homodimers α isoform is nononcogenic as wild-type protein and of enhanced stability with the wild-type proteins. Expres- requires a gain-of-function mutation to become trans- sion of this Max mutant made cells resistant to Myc- forming. The oncogenicity of the wild-type non-α iso- induced transformation by keeping Max in homodimers, forms of PI3K raises the possibility that these isoforms unavailable for dimerization with Myc. We are cur- could be involved in human cancer. Increased levels of rently performing additional screens for small-molecule expression of non-α isoforms have been found in specific stabilizers of Max and will analyze their effects on Myc- cancers, and the role of the isoforms in determining the dependent transcription and oncogenic transformation. oncogenic phenotype deserves further study. PUBLICATIONS SMALL-MOLECULE REGULATORS OF THE Bader, A.G., Kang, S., Vogt, P.K. Cancer-specific mutations in PIK3CA are onco- MYC NETWORK genic in vivo. Proc. Natl. Acad. Sci. U. S. A. 103:1475, 2006.

Myc is a transcriptional regulator that can strongly Bader, A.G., Kang, S., Zhao, L., Vogt, P.K. Oncogenic PI3K deregulates transcrip- stimulate cell proliferation. Increased levels and enhanced tion and translation. Nat. Rev. Cancer 5:921, 2005. function of Myc are common in cancer. They result from Bader, A.G., Vogt, P.K. Leucine zipper transcription factors: bZIP proteins. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Gan- gene amplification, elevated levels of transcription, and ten, D., Ruckpaul, K. (Eds.). Springer, New York, in press.* 278 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE

Bader, A.G., Vogt, P.K. Protein synthesis and cancer. In: Nutritional Genomics: myeloid gene, the gene for the M-SCF receptor, with 2 Impact on Health and Disease. Brigelius-Flohé, R., Joost, H.-G. (Eds.). Wiley-VCH, New York, 2006, p. 180. other important transcription factors, C/EBP and PU.1. To study the effect of AML1-ETO on hematopoiesis, Harada, J.N., Bower, K.E., Orth, A.P., Callaway, S., Nelson, C.G., Laris, C., Hogenesch, J.B., Vogt, P.K., Chanda, S.K. Identification of novel mammalian we produced various mouse models in which wild-type growth regulatory factors by genome-scale quantitative image analysis. Genome AML1 was replaced by AML1-ETO. Currently, we are Res. 8:1136, 2005. identifying cofactors involved in the synergy among Kang, S., Denley, A., Vanhaesebroeck, B., Vogt, P.K. Oncogenic transformation various transcription factors and in AML1-ETO–associ- induced by the p110β, γ, and δ isoforms of class I phosphoinositide 3-kinase. Proc. Natl. Acad. Sci. U. S. A. 103:1289, 2006. ated development of leukemia. A NOVEL UBIQUITIN-SPECIFIC ENZYME, UBP43 Vogt, P.K., Bader, A.G. Jun: stealth, stability, and transformation. Mol. Cell 19:432, 2005. In studying genes differentially expressed in AML1- ETO mice, we isolated the gene for a novel enzyme Vogt, P.K., Bader, A.G. Oncogenes and proto-oncogenes: jun oncogenes. In: Ency- clopedia of Respiratory Medicine. Laurent, G.J., Shapiro, S.D. (Eds.). Academic UBP43 (USP18), which belongs to a family of ubiqui- Press/Elsevier, Philadelphia, 2006, p. 241. tin-specific proteases. Like phosphorylation and dephos- Vogt, P.K., Bader, A.G., Kang, S. Phosphoinositide 3-kinase: from viral oncoprotein phorylation, ubiquitylation and deubiquitylation are to drug target. Virology 344:131, 2006. mechanisms for protein modification. Recently, we Vogt, P.K., Bader, A.G., Kang, S.K. PI 3-kinases: hidden potentials revealed. Cell showed that UBP43 is the only currently known enzyme Cycle 5:946, 2006. that removes a ubiquitin-like modifier, ISG15, from Vogt, P.K., Kang, S.K. Kinase inhibitors: vice becomes virtue. Cancer Cell 9:327, ISG15 conjugates. In mice that lacked the gene for 2006. UBP43, UBP43-deficient bone marrow cells were Xu, Y., Shi, J., Yamamoto, N., Moss, J.A., Vogt, P.K., Janda, K.D. A credit-card hypersensitive to treatment with type I interferon and library approach for disrupting protein-protein interactions. Bioorg. Med. Chem. 14:2660, 2006. died via apoptosis in the presence of interferon. Most important, in UBP43-deficient cells, interferon induced a prolonged Stat1 tyrosine phosphorylation, DNA bind- Molecular Mechanisms of ing, and interferon-mediated gene activation. UBP43- deficient mice are resistant to certain viral and bacterial Leukemia Development and infections. Currently, we are analyzing molecular pathways affected by UBP43. Protein Modification by a ROLE OF ISG15 CONJUGATION IN IMMUNE Ubiquitin-Like Modifier RESPONSES The gene for ISG15 was originally cloned as a gene highly upregulated by interferon and encodes a small D.-E. Zhang, O.A. Malakhova, L.F. Peterson, M. Yan, ubiquitin-like protein. Unlike ubiquitin and other ubiq- A. Boyapati, J.-K. Luo, W. Zou, J.R. Biggs, J.-H. Kim, uitin-like modifiers, ISG15 is not present in lower E.-Y. Ahn, J. Wang, A.J. Okumura, F. Okumura, B. Yeung, eukaryotes, such as yeast, indicating that it may be B. Abdulla, X. Yin, M.-C. Lo associated with specialized functions in higher eukary- AML1 AND ITS FUSION PROTEIN AML1-ETO IN otic cells. Upon viral infection, bacterial infection, or BLOOD CELL DIFFERENTIATION other stress stimulation, ISG15 can be detected in cells cute myeloid leukemia is a major hematopoietic both in free and in conjugated form (ISGylation). Using malignant neoplasm characterized by the prolifer- high-throughput Western blot analysis, we identified 4 A ation of a malignant clone of myeloid progenitor ISGylated proteins: Stat1, Jak1, Erk1, and PLCγ1. We cells. One of the most common targets of chromosomal also found that Ubc8 is an ISG15-conjugating enzyme translocations that have been implicated in this neo- and that Efp is an ISG15 ligase. Regulation of protein plasm is the gene AML1 (RUNX1). The gene was isolated ISGylation may provide valuable treatments to control through a study of t(8;21) chromosomal translocation; cell function and survival. We are using techniques the results revealed that the runt homology domain of such as gene depletion, protein interaction, biochemi- AML1 is fused to a gene termed ETO (MTG8) to form a cal purification, and gene regulation to study the bio- fusion protein called AML1-ETO. Subsequent studies logical function of this interesting protein modification. indicated that the protein AML1 is crucial for normal PUBLICATIONS hematopoiesis. We previously discovered that AML1 Biggs, J.R., Zhang, Y., Peterson, L.F., Garcia, M., Zhang, D.-E., Kraft, A.S. Phos- phorylation of AML1/RUNX1 regulates its degradation and nuclear matrix associa- synergistically activates the expression of a critical tion. Mol. Cancer Res. 3:391, 2005. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 279

Giannakopoulos, N.V., Luo, J.-K., Papov, V., Zou, W., Lenschow, D.J., Jacobs, producing organ-specific disease. We use 3 major B.S., Borden, E.C., Li, J., Virgin, H.W., Zhang, D.-E. Proteomic identification of proteins conjugated to ISG15 in mouse and human cells. Biochem. Biophys. Res. approaches: animals transgenic for the human protein Commun. 336:496, 2005. transthyretin, cell cultures to determine how the mis-

Kim, K.I., Malakhova, O.A., Hoebe, K., Yan, M., Beutler, B., Zhang, D.-E. folded proteins injure their cellular targets, and genetic Enhanced antibacterial potential in UBP43-deficient mice against Salmonella epidemiology to identify potential disease carriers and typhimurium infection by up-regulating type I IFN signaling. J. Immunol. 175:847, 2005. assess the effects of other hereditary and environmental factors on the disease. Kim, K.I., Yan, M., Malakhova, O.A., Luo, J.-K., Shen, M.-F., Zou, W., de la Torre, J.C., Zhang, D.-E. Ube1L and protein ISGylation are not essential for α/β Our studies of the clinical impact of the transthyretin interferon signaling. Mol. Cell. Biol. 26:472, 2006. mutation Val122Ile, an allele carried by 3%–4% of Kim, K.I., Zhang, D.-E. UBP43, an ISG15-specific deconjugating enzyme: expres- African Americans, have revealed that 10% of African sion, purification, and enzymatic assays. Methods Enzymol. 398:491, 2005. Americans more than 60 years old who have severe Peterson, L.F., Boyapati, A., Ranganathan, V., Iwama, A., Tenen, D.G., Tsai, S., heart failure are carriers of the amyloidogenic allele. Zhang, D.-E. The hematopoietic transcription factor AML1 (RUNX1) is negatively regulated by the cell cycle protein cyclin D3. Mol. Cell. Biol. 25:10205, 2005. These data are consistent with our finding of an age- associated decrease in the prevalence of the allele in Zou, W., Papov, V., Malakhova, O.A., Kim, K.I., Dao, C.T., Li, J., Zhang, D.-E. ISG15 modification of ubiquitin E2 Ubc13 disrupts its ability to form thioester African Americans, which suggests that the allele has bond with ubiquitin. Biochem. Biophys. Res. Commun. 336:61, 2005. a discrete mortality effect. Our projections, based on

Zou, W., Zhang, D.-E. The interferon-inducible ubiquitin-protein isopeptide ligase prevalence and demographic data, indicate that at any (E3) EFP also functions as an ISG15 E3 ligase. J. Biol. Chem. 281:3989, 2006. moment as many as 100,000 to 150,000 persons in the United States may have this form of heart disease. Population genotyping in various African locales suggests DIVISION OF RHEUMATOLOGY an origin in West Africa with maintenance of the allele in the United States via a founder effect in the original RESEARCH slave population. Our current studies are designed to

W.M. Keck Autoimmune Disease Center precisely define the disease-producing effects of the allele, which behaves as an autosomal dominant with Joel N. Buxbaum, M.D., Division Head age-dependent penetrance, and the development of signs and symptoms. In collaboration with D.R. Salomon, Department of Pathogenesis of Late-Onset Molecular and Experimental Medicine, we have contin- Genetic Diseases Related ued our studies in transthyretin transgenic mice. We are using microarray techniques to analyze the tran- to Abnormalities of scriptional patterns of (1) tissues that are the targets for transthyretin deposition and (2) the hepatocytes that Protein Conformation synthesize transthyretin. We have identified groups of genes that are associated with resistance to, or lack J.N. Buxbaum, N. Reixach, Z. Ye, L. Friske, M.J. Saraiva,* of, deposition and have defined changes in the hepatic N. Schork,** D. Jacobson,*** G. Gallo,**** C. Tagoe,***** site of transthyretin synthesis that seem to influence O. Suhr† whether or not deposition occurs in distant tissues. Our * Institute of Cell and Molecular Biology, Oporto, Portugal current hypothesis is that the quality of the hepatic ** University of California, San Diego, California response to a misfolded protein determines how much *** Boston University School of Medicine, Boston, Massachusetts abnormal conformer with fibril-forming potential gets **** NYU School of Medicine, New York, New York into the circulation. These results shed light on the recent ***** Albert Einstein College of Medicine, Bronx, New York observations in recipients of normally functioning liver † Umeå University, Umeå, Sweden transplants from donors with transthyretin mutations; the e are studying the pathogenesis of a group of recipients experienced tissue deposition of transthyretin hereditary human diseases, the transthyretin in a shorter time than was anticipated on the basis of W amyloidoses, that are the result of protein studies of persons who carry these mutations. misfolding. The misfolded molecules aggregate and are We have extended our tissue culture studies designed deposited in the heart, kidney, and peripheral nerves, to determine how oligomeric aggregates of transthyretin 280 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE produce damage to heart and nerve cells. We have now Oxidative Stress, Protein defined the pathways of transit of transthyretin in cells that are either sensitive to or resistant to the toxic Oxidation, and Disease effects of aggregated transthyretin and the pathways taken by toxic and nontoxic proteins. J.S. Friedman, R. Gabriel, F.M. Martin, J. Yi In collaboration with J. Kelly, Department of Chem- e are investigating how loss of superoxide istry, and investigators in Boston; Rochester, Minnesota; dismutase 2 (SOD2) affects blood cells. London; Umeå, Sweden; Portugal; and Japan, we orga- W SOD2 deficiency in murine blood cells results nized a clinical trial of diflunisal, a small molecule capa- in an anemia that is similar to sideroblastic anemia ble of stabilizing the native structure of transthyretin. in humans. During the past year, we developed and pub- The proposal recently received funding from the National lished a novel method for purification of iron-overloaded Institutes of Health, and we have begun recruiting cells from SOD2-deficient mice. This method relies on patients for the trial. magnetic purification of iron-loaded cells (Fig. 1). We In collaboration with J. Waalen, Department of Molecular and Experimental Medicine, and T. Bartfai, Molecular and Integrative Neurosciences Department, we performed an epidemiologic analysis of the prevalence of various forms of arthritis in persons with extreme obesity (i.e., body mass index >30). We found that the prevalence of rheumatoid arthritis, but not osteoarthritis, was reduced in persons who are extremely obese. However, examination of other data sets indicates that once rheumatoid arthritis develops, it is more severe in persons who are obese than in persons who are thinner. These data suggest that adipose tissue may be a significant source of anti-inflammatory cytokines, sufficient to suppress the initial development of rheumatoid arthritis in the presence of an inflammatory trigger. Once the inflammatory threshold is breached, proinflammatory cytokines derived from adipose tissue add to the total level of inflammation.

PUBLICATIONS –/– Buxbaum, J.N. Meeting report: VIth International Symposium on Familial Amy- Fig. 1. Purification of SOD2 siderocytes. A, Inset, iron-laden loidotic Polyneuropathy and Other Transthyretin Disorders and the Vth International SOD2+/+ (left) and SOD2–/– (right) cells purified from red blood Workshop on Liver Transplantation in Familial Amyloidotic Polyneuropathy, August cells (RBCs). Scale bar = 1 cm; CBF, column-bound fractions. Dot 24-26, 2005, La Jolla, California, USA. Amyloid, in press. plot shows a significant (>25-fold) enrichment of magnetic iron- Buxbaum, J.N. Transthyretin and the transthyretin amyloidoses. In: Protein Misfold- laden red blood cells purified from SOD2–/– (n = 22) and SOD2+/+ ing, Aggregation and Conformational Diseases. Uversky, V.N., Fink, A.L. (Eds.). (n = 18) cell suspensions. ***P < .001 by unpaired 2-tailed t Springer, New York, in press. Vol. 4 in Protein Reviews. Atassi, M.A. (Series Ed.). test.B, Perl stain of SOD2–/– magnetically purified siderocytes; inset Buxbaum, J.N. Treatment and prevention of the amyloidoses: can the lessons shows deposition of cellular iron. Original magnifications x63 and learned be applied to sporadic inclusion-body myositis? Neurology 66(2 Suppl. 1): x100, respectively. S110, 2006.

Buxbaum, J.N., Jacobson, D.R., Tagoe, C., Alexander, A., Kitzman, D., Green- berg, B., Thaeemit-Chen, S., Lavori, P. Transthyretin V1221 in African Americans have adapted the method to purify abnormal cells from with congestive heart failure. J. Am. Coll. Cardiol. 47:1724, 2006. the bone marrow of patients with sideroblastic anemia, an advance that will facilitate both gene expression and proteomic analyses in this disease. Using this method, we are analyzing protein oxidation and identifying protein components most closely associated with magnetically susceptible iron, that is, biological iron that is attracted to a magnet. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 281

One of our initial findings is that iron-positive sub- genes. We wish to understand the mechanism of cellular fractions from SOD2-deficient cells or cells from siRNA-mediated transcriptional gene silencing (TGS) patients with sideroblastic anemia are enriched in mito- in human cells and to use conditionally replicating lentivi- chondria. We will use the magnetic purification method ral vectors to apply RNA interference to treat infection to characterize mitochondria from patients with sider- with HIV type 1. oblastic anemia for functional defects or mutations in Currently, most investigators use siRNAs to target mitochondrial DNA that may play a role in pathogenesis and inactivate a particular gene transcript in a proce- of the anemia or the related disorder myelodysplasia. dure termed posttranscriptional gene silencing. In Because protein turnover is slow or absent in mature human cells, as in plants and the fission yeast Saccha- red cells, SOD2-deficient cells and iron-loaded cells in romyces pombe, siRNAs mediate TGS by targeting the patients with sideroblastic anemia accumulate oxidatively gene promoter. The observation that siRNAs targeted damaged protein. Using SOD2-deficient cells, we devel- to a gene’s promoter can specifically silence that gene oped 2 novel methods for enriching and identifying oxi- were exciting, but the fundamental mechanism of this dized proteins that can be used in 2-dimensional gel silencing remained unknown. During the past year, we electrophoresis. Use of these methods will allow more have uncovered many of the mechanistic interactions detailed comparison of changes in oxidized proteins involved in siRNA-mediated TGS in human cells. that may accompany aging, inflammatory processes, We found that siRNAs targeted to promoter regions and neurodegenerative disease. can cause silent modifications in chromatin, such as The first method involves the use of multiple fluoro- methylation of histone 3, lysine 9, and lysine 27. We phores (e.g., Cy-2, Cy-3, Cy-5) that can form derivatives also discovered that DNA methyltransferase 3A is of carbonylated proteins by using a hydrazide moiety. involved in a putative transcriptional silencing complex Individual samples are labeled with distinct fluorophores that is directed to the targeted promoter by the antisense and then are combined for comparative 2-dimensional strand of the siRNA. Moreover, the protein Argonaute 1 gel analysis. The second method involves the use of a appears to be involved, possibly in unwinding the siRNAs biotin “hook” to obtain oxidized proteins from more com- and presenting the antisense strand to the complex. plex protein mixtures. Using these techniques, we can Although much has been learned about the putative enrich, identify, and quantitatively compare oxidized transcriptional silencing complex in human cells, it has proteins in experimental samples. We think that these remained unclear whether siRNAs, specifically the techniques will be useful in probing the role of protein antisense strand of the siRNA, can recognize and bind oxidation in signal transduction and will help identify directly to DNA or to an uncharacterized RNA that over- specific oxidation-sensitive proteins important in the laps the targeted promoter. pathogenesis of sideroblastic anemia and perhaps, more We now have direct evidence that a low-copy RNA generally, targets of oxidation in age-related degenera- is transcribed through RNA polymerase II promoters for tive disease. 6 different genes. These promoter-specific RNAs are initially detected by the antisense strand of promoter- PUBLICATIONS Martin, F.M., Bydlon, G., Welsh, M.L., Friedman, J.S. A method for rapid mouse directed siRNA, and their expression is reduced along siderocyte enrichment. Exp. Hematol. 33:1493, 2005. with that of the corresponding promoter-expressed

Martin, F.M., Friedman, J.S. SOD2 deficiency anemia and RBC oxidative stress. mRNA. Additionally, when antisense phosphorothioate Antioxid. Redox Signal., in press. oligodeoxynucleotides are used to block the siRNA target site in the promoter-specific RNA, siRNA-mediated TGS is abrogated. These data suggest that low levels Transcriptional Gene Silencing of promoter-specific RNAs are present in RNA polymerase II promoters that act either in trans (Fig. 1A) by Small Interfering RNA during transcription or in cis (Fig. 1B) as a local scaffolding recognition motif for the antisense strand of the K.V. Morris, J. Han, J.P. Delacruz siRNA to bind the targeted promoter and mediate TGS. NA interference via small interfering RNAs (siRNAs) is a new experimental method for PUBLICATIONS Kawasaki, H., Taira, K., Morris, K.V. siRNA induced transcriptional gene silencing R knocking out (i.e., inactivating or suppressing) in mammalian cells. Cell Cycle 4:442, 2005. 282 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE Autoimmunity Induced by Xenobiotics

K.M. Pollard, D. Cauvi, G. Cauvi

e focus on how interactions between the environment and genetics affect induction of W autoimmune diseases. We use murine models of systemic autoimmunity in which disease is elicited by exposure to xenobiotics. An important aspect of our research is a comparison of the similarities and differences between induced systemic autoimmunity and idiopathic systemic autoimmunity, such as systemic lupus erythematosus, in mice and humans. Fig. 1. Two models for siRNA-mediated TGS in human cells. A, DOWNREGULATION OF DECAY-ACCELERATING FAC- In the trans model, the promoter-specific RNA (pRNA) is recognized by TOR AND ACTIVATION OF CD4+ T CELLS IN the antisense strand of the siRNA during RNA polymerase II INDUCED SYSTEMIC AUTOIMMUNE DISEASE (RNAPII)–mediated transcription of the siRNA targeted promoter. The Decay-accelerating factor (DAF/CD55) is a regula- antisense strand of the siRNA guides a putative transcriptional silencing complex (possibly composed of DNMT3A, Ago-1, HDAC-1, tory protein that protects cells from attack by autologous and/or EZH2) to the targeted promoter where histone modifications complement proteins. DAF deficiency exacerbates auto- leading to initial gene silencing would occur. B, In the cis model, a immunity, most likely by acting as a regulator of T-cell pRNA acts as a scaffolding by overlapping the entire targeted pro- immunity. Therefore, modulation of DAF expression on moter, possibly as part of the local chromatin structure. The pRNA T cells may contribute to the development of autoim- then acts as a scaffolding for the antisense strand of the siRNA to munity. To test this idea, we examined DAF expression bind the targeted promoter along with the putative transcriptional silencing complex or to recruit the complex. in murine mercury-induced autoimmunity. In B10.S mice, which are susceptible to mercury- Morris, K., Castanotto, D., Al-Kadhimi, Z., Jensen, M., Rossi, J.J., Cooper, L.J.N. induced autoimmunity, exposure to mercury resulted Enhancing siRNA effects in T cells for adoptive immunotherapy. Hematology 10:461, 2005. in reduced expression of DAF mRNA (Fig. 1). Expres- sion of the protein was reduced on CD4+ T cells, par- Morris, K.V. siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code. Cell Mol. Life Sci. 62:3057, 2005. ticularly those with an activated/memory phenotype

Morris, K.V. Therapeutic potential of siRNA-mediated transcriptional gene silencing. Biotechniques 40(Suppl.):S7, 2006.

Morris, K.V. VIR-496(VIRxSYS). Curr. Opin. Investig. Drugs 6:209, 2005.

Morris, K.V., Looney, D.J. Characterization of human immunodeficiency virus (HIV)-2 vector mobilization by HIV-1. Hum. Gene Ther. 16:1463, 2005.

Morris, K.V., Rossi, J.J. Antiviral applications of RNAi. Curr. Opin. Mol. Ther. 8:115, 2006.

Morris, K.V., Rossi, J.J. Lentivirus-mediated RNA interference therapy for human immunodeficiency virus type 1 infection. Hum. Gene Ther. 17:479, 2006.

Weinberg, M.S., Villeneuve, L.M., Ehsani, A., Amarzguioui, M., Aagaard, L., Chen, Z.X., Riggs, A.D., Rossi, J.J., Morris, K.V. The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 12:256, 2006. Fig. 1. Exposure to mercury reduced the expression of DAF1 mRNA in the spleens of B10.S mice, which are susceptible to mercury-induced autoimmunity. DBA/2, B10.S, and NZB mice were injected with mercury (filled bar) or phosphate-buffered saline (open bar) twice a week for 4 weeks. Total RNA isolated from spleens was analyzed for DAF1 mRNA. DAF1 levels are expressed relative to cyclophilin A. Data are expressed as mean ± SEM, with n = 4–5 mice per group. *P < .05. MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE 283

(CD44hi) that accumulate as a result of exposure to Pollard, K.M., Hultman, P. Fibrillarin autoantibodies. In: Textbook of Autoantibodies. Shoenfeld, Y., Meroni, P.L., Gershwin, M.E. (Eds.). Elsevier, Philadelphia, in press. mercury. In contrast, DBA/2 and INF-γ–deficient B10.S mice, which are resistant to mercury-induced autoimmunity, had no increase in the number of activated/ Expression and Function of the memory CD4+ T cells and no change in DAF expression after exposure to the metal. These findings sug- High-Affinity Receptor for IgE gest that development of autoimmunity is linked to a reduction in DAF expression on an expanded or long- M.W. Robertson, Z. Wang, X. Tian lived population of activated/memory CD4+ T cells. he high-affinity receptor for IgE (FcεRI) is a mul- CONTROL OF CONSTITUTIVE EXPRESSION OF DAF tisubunit membrane complex that is critically BY SP1 Modulation of murine DAF expression during the T involved in the pathology of the allergic response. development of autoimmunity suggests that DAF con- The receptor is highly expressed by mast cells and tributes to CD4+ T-cell activity, and this idea is sup- basophils. Upon stimulation by IgE-antigen complexes, ported by the hyperactivity of T cells in DAF-deficient these cells secrete histamine and other mediators of mice, as reported by others. Modulation of DAF expres- hypersensitivity, leading to the clinical signs and symp- sion could therefore be a critical regulatory mechanism toms of allergy. Our main focus is defining the molecu- in both innate and adaptive immune responses. To lar basis of FcεRI assembly and expression and the role identify and characterize key transcriptional regulatory of FcεRI structure in initiating or propagating IgE-medi- elements that control DAF expression in mice, we cloned ated cellular activation. a 2.5-kb fragment corresponding to the 5′ flanking region FCε RI ASSEMBLY AND TRANSPORT of Daf1, the mouse gene for DAF. One of our goals is to understand how FcεRI αγ2 and Sequence analysis showed that the mouse Daf1 αβγ2 isoforms assemble and traffic in cells. Recently, we promoter lacks conventional TATA and CCAAT boxes assessed the structural basis of transport of the α-chain and has a high guanine-cytosine content. Rapid ampli- of the FcεRI from the endoplasmic reticulum. We found fication of cDNA ends was used to identify 1 major and that a previously defined endoplasmic reticulum reten- 2 minor transcription start sites 47, 20, and 17 bp tion signal located near the C terminus of the α-chain is only weakly functional in regulating steady-state trans- upstream of the translational codon. Positive and neg- port of the receptor. We also found that a membrane- ative regulatory regions were identified by transiently proximal dilysine sequence in the cytoplasmic domain of transfecting sequential 5′ deletion constructs of the 5′ the α-chain regulates transport, and we determined that flanking region into NIH/3T3, M12.4, and RAW264.7 the new motif functions synergistically with the C-termi- cells. Mutational analyses of the promoter region com- nal retention signal to stringently retain the FcεRI sub- bined with an enzyme-linked immunosorbent assay unit in the endoplasmic reticulum. specific for the transcription factor Sp1 indicated that In another study, we investigated the structural basis Sp1 is required for basal transcription and lipopoly- of the assembly of the αγ subunit, a process previously saccharide-induced expression of Daf1. These findings thought to occur exclusively through interaction of the provide new information on the regulation of the mouse subunit transmembrane domains. Our data revealed Daf1 promoter and will facilitate further studies on that the cytoplasmic domain determinants of each the expression of Daf1 during immune responses. subunit contribute significantly to optimal αγ associa-

PUBLICATIONS tion and to FcεRI-dependent function in transfected Cauvi, D.M., Cauvi, G., Pollard, K.M. Constitutive expression of murine decay- mast cells. accelerating factor 1 (DAF1) is controlled by the transcription factor Sp1. J. Immunol., in press. INHIBITION OF FCε RI-MEDIATED CELLULAR ACTIVA TION BY A MONOCLONAL ANTIBODY TO Hultman, P., Taylor, A., Yang, J.M., Pollard, K.M. The effect of xenobiotic exposure on spontaneous autoimmunity in (SWR x SJL)F1 hybrid mice. J. Toxicol. Environ. THE FCε RI α -CHAIN Health A 69:505, 2006. Previously, we showed that 5H5F8, a monoclonal Lynes, M.A., Fontenot, A.P., Lawrence, D.A., Rosenspire, A.J., Pollard, K.M. antibody to the α-chain of FcεRI, inhibits IgE-depen- Gene expression influences on metal immunomodulation. Toxicol. Appl. Pharmacol. 210:9, 2006. dent activation in mast cells and basophils by a unique mechanism that does not involve perturbation of the Pollard, K.M. (Ed.). Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease. Wiley-VCH, New York, 2006. IgE-binding site. The 5H5F8 epitope has been mapped 284 MOLECULAR AND EXPERIMENTAL MEDICINE 2006 THE SCRIPPS RESEARCH INSTITUTE to the linear membrane proximal region, and the epitope assignment has now been confirmed from the crystallographic structure of a complex formed between 5H5F8 and a synthetic peptide representing the 5H5F8 epitope. We hypothesized that the membrane proximal region of the FcεRI α-chain may be critically important in initiating or propagating FcεRI-dependent signaling. In recent mutagenesis studies, we found that replacing the native membrane proximal region with a series of different sequences significantly enhanced cell-surface expression of the receptor but also resulted in loss of FcεRI-dependent function. On the basis of these findings, we propose that the membrane proximal region is a multifunctional site that plays a critical role in both FcεRI cell transport and transmembrane signaling.

PUBLICATIONS Cauvi, D.M., Tian, X., von Loehneysen, K., Robertson, M.W. Transport of the IgE receptor α-chain is controlled by a multicomponent intracellular retention signal. J. Biol. Chem. 281:10448, 2006. Molecular and Integrative Neurosciences

(Top) Transgenic mice (tg) engineered to overexpress the uncoupling protein-2 in the hypocretin neurons have a modest reduction of core body temperature in the dark/active part of the day. (Bottom) Fed ad libitum, these mice have a prolonged median lifespan compared to their wild- type littermates (wt). Work done by Bruno

Conti, Ph.D., in the laboratory of Tamas

Bartfai, Ph.D. Marisa Roberto, Ph.D.

Assistant Professor

Molecular and Integrative

Neurosciences Department MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 287

MOLECULAR AND Hermann H. Gram, Ph.D. Thomas Nelson, Ph.D. Claes Wahlestedt, M.D., INTEGRATIVE Adjunct Associate Professor Assistant Professor Ph.D. Adjunct Professor NEUROSCIENCES Donna L. Gruol, Ph.D. Benjamin Neuman, Ph.D. DEPARTMENT Associate Professor Assistant Professor Tammy Wall, Ph.D. Adjunct Associate Professor STAFF Steven J. Henriksen, Ph.D. Michael B.A. Oldstone, M.D. Adjunct Professor Professor Friedbert Weiss, Ph.D. Tamas Bartfai, Ph.D. Professor Professor and Chairman Paul L. Herrling, Ph.D. Shirley M. Otis, M.D. Adjunct Professor Adjunct Professor J. Lindsay Whitton, M.B. Serge Ahmed, Ph.D. Ch.B., Ph.D. Adjunct Assistant Professor Tomas Hokfelt, M.D., Ph.D. Loren Parsons, Ph.D. Professor Adjunct Professor Associate Professor Etienne Baulieu, Ph.D. Eric Zorilla, Ph.D. Adjunct Professor Danny Hoyer, Ph.D. Tommy Phillips, Ph.D. Assistant Professor Adjunct Professor Adjunct Assistant Professor Floyd E. Bloom, M.D. Professor Emeritus Koki Inoue, Ph.D. John Polich, Ph.D. STAFF SCIENTISTS Adjunct Associate Professor Associate Professor Jason Botten, Ph.D. Walter Francesconi, Ph.D. Assistant Professor George F. Koob, Ph.D. Luigi Pulvirenti, M.D. Professor Adjunct Associate Professor Bumsuk Hahm, Ph.D. Benjamin Boutrel, Ph.D. Harvey Karten, M.D. Teresa Reyes, Ph.D. Professor Emeritus Salvador Huitrón-Reséndiz, Adjunct Professor Adjunct Assistant Professor Ph.D. Karen T. Britton, M.D., Ph.D. Henri Korn, M.D., Ph.D. Catherine Rivier, Ph.D. Adjunct Associate Professor Xiaoying Lu, Ph.D. Adjunct Professor Adjunct Professor Michael Buchmeier, Ph.D. M. Cecilia Marcondes, Ph.D. Professor Stefan Kunz, Ph.D. Marisa Roberto, Ph.D. Assistant Professor Assistant Professor Remi Martin-Fardon, Ph.D. Iain L. Campbell, Ph.D, Thomas Krucker, Ph.D. Amanda Roberts, Ph.D. Adjunct Professor Zhiguo Nie, Ph.D. Adjunct Assistant Professor Assistant Professor Kathleen Cashman, Ph.D. Robert Purdy, Ph.D. Cary Lai, Ph.D. Michael G. Rosenfeld, M.D. Professor Emeritus Associate Professor Adjunct Professor Mitra Rebek, Ph.D. Zhen Chai, Ph.D. Ulo Langel, Ph.D. Pietro P. Sanna, M.D. Adjunct Assistant Professor Heather Richardson, Ph.D. Adjunct Professor Associate Professor Svetlana Semenova, Ph.D. Jerold Chun, M.D., Ph.D. Michel Le Moal, M.D., Ph.D. Paul Schweitzer, Ph.D. Adjunct Professor Adjunct Professor Associate Professor SCIENCE ASSOCIATES Bruno Conti, Ph.D. Jan O. Lundstrom, Ph.D. George R. Siggins, Ph.D. Assistant Professor Adjunct Professor Professor Elena Crawford

Jose Criado, Ph.D. Athina Markou, Ph.D. Craig Slawecki, Ph.D. Caroline Lanigan, Ph.D. Adjunct Assistant Professor Associate Professor Assistant Professor Rong-Sheng Lee, Ph.D. Juan Carlos de la Torre, Ph.D. Barbara J. Mason, Ph.D. Antoine Tabarin, Ph.D. Associate Professor Professor Adjunct Associate Professor Hanna Lewicki

Cindy L. Ehlers, Ph.D. Dorian McGavern, Ph.D. Michael A. Taffe, Ph.D. John Light Associate Professor Assistant Professor Assistant Professor Sam Madamba Howard S. Fox, M.D., Ph.D. Madis Metsis, Ph.D. Lars Terenius, Ph.D. Associate Professor Adjunct Associate Professor Adjunct Professor Antoinette Tishon 288 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

RESEARCH ASSOCIATES Olivier George, Ph.D. Sunmee Wee, Ph.D. Noemi Sevilla, Ph.D. Universidad Autonoma de Mehrdad Alirezaei, Ph.D. Sandy Ghozland, Ph.D. Jason Whitmire, Ph.D. Madrid Madrid, Spain Harinder Aujla, Ph.D. Nicholas Gilpin, Ph.D. Manisha Yadav, Ph.D. Christina Spiropoulou, Ph.D. Michal Bajo, M.D., Ph.D. Thomas Greenwell, Ph.D. Ge Ying, Ph.D. Centers for Disease Control and Prevention Hilda Bajova, D.V.M. Hazuki Hagihara, Ph.D. Yu Zhao, Ph.D. Atlanta, Georgia

Marco A. Baptista, Ph.D. Peter James, Ph.D. Elina Zuniga, Ph.D. Persephone Tough, M.D. Edward Jenner Institute for Fulvia Berton, Ph.D. Paul John Kenny, Ph.D. Vaccine Research VISITING INVESTIGATORS Compton, England David Brooks, Ph.D. Izabella Klein, Ph.D. Hedieh Badie, Ph.D. Genomics Institute of the Adriaan Bruijnzeel, Ph.D. Henning Lauterbach, Ph.D. Novartis Research Foundation San Diego, California Tricia Burdo, Ph.D. Dusan Lekic, M.D., Ph.D. Roberto Ciccocioppo, Ph.D. Renaud Jean Burrer, Ph.D. Matthias Liechti, Ph.D. University of Camerino Camerino, Italy Vez Repunte Canonigo, Ph.D. Li Ying Liou, Ph.D. Urs Christen, Ph.D. Althea Capul, Ph.D. Fei Lu, Ph.D. La Jolla Institute for Allergy and Immunology Roberto Cervera, Ph.D. Chitra Mandyam, Ph.D. La Jolla, California

Zhifeng Chen, Ph.D. Monica Mendez-Diaz, Ph.D. Jean E. Gairin, Ph.D. CNRS Irene Yoon-Jin Choi, Ph.D. Victor Mendoza-Fernandez, Toulouse, France Ph.D. Christopher Cornell, Ph.D. Karine Guillem, Ph.D. Covadonga Paneda, Ph.D. University of Pennsylvania Cromwell Cornillez-Ty, Ph.D. Philadelphia, Pennsylvania Neil Paterson, M.D. Rebecca Crean, Ph.D. Dirk Homann, M.D., Ph.D. Gurudutt Pendyala, Ph.D. University of Colorado Stephen J. Crocker, Ph.D. Health Sciences Center Jilla Sabeti, Ph.D. Denver, Colorado Chris Davis, Ph.D. Valentina Sabino, Ph.D. Shinchi Iwasaki, M.D., Ph.D. Christopher Dayas, Ph.D. Osaka City University Ana Sanchez, Ph.D. Medical School Andre Der-Avakian, Ph.D. Osaka, Japan Manuel Sanchez-Alavez, M.D, Toby Escher, Ph.D. Ph.D. Rolf Kiessling, Ph.D. Karolinska Institutet Kurt Edelmann, Ph.D. Lisa Sharkey, Ph.D. Stockholm, Sweden

Ralph Feuer, Ph.D. Nimish Sidhpura, Ph.D. Denise Naniche, Ph.D. Universitat de Barcelona Cindy Funk, Ph.D. Iustin Tabarean, Ph.D. Barcelona, Spain

Lucile Garidou, Ph.D. Matthew Trifilo, Ph.D. Laura O’Dell, Ph.D. University of Texas Peter Gaskill, Ph.D. Brendan Walker, Ph.D. El Paso, Texas MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 289

ciated with the consumption of oxygen and nutrients required to produce ATP. This is what Lars Ernster called oxygen toxicity, and as long as we live on earth it can only be reduced by reducing our energy requirement. One way to achieve this reduction is through calorie restriction (CR), a controlled dietary regimen that prolongs lifespan and delays the onset of certain diseases. CR is associated with a reduction in core body temperature (CBT), most likely an adaptive mechanism for coping with limited food resources. Considering that in homeotherms, most calorie intake is used to keep a constant CBT against a normally lower ambient temperature, Dr. Conti set out to test whether reduction of CBT per se could contribute to longevity in the absence of CR. To achieve this goal, he has relied on data showing that CBT is controlled centrally by temperature-sensitive neurons in the hypothalamic preoptic area, where the “core body thermostat” resides. By generating transgenic mice that produce heat locally in the vicinity of

Tamas Bartfai, Ph.D. the preoptic area, he mimicked an increase of CBT. This triggered a thermoregulatory response that resulted in a modest but prolonged reduction of CBT. Fed ad libitum, Chairman’s Overview these transgenic mice have similar calorie intake to that of their wild-type littermates but show increased metabolic his has been a scientifically productive year for efficiency and prolonged median lifespan, demonstrat- the researchers in the Molecular and Integrative ing that a reduction of CBT can contribute to longevity T Neurosciences Department. Our members have independent of CR. These mice will serve as a model published 192 papers in top-tier journals and 2 acclaimed for investigating thermo and metabolic regulation in textbooks: Neurobiology of Addiction by George Koob mammals and the effects of CBT on aging. and Michel Le Moal and Drug Discovery by Tamas The SARS virus has scared the world and has also Bartfai and Graham Lees. given us an incredible dress rehearsal for how to rapidly We have made important breakthroughs in work on identify pathogenic microbes before catastrophic pan- viruses such as severe acute respiratory syndrome (SARS), demics take millions of lives. In a consortium that has uti- Lassa, and lymphocytic choriomeningitis virus and their lized the best structural chemistry and nuclear magnetic interactions with different cell types in the brain. We have resonance expertise at Scripps Research, Drs. Buchmeier learned more about how viral infections are cleared and and Neuman have shown that they can rapidly solve the what strategies viruses use to remain dormant in neurons. structure of multiple surface proteins of new viruses and George Siggins and Marisa Roberto have identified thereby identify targets for vaccine development in a very the mechanism of action of the neuropeptide orphanin short timeframe. In collaboration with Ron Milligan and on neurons in the amygdala, and have drawn important Mark Yeager, they have used electron cryomicroscopy to conclusions about the contribution of this peptide to anxi- investigate the supramolecular architecture of the SARS ety disorders and alcohol addiction. Bruno Conti’s work virus. Working with Peter Kuhn, they have used x-ray on longevity and the work of Michael Buchmeier and crystallography to solve the structure of the SARS Benjamin Neuman on SARS are discussed below. nsp10 protein, and have discovered a novel structural Aging is considered by many scientists to be the motif: a previously unknown fold containing 2 zinc-bind- result of the accumulation of free radicals–mediated ing motifs. Finally, in collaboration with Kurt Wüthrich, cellular damage. Thus, a reduction in the formation of the Nobel Prize–winning nuclear magnetic resonance free radicals should slow aging and possibly prolong researcher, they have solved the dynamic structure of a lifespan. Generation of free radicals is inherently asso- key SARS protein, the protein nsP7. 290 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

These investigators have been able to identify key functions in emerging viruses and have obtained a degree of detail on their structure that permits either the rapid synthesis of chemicals to combat viral replication or the selection of antibodies for passive immunization to treat the infected. Their research also permits identification of components for protective vaccines. Our hope is to be able to obtain the same type of information for the bird flu virus. MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 291

INVESTIGATORS’REPORTS Studies of Severe Acute Respiratory Syndrome Virus and Other Coronaviruses

B.W. Neuman, R.J. Burrer, R. Aur, J.P.C. Ting, B.D. Adair, C. Yoshioka,* J. Quispe,* R.A. Milligan,* M. Yeager,* M.J. Buchmeier Fig. 1. Reconstruction of coronavirus spike and nucleoprotein * Department of Cell Biology, Scripps Research molecules. A, Formalin-fixed feline coronavirus particles were imaged by using low-dose electron cryomicroscopy. Scale bar at lower right oronaviruses are an important family of human indicates 10 nm. B, Three-dimensional reconstructions of the spike and veterinary pathogens that cause a wide range (top) and nucleoprotein (bottom) densities were obtained by using C of diseases. The emergence of the coronavirus single-particle image analysis. C, A schematic interpretation of the that causes severe acute respiratory syndrome (SARS) membrane proximal region shows spikes (top) in contact with membrane-embedded matrix protein, which in turn interacts with nucleo- highlighted a need for structural information on coron- protein-RNA complexes of the viral core (bottom). avirus proteins and effective antiviral treatments. ARCHITECTURE OF CORONAVIRUS PARTICLES ANTISENSE ANTIVIRAL AGENTS Coronaviruses derive their name from the protruding Coronaviruses are the cause of important and transmembrane spike glycoproteins, which are seated emerging enzootic infectious diseases in many parts of in the viral membrane via interactions with the 3-pass the world. To design a rational and consistent approach transmembrane matrix glycoprotein.A core containing to combat these viruses, we used peptide-conjugated nucleoprotein and the single-stranded RNA genome of antisense morpholino oligomers (P-PMOs) designed to approximately 30 kb is incorporated into virions at mem- bind to duplex-specific sequences in the genomes of branes of the endoplasmic reticulum–Golgi complex inter- SARS virus and mouse hepatitis virus, a murine coron- mediate in a process mediated by interactions between avirus. P-PMOs directed against regions in the 5′ untrans- the nucleoprotein and the matrix glycoprotein.We used lated regions of the genomes reduced virus-associated electron cryomicroscopy and image analysis to examine cytopathologic changes and spread by decreasing viral the supramolecular structure of coronaviruses. amplification. Random-sequence control P-PMOs had We found that coronavirus particles are enveloped, low antiviral activity against both viruses. During pro- pleomorphic, and about 85 nm in diameter (Fig. 1A). longed treatment, the SARS virus developed contiguous The surface spikes consist of a globular head supported point mutations at a P-PMO binding site, producing by a slender stalk. The predicted volume for the spike resistant but severely growth-attenuated virus. ectodomain is a close match for a homotrimer of spike In mice, treatment with P-PMOs reduced the lev- molecules. A layer of density directly apposed to the els of virus and cytopathologic changes in the liver inner bilayer leaflet and tightly packed intramembrane and weight loss associated with infection.Treatment densities are ascribed to the matrix protein molecules. postponed death in animals infected with mouse hep- Punctate densities of about 5 nm are present at the atitis virus 3 and reduced mortality in animals infected underside of the viral membrane, distributed through- with mouse hepatitis virus Alb139. These results sug- out the interior of the virion, and ascribed to complexes gest P-PMOs have powerful therapeutic and investiga- consisting of the nucleoprotein and RNA. The spike and tive potential in coronavirus infections. nucleoprotein molecules closest to the viral membrane form overlapping 2-dimensional lattices, most likely due to protein-protein interactions during assembly. Using single-particle techniques, we reconstructed 3-dimensional models of spike and nucleoprotein densities (Figs. 1B and 1C) at a resolution of approximately 3 nm. 292 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE Vaccination for Severe Acute Respiratory Syndrome – Associated Coronavirus

C.T. Cornillez-Ty, R.J. Burrer, B.W. Neuman, J.P.C. Ting, A. Sette,* J. Sidney,* M.J. Buchmeier * La Jolla Institute for Allergy and Immunology, San Diego, California

n an effort to develop a multiepitope vaccine against severe acute respiratory syndrome–associ- I ated coronavirus (SARS-CoV), we are collaborating with investigators at the La Jolla Institute of Allergy Fig. 1. IFN-γ enzyme-linked immunospot assay to determine the and Immunology. We are attempting to identify epitopes MHC I–restricted epitopes of SARS-CoV. A, HLA transgenic mice within the 4 structural proteins (nucleoprotein, matrix, are immunized with a candidate 9-mer peptide. B, Spleens from + envelope, and spike) of the virus that can be presented immunized mice are harvested and CD8 T cells are isolated. C, One day before isolation of CD8+ T cells, antigen-presenting cells on human MHC class I molecules. Although SARS-CoV are infected with a recombinant vaccinia virus that expresses 1 of contains at least 14 open reading frames, the 4 struc- the 4 SARS-CoV structural proteins. Antigen processing of the tural proteins have the highest level of expression in SARS-CoV protein will result in loading of certain epitopes onto cells infected with the virus. Hence, in a natural infec- MHC class I molecules. If the peptide used to immunize HLA trans- tion, these 4 proteins are the ones most likely to be genic mice corresponds to an epitope (light gray region) that can processed and presented on MHC class I molecules. be processed and loaded onto an MHC class I molecule, exposure of isolated CD8+ T cells to the vaccinia-infected antigen-presenting On the basis of a predictive algorithm, several poten- cells will elicit a memory response. D, A memory response can be tial epitopes within these 4 proteins have been identi- determined by assaying for secretion of IFN-γ. fied. These predicted epitopes have been synthesized as 9-mer peptides and have been tested for in vitro to a lesser extent CD4+,T cells results in an impaired binding affinities to MHC class I molecules of the A1, ability to survive sublethal doses of the virus. Innate A2, A3, A24, B7, and B44 supertypes. This process immunity does play a protective role, and previous stud- has yielded a pool of peptides that must be further tested ies by us and by others have highlighted the impor- for their ability to elicit a CD8+ T-cell response in vivo. tance of various chemokines in the immune response To determine whether these peptides are immuno- to MHV. genic in vivo, we will use IFN-γ enzyme-linked immuno- Toll-like receptors (TLRs) recognize specific molecular spot assays (Fig. 1). Construction of recombinant vaccinia patterns that are usually associated with the presence of virus that expresses the 4 structural proteins of SARS- viruses or bacteria. Upon recognition of microbial patho- CoV has been completed, and immunization of HLA gens, the receptors shape both the innate and the transgenic mice with the various peptides are under way. adaptive immune responses by initiating the release of inflammatory cytokine and chemokine mediators and upregulating costimulatory molecules on dendritic Pathogenesis of Coronavirus- cells, respectively. Each TLR interacts with a specific combination of adapter proteins to trigger intracellular Induced Demyelination signaling pathways that result in the activation of transcription factors. R.J. Burrer, C.T. Cornillez-Ty, L. Breakwell, M.J. Buchmeier To determine if TLRs are required to survive MHV ouse hepatitis virus (MHV) causes acute in the CNS, we monitored the outcome of a sublethal encephalomyelitis; in mice that survive, a infection in wild-type (control) mice and in mice defi- M demyelinating disease resembling the human cient in 2 of the adapter proteins, MyD88 and Trif/lps2, disease multiple sclerosis develops. An adaptive immune because each of the TLRs signals through 1 or both of response is required to survive the acute phase of MHV these proteins. Mortality was similar in wild-type mice infection in the CNS, because the lack of CD8+, and and mice deficient in Trif/lps2 and greatly increased MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 293 in mice deficient in MyD88. We are determining which recombinant vaccinia virus construct that expressed TLRs and intracellular pathways are involved in the the glycoprotein precursor. response to MHV infection. We are now applying this epitope identification approach to the remaining pathogenic arenaviruses. For LCMV, we have identified novel HLA-A2 supertype– HLA-Restricted Epitope restricted epitopes from the nucleoprotein, glycoprotein precursor, zinc-binding protein, and viral polymer- Discovery From Pathogenic ase. Immunization of HLA-A2 transgenic mice with 2 of these epitopes led to significant reductions in viral Arenaviruses titer after challenge with LCMV. Last, a subset of the identified Lassa virus and LCMV epitopes cross-react J.W. Botten, P.A. Barrowman, J.P.C. Ting, A. Sette,* with the corresponding determinants in the remaining J.L. Whitton,** M.J. Buchmeier pathogenic arenaviruses. * La Jolla Institute for Allergy and Immunology, San Diego, California The epitopes identified in these studies are poten- ** Molecular and Integrative Neurosciences Department, Scripps Research tial diagnostic reagents and candidates for inclusion in renaviruses are rodent-borne pathogens that genetically engineered or epitope-based vaccine con- cause significant morbidity and mortality in structs. Our approach is applicable to any pathogen with A humans. Pathogenic arenaviruses include Lassa, existing sequence data, does not require manipulation lymphocytic choriomeningitis (LCMV), Junin, Machupo, of the virulent pathogen or access to immune human Guanarito, Sabia, and Whitewater Arroyo viruses. Our donors, and should therefore be generally applicable to understanding of the human immune response to are- category A-C agents (pathogens that pose a risk to navirus infection is limited. In the case of Lassa virus, national security) and other emerging pathogens. infected individuals generate poor neutralizing antibody responses, indicating that cellular immunity most likely plays a primary role in viral clearance and pro- Structure and Function of the tective immunity. Because of its central role, sensitive reagents are needed to measure the cell-mediated Arenavirus Signal Peptide immunity that develops in response to naturally occur- A.A. Saunders, B.W. Neuman, J.P.C. Ting, M.J. Buchmeier ring infections with Lassa virus or to vaccines. The development of diagnostic assays requires identifica- he signal peptide of the surface glycoprotein of tion of HLA-restricted class I and class II epitopes of lymphocytic choriomeningitis virus (LCMV) has the virus. These epitopes could be used not only to T several unique characteristics. It is unusually diagnose Lassa virus infection but also to determine long at 58 amino acids, it contains 2 hydrophobic the quality of immune responses, define correlates of domains, and its sequence is highly conserved among protection and immunopathologic changes, and ulti- both Old and New World arenaviruses. To better under- mately guide the selection of possible vaccines. stand the functions of the peptide, we created a panel We have established a model system for identify- of point and deletion mutants targeting many of the ing human CD8+ T-cell epitopes from pathogenic are- highly conserved elements within the peptide. In char- naviruses; we use Lassa virus as our test virus. In this acterizing our mutant signal peptides, we discovered system, bioinformatic predictions are used to identify that in addition to translocation of the viral surface gly- possible epitopes, in vitro MHC-binding assays and in coprotein precursor into the lumen of the endoplasmic vivo immunogenicity studies in HLA transgenic mice reticulum, the signal peptide is involved in glycoprotein are used to validate epitopes, and recombinant vac- expression, cleavage, and cell-surface localization; gly- cinia virus–based challenge studies are used to evalu- coprotein incorporation during assembly of viral parti- ate whether epitopes protect against viral challenge. cles; and glycoprotein-mediated fusion with a host cell. Using this approach, we identified 4 HLA-A2 PUBLICATIONS supertype–restricted epitopes encoded by the gene for Botten, J., Alexander, J., Pasquetto, V., Sidney, J., Barrowman, P., Ting, J., the Lassa virus glycoprotein precursor. Two of these Peters, B., Southwood, S., Stewart, B., Rodriguez-Carreno, M.P., Mothe, B., Whitton, J.L., Sette, A., Buchmeier. M.J. Identification of protective Lassa virus epitopes protected mice against challenge with a epitopes that are restricted by HLA-A2. J. Virol. 80:8351, 2006. 294 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

Joseph, J.S., Saikatendu, K.S., Subramanian, V., Neuman, B.W., Brooun, A., function, and molecular mechanisms. Initial brain inva- Griffith, M., Moy, K., Yadav, M.K., Velasquez, J., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Crystal structure of nonstructural protein 10 from the severe acute respi- sion by virus occurs early, by the second week after ratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J. infection. At this time, a self-limited illness affects Virol. 80:7894, 2006. many physiologic systems, including the CNS. In addi- Neuman, B.W., Adair, B.D., Yoshioka, C., Quispe, J.D., Orca, G., Kuhn, P., Milli- tion, an innate immune response occurs in the brain, gan, R.A., Yeager, M., Buchmeier, M.J. Supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy. J. with an upregulation of genes induced by interferon Virol. 80:7918, 2006. and IL-6. The adaptive immune response is beginning. + Nussbaum, A.K., Rodriguez-Carreno, M.P., Benning, N., Botten, J., Whitton, J.L. During the next 3 months, the brain CD8 T-cell phe- Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to notype switches from a surveillance mode to an effec- lymphocytic choriomeningitis virus infection and DNA vaccination. J. Immunol. + 175:1153, 2005. tor mode, SIV-specific CD8 T cells can be detected in the brain, and level of virus in the brain decreases Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson, M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural geno- by 100-fold. mics of the severe acute respiratory syndrome coronavirus: nuclear magnetic reso- This chronic, relatively asymptomatic phase can nance structure of the protein nsP7. J. Virol. 79:12905, 2005. last years. However, in both humans and rhesus mon- Reignier, T., Oldenburg, J., Noble, B., Lamb, E., Romanowski, V., Buchmeier, keys, behavioral testing reveals cognitive alterations, M.J., Cannon, P.M. Receptor use by pathogenic arenaviruses. Virology, in press. and neurophysiologic analysis reveals abnormal CNS Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K., Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structural function. We found that 2 years after infection, the basis of severe acute respiratory syndrome coronavirus ADP-ribose-1′′-phosphate concentration of SIV in the brain remained low, but dephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005. the virus was still present and the increased number of CD8+ T cells in the brain that occurred early after infection was preserved. Molecular analysis confirmed Chronic Virus-Host Interaction an active immune interaction in the brain. Intriguingly, in the CNS the level of the chemokine RANTES/CCL5, which has numerous effects on neurons and on immune cells, was increased, and expression of the chemokine was H.S. Fox, M. Alirezaei, J. Boyd, C. Flynn, P. Gaskill, localized to the brain-infiltrating cytotoxic T cells. S. Huitrón-Reséndiz, C. Lanigan, C. Marcondes, R. Ojakian, Thus, rather than being a quiescent state, the G. Pendyala, D. Watry, M. Yadav, C. York-DeFalco, chronic phase is an important stage of the viral-host M. Zandonatti interaction. The original purpose of this interaction is he brain is a unique organ, not only functionally to protect the brain from the virus, but in the long-term, but also in terms of host response to events such the interaction can lead to brain damage. T as infection. We study processes in which this Late in the disease course, the adaptive immune response leads to brain dysfunction; we have mostly response wanes, and the amount of virus again increases focused on a degenerative and dementing condition that in the brain. This increase is accompanied by a number occurs after a known stimulus, infection with HIV. of indications of CNS dysfunction, and an influx of The HIV pandemic continues worldwide. In the macrophages into the infected brain occurs, leading to United States and certain other countries, antiviral SIV encephalitis. At the molecular level, genes associated treatment is available, leading to greatly prolonged with innate immune responses are again upregulated, survival. However, as HIV infection has turned into a and macrophages as well as brain glia are activated. chronic disease, the CNS disorders caused by the virus Currently, we are focusing on the chronic stage of (neuroAIDS) continue to affect a significant proportion SIV infection, modeling the effects of patients who are of those who are infected. Using infection of rhesus infected with HIV but have not progressed to AIDS. We monkeys with simian immunodeficiency virus (SIV) as are investigating immune specificity in the brain vs the a model of neuroAIDS in humans, we are studying the rest of the body and the mechanisms responsible for virology, immunology, pathology, and neurobiology of these differences. We are also examining the effects of the resulting CNS disease. highly active antiretroviral therapy on the interaction We have defined the different stages of SIV disease between the virus and the immune system in the brain. in the CNS in terms of interactions between the virus Understanding the mechanisms of dysfunction in the and the immune system, pathologic changes in CNS brain will define the pathogenesis of CNS HIV infec- MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 295 tion, as well as potentially other CNS disorders, and Other interests include dissecting how viruses and lead to preventive or therapeutic strategies. immune cells traffic to the brain and interact there; how viruses are cleared from the brain; and how viruses alter PUBLICATIONS the differentiation processes of cells they persistently Burdo, T.H., Marcondes, M.C., Lanigan, C.M., Penedo, M.C., Fox, H.S. Suscepti- bility of Chinese rhesus monkeys to SIV infection. AIDS 19:1704, 2005. infect, thereby disturbing homeostasis and causing disease. We also are investigating how viruses induce Everall, I., Salaria, S., Roberts, E., Corbeil, J., Sasik, R., Fox, H.S., Grant, I., Masliah, E., HNRC Group. Methamphetamine stimulates interferon inducible autoimmune disease or induce immunosuppression, genes in HIV infected brain. J. Neuroimmunol. 170:158, 2005. and we are designing therapies to control viral infec- Roberts, E.S., Huitrón-Reséndiz, S., Taffe, M.A., Marcondes, M.C., Flynn, C.T., tions. Because different viruses have different lifestyles, Lanigan, C.M., Hammond, J.A., Head, S.R., Henriksen, S.J., Fox, H.S. Host we focus on 3 RNA negative-stranded viruses: Borna response and dysfunction in the CNS during chronic simian immunodeficiency virus infection. J. Neurosci. 26:4577, 2006. disease virus, lymphocytic choriomeningitis virus, and measles virus. We also investigate the mechanism by which infectious agents cause transmissible spongi- Viral-Immunobiology Laboratory form encephalopathies.

M.B.A. Oldstone, J.C. de la Torre, S. Kunz, D.B. McGavern, B. Hahm, D. Brooks, A. Capul, R. Clemente, K. Edelmann, Resurrection of Nonfunctional L. Garidou, H. Lauterbach, A. Lee, L. Liou, A. Sanchez, M. Trifilo, G. Ying, E. Zuniga, A. Tishon, H. Lewicki, E. Buset, T Cells During Persistent A. Gundersen,P. Borrow,* E. Domingo,** J.E. Gairin,*** Viral Infection R. Kiessling,**** N. Sevilla,** Christina Spiropoulou***** * Edward Jenner Institute for Vaccine Research, Compton, England D. Brooks, D.B. McGavern, M.B.A. Oldstone ** Universidad Autonoma de Madrid, Madrid, Spain ersistent viral infections such as HIV disease *** CNRS, Toulouse, France and hepatitis C are major health problems. A **** Karolinska Institutet, Stockholm, Sweden fundamental obstacle in control of these infections ***** Centers for Disease Control and Prevention, Atlanta, Georgia P is the functional inactivation of antiviral T cells. After he Viral-Immunobiology Laboratory encompasses a persistent infection is established, both CD4+ and the programs of 4 faculty members: Juan Carlos CD8+ T cells rapidly lose their antiviral and immuno- T de la Torre, Stefan Kunz, Dorian B. McGavern, stimulatory functions. Although this phenomenon has and Michael B.A. Oldstone. Each program is indepen- been recognized for years, the pertinent question is dent, but the interactions between the researchers and whether the immune response is programmed to fail the use of different technologies provide an intellectual or can be fixed to eliminate infection. Also unclear are sum greater than any single part. Our studies of both the molecular events or factors involved. viral and transmissible spongiform encephalopathies We found that in contrast to T-cell expansion, which (e.g., prion diseases, scrapie) include basic analysis is hardwired during priming, T-cell functional responses of the mechanisms by which viruses persist, escape are malleable and rely on continuous signals from the immune recognition, and cause disease. Integral parts cells’ antigenic environment. In accordance with this of the programs are understanding how viruses infect plasticity, function can be restored to nonresponsive cells; defining the cellular receptors used by viruses; CD4+ and CD8+ T cells during persistent infection by and mapping the trafficking of viruses into cells and treatment with the antiviral drug ribavirin.Treatment the subsequent viral uncoating, replication, assembly, that reduced the concentration of virus by as little as exit, and spread. Because the immune system has one log led to the removal of T-cell suppression factors. evolved to recognize, attack, and remove these foreign Removal of IL-10 initiated by viral infection resulted in substances, we evaluate the immune response against restoration of T-cell function. viruses, probe how viruses subvert this response to During persistent infection, CD8+ T cells with the provide a selective advantage for their survival, and highest affinity for viral antigens are physically deleted. study how the host can correct this subversion to allow Removal of these high-affinity cells results in the deple- termination of viral persistence. tion of the effector population best equipped to fight 296 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE infection, thereby limiting the breadth, magnitude, and combination and used fluorescence methods to identify efficacy of the antiviral response. We found that dele- and measure the trafficking of these specific T cells to tion of high-affinity CD8+ T cells during persistent viral the lung (Fig. 1). We also examined the effects of vari- infection is a direct result of the inactivation of virus- specific CD4+ T cells. The deletion of high-affinity CD8+ T cells could be averted by therapeutically rescuing CD4+ T-cell activity in vivo, resulting in long-term cytolytic activity of CD8+ T cells against virus-infected cells and control of infection.

Dissecting the Molecular Role of CD4+ and CD8+ T Cells in Control of Acute Viral Infections

Fig. 1. Top, The genomic structure of LCMV and its immun- A. Tishon, H. Lewicki, K. Edelmann, M.B.A. Oldstone odominant CD8+ (GP 33) and CD4+ (GP 65) epitopes. The GP 33 easles virus is one of the most infectious of and GP 65 sequences are inserted into the neuraminidase gene of influenza WSN virus. Bottom, Infiltration into the lung of fluores- human pathogens and today still infects more cently labeled GP 33–specific T cells after intranasal inoculation M than 30 million persons each year, killing more with 1 x 105 plaque-forming units of WSN-LCMV influenza virus. than 500,000 persons annually. Death is primarily due to secondary microbial infections associated with ous therapies on trafficking of these cells to the lung immunosuppression or to CNS disease. and control of the resultant immunopathologic injury. We used a transgenic mouse model to express receptors for measles virus in neurons in the CNS. Infecting the transgenic mice with measles virus in concert with Prion-Induced Amyloid CNS and depleting and reconstituting individual T-cell subsets and B cells alone or in combination revealed that nei- Heart Disease With High Levels ther CD8+ nor CD4+ nor B cells alone can control acute of Infectivity in Blood and a measles virus infection. Combinations of either (1) CD4+ cells and B cells or (2) CD4+ and CD8+ T cells were Transgenic Model for Chronic required, but CD8+ T cells with B cells were not effective. Both IFN-γ and neutralizing antibodies, but not Wasting Disease perforin or TNF-α, were associated with clearance of M.J. Trifilo, G. Ying, M.B.A. Oldstone the virus. Interestingly, lack of IFN-γ but not lack of TNF-α led to persistent measles virus infection. ransmissible spongiform encephalopathies, or Influenza virus remains a major concern for a prion diseases, are a group of infectious diseases returning infection with potential devastating results T due to abnormal folding of the normal cellular for the human population.To better understand how protein PrP. More than 98% of PrP exists as a mem- to control influenza virus infection of the lung, in col- brane-bound, glycosylphosphatidylinositol-anchored laboration with Y. Kawaoka, University of Wisconsin, protein. In collaboration with B. Chesebro, Rocky Moun- Madison, we used reverse genetics to insert the known tain Laboratories, Hamilton, Montana, we produced H-2b–restricted immunodominant CD8+ T-cell epitope transgenic mice in which the C-terminal 21 amino (GP 33–41) and the CD4+ T-cell epitope (GP 61–80) acids of PrP are not transcribed; in these mice more of lymphocytic choriomeningitis virus (LCMV) into the than 98% of PrP exists in an anchorless, non–mem- neuraminidase gene for WSN influenza virus. We then brane-bound form. adoptively transferred fluorescently labeled LCMV-specific When these transgenic mice were inoculated intrac- GP 33 CD8+ cells or GP 61 CD4+ T cells alone or in erebrally with the agent that causes murine scrapie, a MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 297 dramatic accumulation of abnormally folded prion pro- and PrPres could be readily detected. These findings tein, PrPres, occurred within the brain (Fig. 1A). PrPres were the first demonstration of PrPres in the blood and indicate that a way exists to determine the precise blood components involved and to detect or remove PrPres to safeguard blood supplies. Additionally, multiple extraneural tissues, including the heart, had PrPres deposition (Fig. 1C). Studies indicated that similar to deposits in the brain, deposits of PrPres in the heart formed amyloid (Fig. 1D) that was infectious. In catheterization studies done in collaboration with K. Knowlton, University of California, San Diego, we found that the deposition of amyloidogenic PrPres within the hearts of infected transgenic mice caused significant alterations in both systolic (reduced compliance) and diastolic (stiffening of the heart) functions of the heart. These results provided the first evidence that prion- mediated disease could occur outside the CNS. Last, in collaboration with Dr. Chesebro, we inserted the gene for normal deer PrP into mouse genes behind the PrP promoter and introduced this construct into mice in which the gene for mouse PrP had been inactivated. When such transgenic mice were inoculated intracere- brally or orally with the agent that causes deer scrapie, clinical and pathologic evidence of prion disease developed and normal cellular deer PrP was biochemically converted into deer PrPres. Thus, we now have an animal model that can be used to investigate the patho- Fig. 1. Deposition of PrPres in the frontal cortex (A) and around genesis and mechanism of spread of chronic wasting endothelial cells (B) of the brain in transgenic mice with anchorless, non–membrane-bound PrP infected with the agent that causes disease in deer. Both of these characteristics are cur- murine scrapie. Both PrPres and infectious material enter the blood rently unknown, although chronic wasting disease is a and are deposited in several extraneural tissues, including the heart major economic problem for those who hunt or breed (C and D). Deposits of PrPres (C) colocalize with amyloid deposits deer and an unknown public health risk for humans. (D), preventing proper function of the heart. was infectious and formed large amyloid plaques in PUBLICATIONS Brooks, D.G., McGavern, D.B., Oldstone, M.B.A. Reprogramming of antiviral T cells the absence of overt clinical disease during observation prevents inactivation and restores T cell activity during persistent viral infection. J. times of up to 720 days. However, the infected mice Clin. Invest. 116:1675, 2006. had learning and memory deficits, including an inabil- Homann, D., Dummer, W., Wolfe, T., Rodrigo, E., Theofilopoulos, A.N., Oldstone, ity to perform cued learning tests and a failure to induce M.B.A., von Herrath, M.G. Lack of intrinsic CTLA-4 expression has minimal effect on regulation of antiviral T-cell immunity. J. Virol. 80:270, 2006. long-term potentiation. In other studies, we showed Kunz, S., Rojek, J.M., Kanagawa, M., Spiropoulou, C.F., Barresi, R., Campbell, that inhibition of learning and memory was associated K.P., Oldstone, M.B.A. Posttranslational modification of α-dystroglycan, the cellular with PrPres binding, upregulating, and signaling through receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus binding. J. Virol. 79:14282, 2005. the γ-aminobutyric acid receptor. These results indicate for the first time that PrP can function as a ligand for Oldstone, M.B.A. Molecular and cellular mechanisms, pathogenesis, and treatment of insulin-dependent diabetes obtained through study of a transgenic model of this receptor. molecular mimicry. Curr. Top. Microbiol. Immunol. 296:65, 2005. PrPres deposits in the brains of the infected trans- Oldstone, M.B.A. Molecular mimicry, microbial infection, and autoimmune disease: genic mice also occurred within and around endothe- evolution of the concept. Curr. Top. Microbiol. Immunol. 296:1, 2005. lial cells lining blood vessels in the brain (Fig. 1B). Oldstone, M.B.A. Viral persistence: parameters, mechanisms and future predic- Examination of blood indicated that both infectivity tions. Virology 344:111, 2006. 298 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

Oldstone, M.B.A., Dales, S., Tishon, A., Lewicki, H., Martin, L. A role for dual are limited to the use of ribavirin, which is only partially viral hits in causation of subacute sclerosing panencephalitis. J. Exp. Med. 202:1185, 2005. effective and often causes severe secondary effects.

Rhode, A., Pauza, M.E., Barral, A.M., Rodrigo, E., Oldstone, M.B., von Herrath, The LCMV reverse genetics system is an excellent plat- M.G., Christen, U. Islet-specific expression of CXCL10 causes spontaneous islet form for investigating novel antiviral strategies to com- infiltration and accelerates diabetes development. J. Immunol. 175:3516, 2005. bat arenavirus infections. We identified sequence and Tishon, A., Lewicki, H., Andaya, A., McGavern, D., Martin, L., Oldstone, M.B.A. structural constraints within the arenavirus genome CD4 T cell control primary measles virus infection of the CNS: regulation is dependent on combined activity with either CD8 T cells or with B cells: CD4, CD8 or B promoter that revealed a new potential target for cells alone are ineffective. Virology 347:234, 2006. aminoglycoside-based drugs against arenaviruses. Trifilo, M.J., Hahm, B., Zuniga, E.I., Edelmann, K.H., Oldstone, M.B.A. Dendritic cell Likewise, we showed that the arenavirus functional inhibition: memoirs from immunosuppressive viruses. J. Infect. Dis., in press. polymerase strictly requires an L-L interaction that is Trifilo, M.J., Yajima, T., Gu, Y., Dalton, N., Peterson, K.L., Race, R.E., Meade- White, K., Portis, J.L., Masliah, E., Knowlton, K.U., Chesebro, B., Oldstone, amenable to disruption by small molecules. We also M.B.A. Prion-induced amyloid heart disease with high blood infectivity in trans- identified the Z protein as the driving force of arenavi- genic mice. Science 313:94, 2006. rus budding. This process is mediated by proline-rich Zuniga, E.I., Edelmann, K.H., Oldstone, M.B.A. Viruses and dendritic cells: a prominent mechanism for subverting the immune response. In: Microbial Subver- late (L) domain motifs similar to those known to control sion of the Host Immune Response. Lachmann, P., Oldstone, M.B.A. (Eds.). Hori- budding of several other viruses, including HIV and zon Scientific Press, London, 2006, p. 211. Ebola virus, via interaction with specific host-cell proteins. We are using genetic and proteomic approaches to identify cellular proteins that interact with Z and Arenavirus Molecular and Cell are required for arenavirus budding; these approaches Biology: Implications for Novel may yield new antiviral strategies for targeting arenavirus budding. Antiviral Therapies LCMV is a Rosetta stone in viral immunology and pathogenesis. We can now generate recombinant LCMVs A.B. Sánchez, A. Capul, B. Cubitt, N. Nguyen, D. Rosario, that have predetermined specific mutations within their J.C. de la Torre genomes, or express additional foreign genes, and ana- renaviruses merit significant attention both as lyze their phenotypic expression in vivo, a novel and model systems for studies of acute and persis- powerful approach for elucidating the molecular mech- A tent viral infections and as important human anisms that underlie arenavirus-host interactions and pathogens, including Lassa virus and several other associated disease. causative agents of severe hemorrhagic fever. More- PUBLICATIONS over, evidence indicates that the prototypic arenavirus Flatz, L., Bergthaler, A., de la Torre, J.C., Pinschewer D.D. Recovery of an arena- lymphocytic choriomeningitis virus (LCMV) is a neglected virus entirely from RNA polymerase I/II-driven cDNA. Proc. Natl. Acad. Sci. U. S. A. 103:4663, 2006. human pathogen of clinical importance. Kunz, S., de la Torre, J.C. Novel antiviral strategies to combat human arenavirus Arenaviruses are enveloped viruses with a biseg- infections. Curr. Mol. Med. 5:735, 2005. mented negative-stranded RNA genome. Each genomic Merkler, D., Horvath, E., Bruck, W., Zinkernagel, R.M., de la Torre, J.C., Pin- RNA segment, L and S, uses an ambisense coding strat- schewer, D.D. “Viral déjà vu” elicits organ-specific immune disease independent of egy to direct the synthesis of 2 polypeptides in oppo- reactivity to self. J. Clin. Invest. 116:1254, 2006. site orientation, separated by an intergenic region. The Sánchez, A.B., de la Torre, J.C. Rescue of the prototypic arenavirus LCMV entirely S RNA encodes the viral glycoprotein and the nucleopro- from plasmid. Virology 350:370, 2006. tein, whereas the L RNA encodes the viral RNA–depen- Sánchez, A.B., Perez, M., Cornu, T., de la Torre, J.C. RNA interference-mediated dent RNA polymerase and the small RING finger protein virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus. J. Virol. 79:11071, 2005. Z. We have developed a reverse genetics system for Sevilla, N., de la Torre, J.C. Arenavirus diversity and evolution: quasispecies in LCMV that provides a novel and powerful approach for vivo. Curr.Top. Microbiol. Immunol. 299:315, 2006. elucidating the role of viral polypeptides and cis-acting sequences in the control of arenavirus RNA replication, gene expression, assembly, and budding and virus-cell protein interactions. No licensed vaccines against arenavirus are available, and current therapies for arenavirus infections MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 299 Virus-Cell Interactions in Persistent Viral Infection Persistently Infected Brains of the CNS

R. Clemente, M. Perez, B. Cubitt, K. Hagiwara, D. Rosario, D.B. McGavern, P. Truong, H. Lauterbach, L. Garidou J.C. de la Torre e focus on developing and understanding ersistent viral infections of the CNS can cause strategies to purge tissues of persistent viral progressive neurologic disorders associated with W infections without killing or damaging the P diverse abnormalities. These findings led to the tissues. Prevention of persistent infections is tradition- hypothesis that viruses can contribute to human men- ally attained via vaccination; however, vaccination is tal disorders of unknown etiology. We use infection often ineffective once a virus establishes persistence. with Borna disease virus (BDV) as a model system to Another factor that must be considered in strategies to investigate virus-cell interactions that underlie non- eradicate a chronic infection is the anatomic compart- lytic persistent viral infections of the CNS and associ- ment in which the virus establishes persistence. The ated disturbances. CNS, for example, resides behind a specialized blood- BDV is an enveloped virus with a nonsegmented brain barrier, lacks standard lymphatic drainage, limits negative-stranded RNA genome. and is the prototypic the expression of antigen-presenting machinery, and member of the virus family Bornaviridae, within the heavily regulates the activity of T cells. The CNS also order Mononegavirales. We established a reverse genet- has an intricate network of nonreplicative cells (neu- ics system for BDV that enables us to investigate the rons) that if targeted could result in dire, potentially mechanisms that control BDV RNA replication and gene irreparable consequences. expression and interactions between the virus and host Because of the considerable challenges associated cells within the CNS. with eradicating persistent viral infections, we are using We found that the ectodomain of the BDV glycopro- a remarkable therapeutic approach referred to as immu- tein p56 is the sole entity responsible for recognition nocytotherapy. In this approach, which has been used of the virus receptor and cell entry, and we developed successfully in a clinical setting, persistently infected reagents to identify cellular receptors of BDV responsi- hosts are given competent, virus-specific memory T cells. ble for the strong tropism of BDV for limbic system Specifically, we exploit a well-established model in which neurons. We also uncovered a receptor-independent mice are persistently infected from birth or in utero cell-to-cell propagation of BDV that may play an impor- with lymphocytic choriomeningitis virus (LCMV). Mice tant role in the biology of the virus. infected in this manner become lifelong carriers of LCMV, Neonatal infection of rats with BDV causes distinct and the virus establishes persistence in every tissue CNS neurodevelopmental and behavioral abnormalities compartment, including CNS neurons. Quite remark- that parallel those reported in certain neuropsychiatric ably, a single injection of LCMV-specific memory T cells disorders in humans We identified changes in host gene into carrier mice achieves systemic eradication of the expression associated with BDV persistence. We are virus as well as clearance of virus from CNS neurons using a variety of cell culture systems and animal mod- without evidence of injury. els to examine the contribution of identified targets to We are developing novel visualization strategies BDV-induced CNS disturbances. to define the precise mechanisms by which adoptively transferred memory T cells purge neurons of a persis- PUBLICATIONS tent viral infection. Recently, we used genetically tagged Perez, M., de la Torre, J.C. Identification of the Borna disease virus (BDV) proteins required for the formation of BDV-like particles. J. Gen. Virol. 86:1891, 2005. populations of memory T lymphocytes specific for LCMV glycoprotein in combination with 2- and 3-dimensional Yanai, H., Kobayashi, T., Hayashi, Y., Watanabe, Y., Ohtaki, N., Zhang, G., de la Torre, J.C., Ikuta, K., Tomonaga, K.A methionine-rich domain mediates CRM1- microscopy to show that LCMV-specific T cells arrive dependent nuclear export activity of Borna disease virus phosphoprotein. J. Virol. in the CNS soon after adoptive immunotherapy. The 80:1121, 2006. T cells then recruit antigen-presenting cells referred to as dendritic cells into the CNS parenchyma. Importantly, dendritic cells are able to present antigen to the memory T cells and preferentially induce the 300 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE production of an antiviral cytokine (TNF-α) required residues of the receptor crucial for binding to the extra- for successful immunotherapy. Finally, we also showed cellular matrix. This finding suggests that the virus mimics through in vivo depletion studies that successful immu- the mechanism of receptor recognition of extracellular notherapy depends on dendritic cells, because in their matrix proteins. As a consequence, Lassa virus effi- absence immunotherapeutic clearance was impeded ciently competes with extracellular matrix proteins and both in the CNS and in the periphery. On the basis of perturbs the normal function of α-dystroglycan. Using these results, we postulate that therapeutically enhanced a combination of biochemical and cell biological tech- recruitment of dendritic cells into the CNS may be an niques, we are studying the impact of binding of the excellent strategy to promote (or accelerate) clearance virus on α-dystroglycan–mediated signal transduction of a persistent viral infection. and cell-matrix adhesion. A hallmark of fatal Lassa virus infection in humans PUBLICATIONS Brooks, D.G., McGavern, D.B., Oldstone, M.B.A. Reprogramming of antiviral T is an overwhelming viral load and subsequent collapse cells prevents inactivation and restores T cell activity during persistent viral infec- of the host’s immune system. Because rapid dissemi- tion. J. Clin. Invest. 116:1675, 2006. nation of the virus critically depends on viral attach- Brooks, D.G., Teyton, L., Oldstone, M.B.A., McGavern, D.B. Intrinsic functional ment and entry into host cells, drugs that target these dysregulation of CD4 T cells occurs rapidly following persistent viral infection. J. Virol. 79:10514, 2005. steps will be of great therapeutic value. A major goal of our current research is to develop novel antiviral drugs Lauterbach, H., Zuniga, E.I., Truong, P., Oldstone, M.B.A., McGavern, D.B. Adop- tive immunotherapy induces CNS dendritic cell recruitment and antigen presentation that can block these initial steps of infection. Using during clearance of a persistent viral infection. J. Exp. Med. 203:1963, 2006. high-throughput screening assays for combinatorial McGavern, D.B. Immunotherapeutic relief from persistent infections and amyloid small-molecule libraries obtained from the laboratory disorders. Neurology 66(Suppl. 1):S59, 2006. of D.L. Boger, Department of Chemistry, we have iden- McGavern, D.B. The role of bystander T cells in CNS pathology and pathogen tified a number of compounds that specifically block clearance. Crit. Rev. Immunol. 25:289, 2005. attachment and entry of Lassa virus into human cells. The most promising compounds are being optimized and pharmacologically characterized, and their exact Interaction Between Lassa mechanism of action is being determined. Virus and Its Receptor and In contrast to the receptor for Lassa virus, the cellular receptors of the highly pathogenic South Ameri- Development of Novel Antiviral can human hemorrhagic fever viruses Junin, Machupo, and Guanarito are currently unknown. Because of the Drugs Against Lassa Fever pivotal role of the virus-receptor interaction for infection and tissue tropism, identifying the cellular recep- J.M. Rojek, A.T. Gundersen, D.L. Boger,* S. Kunz tors used by these emerging viruses will substantially * Department of Chemistry, Scripps Research contribute to the understanding of their pathogenesis assa fever is the second most important viral and provide promising new targets for antiviral strate- hemorrhagic fever in humans; it accounts for gies. After initial biochemical characterization of the L more than 200,000 infections and several thou- receptors for these severe human pathogens, we will sand deaths each year.We focus on the earliest steps use a combination of genetic and biochemical tech- of Lassa virus infection, the binding of the virus to its niques to identify these cellular receptor molecules. cellular receptor, and combine studies of fundamental PUBLICATIONS mechanisms of virus–host cell interaction with the Kunz, S., de la Torre, J.C. Novel antiviral strategies to combat human arenavirus development of novel antiviral strategies against this infections. Curr. Mol. Med. 5:735, 2005. devastating disease. Kunz, S., Rojek, J.M., Kanagawa, M., Spiropoulou, C.F., Barresi. R., Campbell, The cellular receptor for Lassa virus is α-dystrogly- K.P., Oldstone, M.B.A. Posttranslational modification of α-dystroglycan, the cellular receptor for arenaviruses, by the glycosyltransferase LARGE is critical for virus can, a cell-surface receptor for proteins of the extra- binding. J. Virol. 79:14282, 2005. cellular matrix that provides a molecular link between Rojek, J.M., Spiropoulou, C.F., Kunz, S. Characterization of the cellular receptors the matrix and the cytoskeleton. Our recent studies for the South American hemorrhagic fever viruses Junin, Guanarito, and Machupo. Virology 349:476, 2006. indicate that binding of Lassa virus to α-dystroglycan involves a high-affinity interaction with specific sugar MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 301

VIRAL PATHOGENESIS Viral Pathogenesis and Our ongoing studies of the molecular biology of Antiviral Immunity coxsackievirus B3 have suggested a new explanation for how this class of virus evades the host cellular J.L. Whitton, N. An, N. Benning, C. Cornell, S. Crocker, immune response: several of the viral proteins target B. Eam, R. Feuer, R. Frausto, S. Harkins, I. Hunziker, F. Liu, the Golgi complex, inhibiting the function of the com- A. Nussbaum, R. Pagarigan, M.P. Rodriguez-Carreno, plex and leading to its eventual dissolution. Coxsack- J. Whitmire ievirus B3 is an important human pathogen that causes ANTIVIRAL T-CELL FUNCTION a variety of clinical syndromes, including myocarditis D8+ T cells play a key role in combating most and pancreatitis. Myocarditis is remarkably common viral infections, either by killing virus-infected (about 1 million cases per year in the United States), C cells or by showering the cells with antiviral currently is not treatable, and can lead to dilated car- cytokines such as IFN-γ. During microbial infection, diomyopathy, which is the most common indicator for epitope-specific CD8+ T-cell responses usually exist heart transplantation in young males. We previously + + as a hierarchy; responses to some epitopes are much showed the importance of CD4 and CD8 T cells in stronger than responses to others. The stronger responses the control of virus-induced myocarditis and in the are termed dominant; the weaker, subdominant. The related immunopathologic changes. hierarchy is regulated by a poorly understood phenom- We are extending our studies of coxsackievirus enon called immunodominance. B–specific immune responses to ask why this virus + We have found that immunodominance depends on does not induce strong CD8 T-cell responses, despite expression of IFN-γ. Our current hypothesis is that the reaching very high concentrations in various tissues. immunodominance hierarchy (i.e., the relative abun- We are also investigating prophylactic measures by dances of the various epitope-specific T-cell populations) evaluating RNA immunization in the coxsackievirus B3 is defined by the rate at which the various epitope-spe- model by using variant viral genomes with directed cific cells can initiate production of IFN-γ; the fastest mutations that are intended to retain immunogenicity cells become the dominant population. Our most recent while reducing virulence. Recently, we found that block- data indicate that expression of receptors for IFN-γ on ade of the protein tissue inhibitor of metalloproteinase 1 CD8+ T cells is tightly regulated and that cells lacking can ameliorate myocarditis and its consequences; this these receptors are at a selective disadvantage. There- observation may be of substantial clinical usefulness. fore, evolution appears to have used IFN-γ to kill two Finally, our studies of coxsackievirus B3 infection of the birds with one stone; the cells that are best suited to CNS in neonates indicated that the virus may prefer- combat viral infection (i.e., the cells that most rapidly entially infect stem cells and be carried into the brain elaborate IFN-γ) are the ones that are preferentially parenchyma by these cells as the cells migrate toward expanded in the host. These studies of T-cell regulation their final destinations. are being extended to include CD4+ T cells. AUTOIMMUNITY In most studies of T-cell function, including ours, Together with colleagues at the University of Utah synthetic peptides are used to stimulate T-cell responses and the La Jolla Institute of Allergy and Immunology, in vitro. We developed a novel method to identify T cells, we are studying the molecular basis of autoimmunity and other cell types, that are actively responding to induced by viral infection. Some autoimmune diseases contact with an authentic antigen in vivo. Using this (e.g., multiple sclerosis) appear to be triggered and/or approach, we showed that the in vivo response of CD8+ exacerbated by a wide variety of viral infections. Two memory T cells to viral infection is explosive. This general mechanisms, molecular mimicry and bystander method not only will be useful for studies of immune activation, have been proposed to explain this phenom- responses to infection but also may facilitate a better enon. We have suggested an alternative explanation understanding of autoimmune disease. In our analysis that is based on changes in antigen presentation that of antigen-specific activation in vivo, we also use in occur during almost all viral infections. situ hybridization, and we have confirmed that the in DNA IMMUNIZATION vivo CD8+ T-cell IFN-γ response to antigen contact is With our colleague M.J. Buchmeier, Molecular and very rapid and is regulated at the transcriptional level. Integrative Neurosciences Department, we are evaluating 302 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

DNA vaccines against the highly pathogenic arenavirus between ethnic groups is a natural variation in the genes Lassa virus. We have developed a DNA vaccine that that encode the structure of the enzymes that metabo- encodes Lassa virus proteins, shown that this vaccine lize alcohol. We were the first to identify a role for genetic induces Lassa virus–specific immune responses in mice, variations in 2 genes, the gene for alcohol dehydro- and shown that these mice are protected against chal- genase (ADH1B*3) and the gene for cytosolic alde- lenge with a related, but less pathogenic, arenavirus. hyde dehydrogenase (ALDH1A*1), in African Americans, Southwest California Indians, and islanders on Trinidad PUBLICATIONS and Tobago. We also showed that the response to alco- Fujinami, R.S., von Herrath, M.G., Christen, U., Whitton, J.L. Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease. Clin. hol in Asians is highly dependent on variants of the Microbiol. Rev. 19:80, 2006. gene for the mitochondrial enzyme aldehyde dehydro- Liu, F., Feuer, R., Hassett, D.E., Whitton, J.L. Peptide vaccination of mice genase (ALDH2*2). In Mexican Americans, the presence immune to LCMV or vaccinia virus causes serious CD8 T cell-mediated, TNF- dependent immunopathology. J. Clin. Invest. 116:465, 2006. of another form of alcohol dehydrogenase (ADH1B*2) can provide some protection from heavy drinking and Nussbaum, A.K., Rodriguez-Carreno, M.P., Benning, N., Botten, J., Whitton, J.L. Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to alcohol dependence. Our recent studies in Trinidad lymphocytic choriomeningitis virus infection and DNA vaccination. J. Immunol. and Tobago have indicated that individuals with the 175:1153, 2005. ADH1C2*2 genotype are more at risk for alcoholism Whitmire, J.K., Benning, N., Whitton, J.L. Cutting edge: early IFN-γ signaling directly and alcoholic liver disease than are individuals with- enhances primary antiviral CD4+ T cell responses. J. Immunol. 175:5624, 2005. out that genotype. Whitmire, J.K., Benning, N., Whitton, J.L. Precursor frequency, nonlinear proliferation, and functional maturation of virus-specific CD4+ T cells. J. Immunol. Although variations in alcohol-metabolizing enzymes 176:3028, 2006. clearly confer protection from or risk for alcoholism, other

Whitton, J.L. Adaptive immune responses. In: Molecular Pathogenesis of Virus genes also add to the genetic variance in risk for the Infections. Digard, P., Nash, A.A., Randall, R.E. (Eds.). Cambridge University disorder. To further evaluate other genetic factors associ- Press, New York, 2005, p. 1. Society for General Microbiology Symposia. Scour- field, M. (Series Ed.). ated with substance dependence, we conducted a genome scan in Southwest California Indian families for Whitton, J.L., Cornell, C.T., Feuer, R. Host and virus determinants of picornavirus pathogenesis and tropism. Nat. Rev. Microbiol. 3:765, 2005. alcoholism and behaviors related to substance abuse. We found that chromosomes 4 and 12 appear to have genes linked to the severity of an individual’s drinking; chro- Laboratory of Translational mosomes 6, 15, 16 have genes linked to a severe form of alcoholism with symptoms of withdrawal; and a locus Neurophysiology and the San on chromosome 5 is linked to “craving” for alcohol. Additional genome scans were conducted for use Diego Substance Abuse and of tobacco, dependence on marijuana, and dependence Minorities Project on stimulants. We discovered that a unique locus on chromosome 14 is linked to marijuana dependence; a C.L. Ehlers, C. Agneta, L. Corey, G. Finerman, D.A. Gilder, locus on chromosome 1, to stimulant dependence; and a J.W. Havstad, P. Lau, S.L. Lopez, E. Phillips, J. Roth, D. Wills locus on chromosome 4 and 8, to use of tobacco. In addition, several sites in the genome are linked not only ates of alcoholism and drug dependence within to use of multiple drugs of abuse but also to body mass. a population are thought to reflect an almost One theoretical assumption concerning Native Amer- R equal combination of sociocultural (environmen- icans is that the long history of dependence on foraging tal) and biological (genetically determined) factors. Our and subsistence agriculture may have led to selective goal is to identify genes that may encode the neuro- enrichment of traits that improve genetic fitness, so- physiologic processes that underlie drug dependence called thrifty or fat-sparing genes. In addition, such genes in selected populations at high risk for drug depen- might influence fat accumulation during food availabil- dence, including Native American Indians, Mexican ity, thus improving survival during times of shortage. Americans, and islanders of Trinidad and Tobago. The same selective pressure may have enriched for The prevalence of drug and alcohol dependence genetic variants that increase the risk for consumption among ethnic groups varies widely. These differences of energy-rich beverages such as alcohol and perhaps provide an opportunity to investigate how genetic vari- for consumption of other drugs of abuse. Taken together, ation may influence substance abuse. One difference our data lend support for the hypothesis that some genes MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 303 confer risk and/or protection for the abuse of specific Extensive studies of VGCCs in cerebellar Purkinje drugs and that other genes influence common behav- neurons from adult animals, a neuronal type that plays a iors associated with addiction such as the consump- critical role in fine motor control, have indicated that tive drive. the channels are essential for dendritic excitability. The primary VGCCs involved in this function are the P/Q- PUBLICATIONS Cook, T.A.R., Luczak, S.E., Shea, S.H., Ehlers, C.L., Carr, L.G., Wall, T.L. Associ- type VGCCs, which are expressed in abundance in Purk- ations of ALDH2 and ADH1B genotypes with response to alcohol in Asian Ameri- inje neurons. Our recent immunohistochemical studies cans. J. Stud. Alcohol 66:196, 2005. revealed that in addition to P/Q-type VGCCs, Purkinje Ehlers, C.L., Slutske, W., Gilder, D.A., Lau, P. Age of first marijuana use and the development of abuse and dependence in Southwest California Indians. Pharmacol. neurons express L-type VGCCs, both in the mature state Biochem. Behav., in press. and at early stages of development. In the mature neu- Ehlers, C.L., Slutske, W., Gilder, D.A., Lau, P., Wilhelmsen, K.C. Age at first rons, the L-type channels were located primarily in the intoxication and the development of alcohol dependence in Southwest California Indians. Alcohol. Clin. Exp. Res., in press. somatic region, whereas the P/Q-type channels were prominent in both the somatic and the dendritic regions. Ehlers, C.L., Wall, T.L., Dixon, M., Corey, L., Lau, P., Gilder, D.A., Wilhelmsen, K.C. Heritability of illicit drug use and transition to dependence in Southwest Cali- To examine the role of L-type VGCCs in Purkinje fornia Indians. Psychiatr. Genet., in press. neurons, we used combined recordings of intracellular Ehlers, C.L., Wilhelmsen, K.C. Genomic screen for loci associated with tobacco calcium and electrical activity in cultured Purkinje neu- usage in Mission Indians. BMC Med. Genet. 7:9, 2006. rons at different developmental stages. The results Ehlers, C.L., Wilhelmsen, K.C. Genomic screen for substance dependence and body mass index in Southwest California Indians. Genes Brain Behav., in press. showed that L-type VGCCs contribute to somatic excitability and calcium signaling both early in development, Gilder, D.A., Lau, P., Dixon, M., Corey, L., Phillips, E., Ehlers, C.L. Comorbidity of select anxiety, affective, and psychotic disorders with cannabis dependence in when each Purkinje neuron consists of just a soma and Southwest California Indians. J. Addict. Dis., in press. fine perisomatic processes, and at mature stages, when Irwin, M.R, Valladares, E., Motivala, S., Thayer, J.F., Ehlers, C.L. Association dendrites are present. Interestingly, L-type VGCCs played between nocturnal vagal tone and sleep depth, sleep quality, and fatigue in alcohol dependence. Psychosom. Med. 68:159, 2006. a prominent role in the somatic excitability in immature Purkinje neurons but only a modest role in mature Venner, K.L., Wall, T.L., Lau, P., Ehlers, C.L. Testing of an orthogonal measure of cultural identification with adult Mission Indians. Cultur. Divers. Ethnic Minor. Purkinje neurons. Psychol., in press. Calcium signaling involving L-type VGCCs is an Wilhelmsen, K.C., Ehlers, C. Heritability of substance dependence in a Native important pathway to gene expression, especially during American population.Psychiatr. Genet. 15:101, 2005. development. Consistent with such a role for L-type channels in immature Purkinje neurons, our recent stud- Cellular and Molecular ies showed that the calcium signals through L-type VGCCs were communicated to the nucleus and were Mechanisms of Neuronal associated with activation of the transcription factors Signaling in the CNS CREB and c-Fos. These results are consistent with a role for activity-dependent gene expression involving D.L. Gruol, T.E. Nelson, J. Cho,* J. Sabeti, H. Bajova, L-type VGCCs in early developing Purkinje neurons. S. Chow, E. Vereyken,** P.N.E. de Graan** REGULATION OF MEMORY MECHANISMS BY * Dongguk University, Gyeong Buk, Korea NEUROACTIVE STEROIDS ** University Medical Center Utrecht, Utrecht, the Netherlands The hippocampus is an integral component of the DEVELOPMENTAL REGULATION OF ION CHANNEL limbic circuitry and plays a central role in memory for- FUNCTION IN CNS NEURONS mation. Through effects on memory-related mechanisms NS neurons express a variety of ion channels with in the hippocampus, exposure to stress can result in specific roles in the generation and regulation of strong intrusive memories that interfere with normal C neuronal properties and functions. Of particular memory processing. A diverse class of biological sig- importance are the voltage-gated calcium channels naling molecules is thought to play a role in stress- (VGCCs). Although several types of these channels are related changes in hippocampal memory processes, expressed by neurons, calcium signaling through L-type including the glucocorticoids (i.e., cortisol in humans VGCCs is particularly important in many fundamental and corticosterone in rodents), which are considered neuronal processes, including neuronal development, the major stress hormones; pregnenolone sulfate; and neuronal excitability, and calcium homeostasis. progesterone and its reduced metabolites. These stress 304 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE steroids are designated neuroactive steroids because Neurobiology of Addiction of their ability to modify neuronal activity via both rapid (i.e., membrane receptor– or second messen- and Stress ger–mediated) and delayed (i.e., genomic) actions. The involvement of glucocorticoids in the brain stress G.F. Koob, M. Le Moal,* S. Ahmed, E. Riley,** L. Stinus,* response has been intensely studied, especially in hip- L. Pulvirenti,*** R. Purdy,**** H. Richardson, K. Inoue,***** † pocampus; considerably less is known about the role A. Tabarin, C. Funk, S. Ghozland, T. Greenwell, B. Walker, of other stress-induced neuroactive steroids. To address S. Wee, N. Gilpin, C. Mandyam, O. George, R. Lintz, this question, we investigated the actions of preg- E. Crawford, R. Schroeder, T. Kimber, M. Cole, M. Arends, M. Brennan, R. Smith, Y. Grant nenolone sulfate on hippocampal long-tem potentia- * Université Victor Ségalen Bordeaux 2, Bordeaux, France tion (LTP), considered the cellular basis for memory ** San Diego State University, San Diego, California and learning, by combining pharmacologic studies with *** Claude Bernard Neuroscience Institute, Pozzilli, Italy electrophysiology. We found that pregnenolone sulfate **** Veterans Affairs Medical Center, San Diego, California selectively facilitated the induction of a slow-developing ***** Osaka City University Medical School, Osaka, Japan LTP that was contingent on high-frequency (100 Hz) † Université Victor Ségalen Bordeaux 2, Hôpital du Haut-Lévêque, Pessac, stimulation of afferent neurons. This LTP was indepen- France dent of activation of receptors for N-methyl-D-aspartate but dependent on L-type VGCCs. Additional studies ADDICTION showed that pregnenolone sulfate produced concentra- n studies on the neurobiology of addiction, we con- tion-dependent inhibition of LTP dependent on recep- tinue to focus on the neuropharmacologic mecha- tors for N-methyl-D-aspartate. These findings indicate I nisms involved in motivated and emotional behavior that pregnenolone sulfate produces opposing actions and how these mechanisms are altered in addiction, on 2 pharmacologically distinct forms of hippocampal stress, and genetic variability. LTP, effects that could be important in the formation We are exploring the role of neurochemical systems of stress-related memories. in the extended amygdala in the neuroadaptations associated with the transition from drug taking to drug PUBLICATIONS dependence that is an integral part of the development Gruol, D.L., Nelson, T.E. Purkinje neuron physiology is altered by the inflammatory factor interleukin-6. Cerebellum 4:198, 2005. of addiction. We are also developing animal models for

Gruol, D.L., Netzeband, J.G, Quina, L.A., Blakely-Gonzalez, P.K. Contribution of excessive drug intake and charting the changes in neu- L-type channels to Ca2+ regulation of neuronal properties in early developing Purk- rocircuitry associated with excessive drug intake. Previ- inje neurons. Cerebellum. 4:128, 2005. ous results established that prolonged access to cocaine Gruol, D.L., Netzeband, J.G., Schneeloch, J., Gullette, C.E. L-type Ca2+ channels can produce progressive increases in drug intake that are contribute to current-evoked spike firing and associated Ca2+ signals in cerebellar Purkinje neurons. Cerebellum 5:146, 2006. paralleled by decreases in reward function. This escalation is paralleled by increased activity in the brain Gruol, D.L., Quina, L.A., Netzeband, J.G., Nguyen, D., Gullette, C.E. Develop- mental changes in Ca2+-regulated functions of early postnatal Purkinje neurons. J. stress system mediated by corticotropin-releasing factor Neurosci. Res. 83:1381, 2006. (CRF). New studies suggest that cocaine also activates Nelson, T.E., Ur, C.L., Gruol, D.L. Chronic intermittent ethanol exposure enhances the neuropeptide hypocretin to produce cocaine-seek- NMDA-receptor-mediated synaptic responses and NMDA receptor expression in hippocampal CA1 region. Brain Res. 1048:69, 2005. ing behavior via an arousal action originating in the hypothalamus. Consistent with this observation, ani- van Gassen, K.L.I., Netzeband, J.G., de Graan, P.N.E., Gruol, D.L. The chemokine CCL2 modulates Ca2+ dynamics and electrophysiological properties of cul- mals that escalate cocaine intake have a remodeling tured cerebellar Purkinje neurons. Eur. J. Neurosci. 21:2949, 2005. of lateral hypothalamic circuitry as measured by gene array studies. These results suggest that the reward dysregulation associated with extended access to drugs of abuse that leads to addiction may depend not only on neuroadaptive changes in basal forebrain systems but also on activation of hypothalamic systems. Studies in animal models of nicotine dependence revealed similar neuropharmacologic adaptations to chronic administration of nicotine. We found that chronic MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 305

exposure to nicotine increased nicotine self-administra- receptor systems and activation of CRF2 receptor systems tion in rats and that this increase could be exaggerated produce anti–anxiety-like effects. These results empha- by intermittent periods of abstinence from nicotine. A size the selectivity of the actions of the brain CRF and

CRF1 antagonist effectively blocked the development urocortin receptor systems and provide a basis for future of anxiety-like responses to precipitated nicotine with- studies of the pathophysiology of stress disorders. drawal. These findings suggest that excessive exposure to nicotine can produce some of the same neuroadap- PUBLICATIONS Ahmed, S.H., Koob, G.F. Transition to drug addiction: a negative reinforcement tive changes in the brain that occur with excessive model based on an allostatic decrease in reward function. Psychopharmacology access to other drugs of abuse. (Berl.) 180:473, 2005. We continue to develop animal models for exces- Ahmed, S.H., Lutjens, R., van der Stap, L.D., Lekic, D., Romano-Spica, V., Morales, M., Koob, G.F., Repunte-Canonigo, V., Sanna, P.P. Gene expression evi- sive drinking of alcohol that will be useful in identifying dence for remodeling of lateral hypothalamic circuitry in cocaine addiction. Proc. compounds with potential as medications for treatment Natl. Acad. Sci. U. S. A. 102:11533, 2005.

of drug addiction. The excessive drinking associated Boutrel, B., Kenny, P.J., Markou, A., Koob, G.F. Hypocretin and brain reward func- with alcohol dependence can be exacerbated by intermit- tion. In: Hypocretins: Integrators of Physiological Functions. de Lecea, L., Sutcliffe, J.G. (Eds.). Springer, New York, 2005, p. 315. tent repeated withdrawal from chronic alcohol exposure. Research with highly selective CRF small molecular Boutrel, B., Kenny, P.J., Specio, S.E., Martin-Fardon, R., Markou, A., Koob, G.F., 1 de Lecea, L. Role for hypocretin in mediating stress-induced reinstatement of antagonists has shown that CRF1 receptors may be cocaine-seeking behavior. Proc. Natl. Acad. Sci. U. S. A. 102:19168, 2005. effective in selectively blocking excessive drinking asso- De Witte, P., Littleton, J., Parot, P., Koob, G. Neuroprotective and abstinence-pro- ciated with dependence but have no effect in nonde- moting effects of acamprosate: elucidating the mechanism of action. CNS Drugs pendent animals. Because exposure to stressors is a 19:517, 2005. major stimulus for relapse in humans with alcoholism, Frantz, K.J., Koob, G.F. The neurobiology of addiction. In: Addiction Counseling these data suggest a potential novel role for the CRF Review: Preparing for Comprehensive, Certification, and Licensing Examinations. Coombs, R.H. (Ed.). Lawrence Erlbaum, Mahwah, NJ, 2005, p. 33. system, via CRF1 receptors, in vulnerability to relapse. Guillem, K., Vouillac, C., Azar, M.R., Parsons, L.H., Koob, G.F., Cador, M., We are refining our conceptual framework that the Stinus, L. Monoamine oxidase inhibition dramatically increases the motivation to neurochemical changes in brain stress neurotransmit- self-administer nicotine in rats. J. Neurosci. 25:8593, 2005.

ter systems lead to an allostatic change in motivated Heinrichs, S.C., Koob, G.F. Application of experimental stressors in laboratory behavior. Consistent with a role for self-medication for rodents. In: Current Protocols in Neuroscience, 2nd ed. Crawley, J.N., et al. (Eds.). Wiley & Sons, New York, 2005, p. 8.4.1. treatment of emotional states in humans with drug addiction, in the allostatic view, individuals, through genetic Izzo, E., Sanna, P.P., Koob, G.F. Impairment of dopaminergic system function after chronic treatment with corticotropin-releasing factor. Pharmacol. Biochem. Behav. vulnerability or environmental events, may use drugs 81:701, 2005. in an attempt to return to a nonstressed state (i.e., to Johnson, B.A., Koob, G.F., Schuckit, M.A., Mason, B.J., Ait-Daoud, N. Under- return to motivational homeostasis). Because of the standing and treating alcohol dependence. Alcohol. Clin. Exp. Res. 30:567, 2005. time lag between cause and effect in the neuroadapta- Kenny, P.J., Boutrel, B., Gasparini, F., Koob, G.F., Markou, A. Metabotropic gluta- tional capabilities of the brain motivational systems, mate 5 receptor blockade may attenuate cocaine self-administration by decreasing however, such individuals do not return to a nonstressed brain reward function in rats. Psychopharmacology (Berl.) 179:247, 2005. state, and the continued use of drugs further exacerbates Koob, G.F. The neurocircuitry of addiction: implications for treatment. Clin. Neu- the situation. Further refinement of this hypothesis was rosci. Res. 5:89, 2005. explored both in the context of a model of negative Koob, G.F., Le Moal, M. Plasticity of reward neurocircuitry and the “dark side” of drug addiction. Nat. Neurosci. 8:1442, 2005. reinforcement for drug seeking and in the context of the conceptual framework of a brain antireward system. O’Brien, C.P., Anthony, J.C., Carroll, K., Childress, A.R., Dackis, C., Diamond, G., Hornik, R., Johnston, L.D., Jones, R., Koob, G.F., Kosten, T., Lerman, C., McLel- NEUROPEPTIDES AND STRESS lan, A.T., Moss, H., Pettinati, H., Spoth, R. Defining substance use disorders. In: We are examining the functional significance of mem- Treating and Preventing Adolescent Mental Health Disorders: What We Know and What We Don’t Know—A Research Agenda for Improving the Mental Health of Our bers of the CRF brain stress neurotransmitter system. Youth. Evans, D.L., et al. (Eds.). Oxford University Press, New York, 2005, p. 335. In vivo tests with rats and defensive burying showed O’Dell, L.E., Purdy, R.H., Covey, D.F., Richardson, H.N., Roberto, M., Koob, G.F. efficacy for highly selective small-molecule CRF1 antag- Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self- administration in rats. Pharmacol. Biochem. Behav. 81:543, 2005. onists, confirming a role for the CRF1 receptor in anxiety-like responses. The CRF2 receptor–selective agonist Qi, L., Yamamoto, N., Meijler, M.M., Altobell, L.J. III, Koob, G.F., Wirsching, P., murine urocortin 3 had similar anxiolytic-like effects in Janda, K.D. ∆9-Tetrahydrocannabinol immunochemical studies: haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled ∆9-tetrahydro- the defensive burying test. Thus, both blockade of CRF1 cannabinol. J. Med. Chem. 48:7389, 2005. 306 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

Sanchis-Segura, C., Grisel, J.E., Olive, M.F., Ghozland, S., Koob, G.F., Roberts, We have 4 areas of primary interest. The first is A.J., Cowen, M.S. Role of the endogenous opioid system on the neuropsychophar- macological effects of ethanol: new insights about an old question. Alcohol. Clin. the roles of the 3 types of NRG-1 in the developing and Exp. Res. 29:1522, 2005. mature nervous system. We developed transgenic mice

Tabarin, A., Chaves, Y.D., Carmona, M.D., Catargi, B., Zorrilla, E.P., Roberts, A.J., that permit the tetracycline-regulated expression of spe- Coscina, D.V., Rousset, S., Redonnet, A., Parker, G.C., Inoue, K., Ricquier, D., cific NRG-1 isoforms. With these mice, we can assess Penicaud, L., Kieffer, B.L., Koob, G.F. Resistance to diet-induced obesity in µ-opioid receptor-deficient mice: evidence for a “thrifty gene.” Diabetes 54:3510, 2005. the distinct biological functions served by each isoform.

Zorrilla, E.P., Inoue, K., Fekete, E.M., Tabarin, A., Valdez, G.R., Koob, G.F. Mea- The second area is neurogenesis and migration. We suring meals: structure of prandial food and water intake of rats. Am. J. Physiol. found that the neuregulin receptor ErbB4 is expressed Regul. Integr. Comp. Physiol. 288:R1450, 2005. by multiple tangentially migrating populations of neu- Zorrilla, E.P., Koob, G.F. The roles of urocortins 1, 2 and 3 in the brain. In: Hand- ronal cells in the developing and mature nervous sys- book of Stress and the Brain, Part 1: The Neurobiology of Stress. Steckler, T., Kalin, N.H., Reul, J.M.H.M. (Eds.). Elsevier Science, New York, 2005, p. 179. tems. ErbB4 is expressed at high levels in the mature Techniques in the Behavioral and Neural Sciences; Vol. 15. subventricular zone and rostral migratory stream, one of the few regions in the brain in rats where neurogenesis occurs in adults. We are searching for the endog- Role of the Neuregulins in enous ligands and are testing the effects of the NRGs on cells derived from the subventricular zone. Our data the Nervous System suggest that ErbB4 influences both the proliferation of neural progenitor cells and the migration of neuroblasts C. Lai, J.L. Weber, J. Tan, A. Dowell, R. Salazar, D. Hom in the rostral migratory stream. he focus of our research is understanding the The third area of interest is the effects of the loss signaling mechanisms that underlie the estab- of ErbB4 function in the mature brain. We are analyz- T lishment and maintenance of mature neuronal ing the phenotype of mice that lack the gene for ErbB4 and glial cell phenotypes. We are studying the roles in the nervous system. These animals have a reduction played by a subfamily of receptor protein-tyrosine in anxiety-like behavior, and we are testing the hypothe- kinases, the ErbBs (EGFR, ErbB2, ErbB3, and ErbB4), sis that the loss of ErbB4 in the amygdala underlies and their ligands, the neuregulins (NRG-1–NRG-4). this defect. NRG-1 was first recognized as the Schwann cell mito- Last, we are developing transgenic tools that allow gen glial growth factor. NRG-1 was also termed ARIA regulated gene expression in specific subsets of neurons. (for acetylcholine receptor inducing activity), which was We developed lines of mice that permit regulated gene thought to regulate expression of acetylcholine receptors expression in cholinergic neurons, and we are evaluating at developing neuromuscular junctions. These distinct similar lines that permit regulated expression in either functions are now thought to be served by discrete types dopaminergic neurons or the medium spiny neurons of NRG-1 (I, II, and III) that arise by alternative splic- of the striatum. These animal models may be useful ing. A primary goal of our research is to understand for investigating addiction and neurodegenerative dis- the specific roles of each of these types of NRG-1 in orders such as Alzheimer’s, Parkinson’s and Hunting- the nervous system. ton’s diseases. NRG-1 supports survival of Schwann cells and regulates the number of premyelinating Schwann cells. PUBLICATIONS Ghashghaei, H.T., Weber, J.L., Pevny, L., Schmid, R., Schwab, M.H., Lloyd, K.C., The results of genetic studies suggested that the type III Eisenstat, D.D., Lai, C., Anton, E.S. The role of neuregulin-ErbB4 interactions on isoform serves in this capacity, and we helped determine the proliferation and organization of cells in the subventricular zone. Proc. Natl. Acad. Sci. U. S. A. 103:1930, 2006. that this isoform also plays a key role in regulating the Ponomareva, O., Ma, H., Dakour, R., Raabe, T.D., Lai, C., Rimer, M. Stimulation thickness of the myelin sheath. The emerging picture of acetylcholine receptor transcription by neuregulin-2 requires an N-box response is that different NRG-1 isoforms serve as signaling element and is regulated by alternative splicing. Neuroscience 134:495, 2005. molecules from neuron to glial cell and from neuron to muscle to carry out distinct biological activities. We are also pursuing the roles of these NRG-1 isoforms in the brain, which became an area of considerable interest after NRG-1 was identified as a susceptibility gene for schizophrenia. MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 307 Neurobiology of Reward, a property of nicotine that contributes to its potential for abuse. In addition, in a study of the reward-facilitat- Motivation, and Emotion in ing effects of nicotine in rats self-administering nicotine, chronic nicotine self-administration led to plastic changes Psychiatric Disorders in NMDA receptor activity reflected by increased sensitivity to NMDA receptor antagonism. A. Markou, K. Fish, S.G. Semenova, N.E. Paterson, P.J. Kenny, We also examined the role of mGlu2/3 receptors, M. Liechti, A. Bruijnzeel, A. Barr, B. Boutrel, B. Henry, which are inhibitory autoreceptors on glutamate-releasing N. Amitai, S. Jonkman, J. Benedict, G. Finnerman, neurons, in nicotine-related behaviors. Activation of these B. Silbaugh, J. Cryan,* W. Froestl,* D. Slattery,* receptors with LY379268, an mGlu2/3 receptor agonist, A. Bespalov,** T. Svensson*** decreased nicotine self-administration at doses that had * Novartis Pharma AG, Basel, Switzerland no effect on responding for food; this effect persisted ** Pavlov Medical University, St. Petersburg, Russia with chronic LY379268 administration for 5 days before *** Karolinska Institutet, Stockholm, Sweden tolerance developed. Further, LY379268 blocked cue- he focus of our research is the neurobiology of induced reinstatement of nicotine-seeking behavior. reward, motivation, and emotion in 3 psychiatric dis- Interestingly, rats chronically treated with nicotine had T orders: drug abuse, depression, and schizophrenia. increased activity of mGlu2/3 receptors; treatment with Two factors that contribute to habitual tobacco smok- the mGlu2/3 receptor agonist LY314582 precipitated ing are the euphorigenic and other reinforcing (e.g., cog- withdrawal-like reward deficits. Consistent with this nitive enhancement, anxiolytic) effects of nicotine and finding, the mGlu2/3 receptor antagonist LY341495 the depression-like symptoms that occur when a per- reversed the reward deficits that occurred during the son quits smoking. The depressive symptoms motivate early phase of spontaneous nicotine withdrawal. the reinitiation of tobacco use to alleviate this negative These data indicate that actions of nicotine on affective state. Because nicotine is the main ingredient glutamate transmission are critically involved in medi- in tobacco that leads to addiction, we are studying the ating several behavioral effects of nicotine related to its neurobiology of nicotine reinforcement and dependence. potential for abuse and its dependence-inducing prop- During the past few years, we have focused on gluta- erties. Blockade of the stimulatory effects of nicotine matergic and γ-aminobutyric acid modulation of nico- on glutamate transmission, through antagonism of tine reinforcement and dependence. excitatory postsynaptic glutamate receptors or activa- Nicotine activates nicotinic acetylcholine receptors tion of inhibitory presynaptic glutamate autoreceptors, on glutamate-releasing neurons, terminals, leading to attenuates the reinforcing effects of nicotine and nico- increased glutamate release. This finding suggests that tine-seeking behaviors. drugs that act on glutamate receptors may alter behav- Further, chronic exposure to nicotine results in adap- ioral effects of nicotine related to dependence. In rats, tations in presynaptic inhibitory and postsynaptic exci- blockade of postsynaptic metabotropic glutamate 5 tatory glutamate receptors, most likely to counteract the (mGlu5) receptors with 2-methyl-6-(phenylethynyl)- acute effects of nicotine on glutamate transmission. Such pyridine hydrochloride, a selective mGlu5 receptor adaptations in the activity of glutamate receptors most antagonist, and blockade of postsynaptic ionotropic likely lead to the behavioral effects associated with N-methyl-D-aspartate (NMDA) receptors with LY235959, dependence on nicotine, such as reward facilitation, an NMDA receptor antagonist, decreased nicotine self- aversive withdrawal signs shortly after cessation of nico- administration at doses that had no effect on respond- tine administration, and increased vulnerability to relapse ing for food. Treatment with 2,3-dihydroxy-6-nitro-7- during extended abstinence. sulfamoyl-benzo(F)quinoxaline, an antagonist of α-amino- These findings suggest several new targets for the 3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate development of antismoking medications. In addition, receptors, did not alter nicotine self-administration. Fur- on the basis of the phenomenologic and neurobiological ther, blockade of mGlu5 receptors decreased incentive- similarities between drug withdrawal and non–drug- motivation for nicotine and nicotine-seeking behavior in induced depressions, we hypothesize that mGlu2/3 rats. In addition, NMDA receptor antagonism reversed receptor antagonists may also have therapeutic antide- the reward-facilitating effects of nicotine, thus blocking pressant properties for non–drug-induced depression. 308 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

In other ongoing work, we are developing and using Slattery, D.A., Markou, A., Froestl, W., Cryan, J.F. The GABAB receptor-positive modulator GS39783 and the GABAB receptor agonist baclofen attenuate the rat and murine models of the cognitive deficits of schiz- reward-facilitating effects of cocaine: intracranial self-stimulation studies in the rat. ophrenia in humans, and we are extending our investi- Neuropsychopharmacology 30:2065, 2005. gations on nicotine dependence to mice, the mostly commonly used species in genetic studies. Medication Development in PUBLICATIONS Barr, A.M., Powell, S.B., Markou, A., Geyer, M.A. Iloperidone reduces sensorimo- tor gating deficits in pharmacological models, but not a developmental model, of Substance Dependence disrupted prepulse inhibition in rats. Neuropharmacology, in press. B.J. Mason, K. Buffkins, K. Coveney, R. Crean, T. Escher, Bespalov, A.Y., Dravolina, O.A., Sukhanov, I., Zakharova, E., Blokhina, E., Zvartau, E., Danysz, W., van Heeke, G., Markou, A. Metabotropic glutamate receptor (mGluR5) J. Light, S. Nash, S. Payton, S. Quello, J. Reiter, J. Diamant,* antagonist MPEP attenuated cue- and schedule-induced reinstatement of nicotine self- administration behavior in rats. Neuropharmacology 49(Suppl. 1):167, 2005. F. Shadan,* M. Kyle,* S. Rao,* M. Adusumalli,* J. Gleason,* D. Drobes** Boutrel, B., Kenny, P.J., Specio, S.E., Martin-Fardon, R., Markou, A., Koob, G.F., de Lecea, L. Role for hypocretin in mediating stress-induced reinstatement of * Scripps Green Hospital, La Jolla, California cocaine-seeking behavior. Proc. Natl. Acad. Sci. U. S. A. 102:19168, 2005. ** University of South Florida, Tampa, Florida Bruijnzeel, A.W., Markou, A. Decreased sensitivity to the effects of dopamine D1- like, but not D2-like, receptor antagonism in the posterior hypothalamic he focus of our research is the clinical evalua- region/anterior ventral tegmental area on brain reward function during chronic tion of medications for treatment of substance exposure to nicotine in rats. Brain Res. 1058:91, 2005. T dependence. Our primary aim is to reduce the Jonkman, S., Markou, A. Blockade of nicotinic acetylcholine or dopamine D1-like risk of relapse and the signs and symptoms of pro- receptors in the central nucleus of the amygdala or the bed nucleus of the stria terminalis does not precipitate nicotine withdrawal in nicotine-dependent rats. Neu- tracted abstinence, such as disturbances of mood and rosci. Lett. 400:140, 2006. sleep, associated with an increased risk for relapse. Kenny, P.J., Chen, S.C., Kitamura, O., Markou, A., Koob, G.F. Conditioned with- Projects range from proof-of-concept early-phase labo- drawal drives heroin consumption and decreases reward sensitivity. J. Neurosci. 26:5894, 2006. ratory studies in humans to long-term, double-blind, placebo-controlled studies of clinical efficacy. Kenny, P.J., Markou, A. Conditioned nicotine withdrawal profoundly decreases the activity of brain reward systems. J. Neurosci. 25:6208, 2005. A critical aspect of our conceptual framework is dynamic feedback from the scientists involved in pre- Kenny,P.J., Markou, A. Nicotine self-administration acutely activates brain reward systems and induces a long-lasting increase in reward sensitivity. Neuropsy- clinical and clinical studies, which are designed to chopharmacology 31:1203, 2006. streamline information and provide converging evidence Lindblom, N., de Villiers, S.H., Semenova, S., Kalayanov, G., Gordon, S., Schilstrom, for ultimate clinical use of medications. We also hope B., Johansson, A., Markou, A., Svensson, T.H. Active immunisation against nicotine blocks the reward facilitating effects of nicotine and partially prevents nicotine with- that the results of the clinical laboratory studies will in drawal in the rat as measured by dopamine output in the nucleus accumbens, brain turn be useful in the preclinical animal studies to fur- reward thresholds and somatic signs. Naunyn Schmiedebergs Arch. Pharmacol. 372:182, 2005. ther refine basic research involving animal models and

Markou, A. Metabotropic glutamate receptor antagonists: novel therapeutics for the neuropharmacologic approach. Using this approach, nicotine dependence and depression? Biol. Psychiatry, in press. we have identified areas of research that are being trans-

Matta, S.G., Balfour, D.J., Benowitz, N.L., Boyd, R.T., Buccafusco, J.J., Caggiula, lated into long-term studies of the clinical efficacy of A.R., Craig, C.R., Collins, A.C., Corrigall, W.A., Damaj, M.A., Donny, E.C., Gar- various medications for treatment of alcohol, nicotine, diner,P.S., Grady, S.R., Heberlein, U., Leonard, S.S., Levin, E.D., Lukas, R.J., Markou, A., Marks, M.J., McCallum, S.E., Parameswaran, N., Perkins, K.A., Pic- and cannabis dependence. ciotto, M.R., Quik, M., Rose, J.E., Rothenflut, A., Schafer, W.R., Stolerman, I.P., CLINICAL RESEARCH Tyndale, R.F., Wehner, J.M., Zirger, J.M. Guidelines on nicotine dose selection for in vivo research. Psychopharmacology (Berl.), in press. Alcohol and nicotine dependence are major public

O’Dell, L.E., Bruijnzeel, A.W., Smith, R.T., Parsons, L.H., Merves, M.L., Gold- health problems that tend to occur together. Existing berger, B.A., Richardson, H.N., Koob, G.F., Markou, A. Diminished nicotine with- treatments address each disorder independently and drawal in adolescent rats: implications for vulnerability to addiction. Psychopharmacology (Berl.) 186:629, 2006. are of limited efficacy. The opioid antagonist naltrexone (ReVia) is approved by the Food and Drug Adminis- Paterson, N.E., Bruijnzeel, A.W., Kenny, P.J., Wright, C.D., Froestl, W., Markou, A. Prolonged nicotine exposure does not alter GABAB receptor-mediated regulation tration for the treatment of alcohol dependence. Some of brain reward function. Neuropharmacology 49:953, 2005. laboratory and clinical studies suggest that administra- Paterson, N.E., Markou, A. Design of animal models and treatments for addiction tion of opioid antagonists also reduces signs and symp- and depression comorbidity. Neurotox. Res., in press. toms of nicotine dependence, but the findings vary. Paterson, N.E., Markou, A. The metabotropic glutamate 5 antagonist MPEP We recently determined the efficacy of naltrexone for decreased break points for nicotine, cocaine and food in rats. Psychopharmacology (Berl.) 179:255, 2005. treatment of outpatients with concurrent nicotine and MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 309 alcohol dependence in a double-blind, placebo-controlled Food and Drug Administration to reduce relapse in early 12-week trial of either a fixed daily dose of naltrexone or a abstinence from alcohol, is highly involved in mediat- daily placebo. In order to control for over-the-counter ing responsivity to affective as well as alcohol cues in availability of nicotine replacement products and to recently abstinent patients with alcoholism. A 5-year examine potential drug-drug interactions, patients who renewal of this project by the National Institute on set a smoking quit date during the first 6 weeks of study Alcohol Abuse and Alcoholism enables us to use the were also randomized to receive a nicotine replacement model to rapidly screen medications that may prevent patch (Nicotrol) or a placebo patch. relapse to drinking. In an interim analysis of the first 53 subjects, compared with the other treatments, treatment with nal- PUBLICATIONS Anton, R.F., O’Malley, S.S., Ciraulo, D.A., Cisler, R.A., Couper, D., Donovan, D.M., trexone was associated with a higher rate of premature Gastfriend, D.R., Hosking, J.D., Johnson, B.A., LoCastro, J.S., Longabaugh, R., Mason, B.J., Mattson, M.E., Miller, W.R., Pettinati, H.M., Randall, C.L., Swift, R., termination of therapy and no beneficial effects on drink- Weiss, R.D., Williams, L.D., Zweben, A., the COMBINE Study Research Group. ing or smoking outcomes. Patients who used the nico- Combined pharmacotherapies and behavioral interventions for alcohol dependence: tine replacement patch stayed in treatment longer and the COMBINE study: a randomized controlled trial. JAMA 295:2003, 2006. had more nonsmoking days, fewer heavy smoking days, Johnson, B.A., Koob, G.F., Schuckit, M.A., Mason, B.J., Ait-Daoud, N. Under- standing and treating alcohol dependence. Alcohol. Clin. Exp. Res. 30:567, 2006. longer time to smoking relapse, less irritability, and more nondrinking days than did patients who did not use the Kranzler, H.R., Mueller, T., Cornelius, J., Pettinati, H.M., Moak, D., Martin, P.R., Anthenelli, R., Brower, K.J., O’Malley, S., Mason, B.J., Hasin, D., Keller, M. Ser- patch. These results suggest that nicotine replacement traline treatment of co-occurring alcohol dependence and major depression. J. Clin. (agonist) therapy may result in longer compliance with Psychopharmacol. 26:13, 2006. treatment and better smoking and drinking outcomes Mason, B.J. Acamprosate in the treatment of alcohol dependence. Expert Opin. than does naltrexone (antagonist) treatment in patients Pharmacother. 6:2103, 2005. with concurrent alcohol and tobacco dependence. Mason, B.J., Goodman, A.M., Chabac, S., Lehert, P. Effect of oral acamprosate on abstinence in patients with alcohol dependence in a double-blind, placebo-con- We are currently conducting a 12-week, double- trolled trial: the role of patient motivation. J. Psychiatr. Res. 40:383, 2006. blind, placebo-controlled dose-ranging study to evaluate gabapentin, an anticonvulsant with favorable side effects, as a treatment for signs and symptoms such as anxi- Chemokine Effects on ety and insomnia that may occur after alcohol withdrawal and that can precipitate a relapse to drinking. Neuronal Physiology The study sample will consist of 150 recently abstinent outpatient volunteers who are alcohol dependent. This T.E. Nelson, D.L. Gruol, H. Bajova, J. Cho* project is funded by the National Institute on Alcohol * Dongguk University, Gyeong Buk, South Korea Abuse and Alcoholism. hemokines are members of the cytokine family We have also received funding from the National of immunoregulators whose primary role is the Institute on Drug Abuse to conduct a 12-week, double- activation and trafficking of leukocytes to sites blind, placebo-controlled study of gabapentin as a poten- C tial treatment for cannabis dependence. We hypothesize of infection or injury. Expression of chemokines in the that gabapentin will improve signs and symptoms of can- CNS is upregulated in a number of neurologic diseases nabis withdrawal and, as a result, facilitate setting a quit and disorders, including HIV-associated dementia, date, promote longer-term abstinence, and decrease risk multiple sclerosis, Alzheimer’s disease, brain tumors, and severity of relapse to cannabis use. CNS trauma, and stroke. In the CNS, chemokines are DEVELOPMENT OF A HUMAN LABORATORY MODEL expressed predominantly by glial cells (astrocytes and We developed a human experimental model of var- microglia), whereas chemokine receptors are expressed ious components of the alcoholism cycle. We use cue by both neurons and glial cells, indicating that the latter reactivity and mood induction techniques in the labo- 2 cell types are potential targets of chemokine actions in ratory and assessment of drinking, mood, and sleep the CNS. In addition to their role in neuroinflammation, under natural conditions. The parameters of the model chemokines are also involved in regulating normal neural are used to evaluate potential treatments for various processes, including neuronal migration, modulation of components of the alcoholism cycle. synaptic activity and plasticity, and neuronal survival. In an important validation study of the model, we Currently, we are using primary organotypic cul- found that acamprosate, a medication approved by the tures of rat hippocampus to investigate the effects of 310 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE acute and chronic chemokine exposure on neuronal function. We have focused on the chemokine CXCL10 Neurochemistry of Addiction (previously known as IFN-γ–inducible protein-10 or IP- L.H. Parsons, L. Alvarez, I. Polis, D. Stouffer 10). Elevated levels of CXCL10 are highly prevalent in the cerebrospinal fluid of patients with HIV type 1 ndogenous cannabinoids such as anandamide infection and correlate strongly with the severity of the and 2-arachidonoylglycerol (2-AG) modulate neurologic disorders associated with the infection. several functions, including memory, emotional Using fluorescence-based calcium imaging and intra- Ebehavior, and pain perception. Endocannabinoids are cellular electrophysiologic recording, we found that acute also involved in motivation and reward processes, and exposure to CXCL10 enhanced ongoing electrical activ- converging evidence from studies in humans and ani- ity of hippocampal neurons maintained in culture and mals implicates the endogenous cannabinoid system that this enhancement coincided with increased activ- in the etiology of drug addiction. ity-dependent elevations of intracellular calcium in these We have developed an in vivo microdialysis method cells. In addition, using immunoblotting and immuno- for monitoring extracellular endocannabinoid levels in histochemical methods, we investigated (1) effects of the brains of rats during ongoing behavioral tasks. Using acute CXCL10 exposure on signal transduction mediated this technique, we found that voluntary self-administra- by extracellular-signal–regulated kinase 1/2 (ERK1/2) tion of ethanol, heroin, and cocaine in rats induces in hippocampal neurons and (2) alterations in expression distinct alterations in extracellular endocannabinoid of neurotransmitter receptors induced in hippocampal levels in the nucleus accumbens, a brain region criti- cultures by chronic exposure to CXCL10. We found cally involved in drug reward. that acute exposure (5 minutes) to CXCL10 activated Oral self-administration of ethanol produced a strong ERK1/2 in hippocampal neurons; after longer exposure increase in extracellular 2-AG levels with no concomi- (15–30 minutes), ERK1/2 activity was decreased rel- tant alteration in extracellular levels of anandamide. ative to control levels. In addition, 30–40 minutes of Conversely, heroin self-administration significantly exposure inhibited ERK1/2 activation mediated by the increased anandamide levels while inducing a slight but N-methyl-D-aspartate (NMDA) subtype of glutamate significant decrease in 2-AG levels. The relative changes receptors. After chronic CXCL10 exposure (7 days), pro- in endocannabinoid levels in the nucleus accumbens tein levels of the NR1, NR2A, and NR2B subunits of induced by ethanol and heroin were significantly cor- the NMDA receptor were increased. related with the amount of drug consumed by each ani- Functional studies indicated that calcium signaling mal, indicating a dose-dependent pharmacologic process. mediated by NMDA receptors in hippocampal neurons In contrast, neither the anandamide level nor the 2-AG was enhanced after chronic CXCL10 exposure, corre- level in the nucleus accumbens was altered by cocaine sponding to the increased expression of the receptor self-administration. subunits. In contrast, expression of the R1 subunit These observations are consistent with our finding of the γ-aminobutyric acid B receptor was reduced by that self-administration of ethanol and of heroin is chronic CXCL10 exposure. Chronic exposure also was reduced by blockade of cannabinoid-1 (CB ) receptors, associated with a decrease in the effect of CGP55845A, 1 whereas cocaine self-administration is not. Collectively an antagonist of the γ-aminobutyric acid B receptor, these findings suggest that the motivational effects of on spontaneous calcium oscillations in the neuronal ethanol and heroin are mediated in part by drug-induced network of hippocampal neurons. Our results indicate formation of endocannabinoids. that chemokines modulate CNS function under normal In support of this hypothesis, we found that phar- physiologic conditions as well as during periods of macologic manipulations of endocannabinoid signal- immune challenge, neurotrauma, or neurologic disease. ing altered ethanol self-administration behavior only

PUBLICATIONS when sufficient blood alcohol levels were achieved. Gruol, D.L., Nelson, T.E. Purkinje neuron physiology is altered by the inflammatory Blood alcohol levels after self-administration were nearly factor interleukin-6. Cerebellum 4:198, 2005. 3-fold higher when rats were presented with a 10% Nelson, T.E., Ur, C.L., Gruol, D.L. Chronic intermittent ethanol exposure enhances NMDA-receptor-mediated synaptic responses and NMDA receptor expression in ethanol solution than when they were presented with hippocampal CA1 region. Brain Res. 1048:69, 2005. a 2% ethanol solution. Lever pressing for a 10% ethanol MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 311 solution was decreased by treatment with the CB recep- PUBLICATIONS 1 Breese, G.R., Criswell, H.E., Carta, M., Dodson, P.D., Hanchar, H.J., Khisti, R.T., tor antagonist rimonabant and increased by treatment Mamelli, M., Ming, Z., Morrow, A.L., Olsen, R.W., Otis, T.S., Parsons, L.H., Pen- with the endocannabinoid clearance inhibitor AM404; land, S.N., Roberto, M., Siggins, G.R., Valenzuela, C.F., Wallner, M. Basis of the GABAmimetic profile of ethanol. Alcohol. Clin. Exp. Res. 30:731, 2006. treatment with these compounds did not alter the self- Caillé, S., Parsons, L.H. Cannabinoid modulation of opiate reinforcement through administration of 2% ethanol. In contrast, treatment the ventral striatopallidal pathway. Neuropsychopharmacology 31:804, 2006. with the synthetic CB receptor agonist WIN 55,212- 1 Frantz, K.J., O’Dell, L.E., Parsons, L.H. Behavioral and neurochemical responses 2 significantly increased lever pressing for both 10% to cocaine in periadolescent and adult rats. Neuropsychopharmacology, in press. and 2% concentrations of ethanol. These data are con- O’Dell, L.E., Manzardo A., Polis, I., Parsons, L.H. Biphasic alterations in 5-HT1B sistent with a dose-dependent effect of ethanol on receptor function during abstinence from extended cocaine self-administration. J. Neurochem., in press. the formation of endocannabinoids and indicate that Purdy, R.H., Fitzgerald, R.L., Alomary, A.A., Parsons, L.H. The analysis of neu- ethanol-induced increases in endocannabinoid formation roactive steroids by mass spectrometry. In: Handbook of Neurochemistry and participate in the regulation of alcohol consumption. Molecular Neurobiology, 3rd ed. Baker, G., et al. (Eds.). Springer, New York, in press. Practical Neurochemistry Methods. We also found evidence for a sensitization of ethanol- induced increases in brain levels of 2-AG after chronic Selvage, D.J., Parsons, L., Rivier, C. Role played by brainstem neurons in regulating testosterone secretion via a direct neuronal pathway between the hypothalamus exposure to ethanol. In ethanol-naive rats, acute intraper- and the testes. Endocrinology 147:3070, 2006. itoneal administration of ethanol increased levels of 2-AG in the nucleus accumbens to approximately 150% of baseline; this effect dissipated within 60 minutes of Brain-Computer Interface: ethanol administration. However, this same dose increased levels of 2-AG in the nucleus accumbens to New Directions approximately 280% of baseline for a period of more B.Z. Allison, J. Polich than 2 hours in rats previously exposed to ethanol for 14 days via a liquid-diet procedure. The potentiation illions of persons have neuromuscular disorders of ethanol-induced formation of endocannabinoids due to central or peripheral nervous system after chronic ethanol exposure suggests a potential M injury, amyotrophic lateral sclerosis, stroke, involvement of endocannabinoids in the development cerebral palsy, muscular dystrophy, multiple sclerosis, of ethanol dependence. Guillian-Barré syndrome, and other causes. Amputees also need alternative interfaces to control prosthetic Finally, we have been testing the influence of endo- devices. Many patients with disabilities cannot use cannabinoid signaling on the vulnerability to drug keyboards, computer mice, or other conventional inter- relapse. We found that intraperitoneal administration faces and rely on alternative means of communication of rimonabant significantly decreased the reinstatement that require voluntary control of movement, such as of drug-seeking behavior induced by presentation of speech, gaze shifting, muscle activity, and breathing. an environmental cue that signals the availability of Some patients cannot use any interface that requires heroin. Several brain regions were implicated in the motor control. mediation of cue-induced drug-seeking behavior, includ- A brain-computer interface (BCI) is a communica- ing the core subregion of the nucleus accumbens, the tion system in which messages or commands that a basolateral nucleus of the amygdala, and the medial person sends to the external world do not pass through prefrontal cortex. Localized administration of rimona- the normal output pathways of peripheral nerves and bant into the core subregion of the nucleus accumbens muscles. The system infers the user’s intent via direct dose-dependently attenuated cue-induced drug seek- measures of brain activity. ing, whereas administration of this CB1 antagonist BCI COMPONENTS into the basolateral amygdala did not. Figure 1 schematically illustrates the 4 BCI compo- Collectively our observations indicate an involve- nents. The first component is signal acquisition. Nearly ment of drug-induced formation of endocannabinoids all BCI systems rely on measures of electroencephalo- in the regulation of ethanol and heroin intake and sug- graphic activity collected via electrodes placed on the gest a possible role for endocannabinoids in the devel- scalp. These noninvasive systems do not require expen- opment of drug dependence and the vulnerability toward sive or bulky equipment, highly trained personnel, drug relapse. surgery, or long preparation times. 312 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

SCRIPPS BCI RESEARCH GROUP A major limitation of current BCIs is poor information throughput compared with that of other assistive interfaces. This difficulty hampers direct communication between patients and other persons, monitors, or devices. Researchers in the Cognitive Electrophysiol- ogy Laboratory in the Molecular and Integrative Neuro- sciences Department have begun a series of studies to improve traditional BCIs that enable spelling and other communication by combining the BCIs with steady-state stimulation to produce highly readable brain wave signals. Initial investigations with these “hybrid BCI” approaches have shown promise for improving performance. These methods are more reliable than conventional systems and are easier to use with different Fig. 1. Schematic illustration of the 4 major BCI components: technologies. If successful, this approach will greatly signal acquisition, signal processing, output device, and operating accelerate development of system applications to criti- protocol. Reprinted from Wolpaw, J.R,. Birbaumer, N., McFarland, D.J., Pfurtscheller, G., Vaughan, T.M. Brain-computer interfaces for cal populations of patients. communication and control. Clin. Neurophysiol. 113:767. Copyright PUBLICATIONS 2002, with permission from International Federation of Clinical Neuro- Cahn, B.R., Polich, J. Meditation states and traits: EEG, ERP, and neuroimaging physiology. studies. Psychol. Bull. 132:180, 2006.

The second component is signal processing, which Combs, L.A., Polich, J. P3a from white noise stimuli. Clin. Neurophysiol. 117:1106, 2006. includes feature extraction and a translation algorithm. Electroencephalographic features of interest are isolated Conroy, M.A., Polich, J. Affective valence and P300 when stimulus arousal level is controlled. Cogn. Emot., in press. and then translated into instructions for the output device. Some signal-processing mechanisms are more Hagen, G.F., Gatherwright, J.R., Lopez, B.A., Polich, J. P3a from visual stimuli: task difficulty effects. Int. J. Psychophysiol. 59:8, 2006. adaptive than others are. An ideal BCI would respond to Polich, J., Corey-Bloom, J. Alzheimer’s disease and P300: review and evaluation initial identification of a user’s brain wave features, of task and modality. Cur. Alzheimer Res. 2:515, 2005. adjust to phasic changes, and update as the user adapts. Polich, J., Criado, J.R. Neuropsychology and neuropharmacology of P3a and P3b. The third component, the output device, implements Int. J. Psychophysiol. 60:172, 2006. the messages or commands conveyed by the translation algorithm. The most common BCI output device is a computer monitor, although other output devices have Cellular Physiology of been developed to control appliances, robotic arms, mobile robots, and functional electrical stimulators. Neuropeptides and Drugs The fourth component is the operating protocol, which reflects how the user and the BCI interact. The of Abuse in the Brain output device is hardware, whereas the operating pro- M. Roberto, P. Schweitzer, G.R. Siggins, L.H. Parsons tocol is software that includes how the user can affect the hardware. Monitor-based BCIs have been developed e study the effects of ethanol on neuronal in which users control a switch, move a cursor in one communication by focusing on the nucleus or more dimensions, directly select one of two or more W of the central amygdala. Our aim is to uncover choices, select items from a scrolling or iterative menu, (1) the physiologic mechanisms that underlie the acute browse the Internet, or navigate a virtual environment. action of ethanol and the involvement of neuropeptides Additional issues include the timing of trials and ses- such as corticotropin-releasing factor (CRF) and noci- sions, feedback, word and sentence completion, and ceptin and (2) the neuroadaptations associated with error-correction mechanisms based on response verifi- ethanol dependence. cation, a backspace option, or event-related brain poten- Using an electrophysiologic approach, we found that tials such as the P300 component. acute exposure to ethanol increases inhibitory trans- MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 313 mission in neurons of the central amygdala in both naive Roberto, M., Bajo, M., Crawford, E., Madamba, S.G., Siggins, G.R. Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amyg- and ethanol-dependent rats, in part by enhancing the dala. Neuropsychopharmacology 31:988, 2006. vesicular release of γ-aminobutyric acid (GABA) in the Roberto, M., Siggins, G.R. Nociceptin/orphanin FQ presynaptically decreases central amygdala. We confirmed these in vitro findings GABAergic transmission and blocks the ethanol-induced increase of GABA release with in vivo microdialysis studies in which we found in central amygdala. Proc. Natl. Acad. Sci. U. S. A. 103:9715, 2006. that both acute and chronic ethanol exposure increased Roberto, M., Treistman, S.N., Pietrzykowski, A.Z., Weiner, J., Galindo, R., Mameli, M., Valenzuela, F., Zhu, P.J., Lovinger, D., Zhang, T.A., Hendricson, GABA release in the central amygdala. In addition, we A.H., Morrisett, R., Siggins, G.R. Actions of acute and chronic ethanol on presy- found a critical mimicry between ethanol and CRF effects naptic terminals. Alcohol. Clin. Exp. Res. 30:222, 2006. on GABAergic transmission that showed marked adap- Siggins, G.R., Roberto, M., Nie, Z. The tipsy terminal: presynaptic effects of tations during the development of ethanol dependence. ethanol. Pharmacol. Ther. 107:80, 2005.

Nociceptin, however, decreased GABA release and Slanina, K.A., Roberto, M., Schweitzer, P. Endocannabinoids restrict hippocampal opposed both ethanol and CRF effects. Chronic ethanol long-term potentiation via CB1. Neuropharmacology 49:660, 2005. exposure enhances the sensitivity of GABAergic systems Woodward, J.J., Ron, D., Winder, D., Roberto, M. From blue states to up states: a in central amygdala to these 2 neuropeptides. On the regional view of NMDA-ethanol interactions. Alcohol. Clin. Exp. Res. 30:359, 2006. basis of this evidence, we hypothesize that nociceptin functions as a “brake” to limit the enhancement of inhibitory transmission in the neural circuitry involved Alcohol and Drug Self- in the reinforcing actions of ethanol. Administration and the We also recently discovered that acute exposure to ethanol inhibits responses in neurons in the central Mouse Behavioral amygdala mediated by receptors for N-methyl-D-aspartate (NMDA), principally at postsynaptic sites. Pro- Assessment Core Facility longed exposure to ethanol postsynaptically increased A.J. Roberts, C.L. Levy, L. Underwood, C. Pañeda the sensitivity of NMDA receptors and increased glutamate release. Importantly, these changes were reversed ur overall goal is to investigate the neural bases with ethanol withdrawal. These physiologic data were of behavior by using mouse models. In partic- correlated with molecular studies showing that chronic O ular, we are interested in motivated behaviors exposure to ethanol increased the levels of messenger such as drug and alcohol self-administration and RNA and protein of selective NMDA receptor subunits exploratory drive. in neurons in the central amygdala, indicating that We are involved in a multisite integrated neuro- ethanol elicits reversible neuroadaptations in synaptic science initiative on alcoholism sponsored by the function, gene expression, and protein composition of National Institute on Alcohol Abuse and Alcoholism NMDA receptors in these neurons. in which the overall goal is to examine the neural basis Understanding the cellular adaptations induced by of excessive alcohol drinking. We developed a model chronic exposure to alcohol will provide insight into of excessive alcohol drinking after a period of abstinence how the transition to alcoholism occurs, and possibly in alcohol-dependent mice that we are using in both treatments for alcoholism. Currently, we are examining genetic and neuropharmacologic experiments. For how these changes contribute to the development of example, recently we found that mice lacking the cor- ethanol dependence and protracted abstinence. ticotropin-releasing factor (CRF) receptor 1 do not have increased ethanol self-administration after alcohol PUBLICATIONS dependence. In parallel, we showed that intra-amyg- Bajo, M., Crawford, E.F., Roberto, M., Madamba, S.G., Siggins, G.R. Chronic dalar injections of a CRF receptor antagonist block morphine treatment alters expression of N-methyl-D-aspartate receptor subunits in the extended central amygdala. J. Neurosci. Res. 83:532, 2006. increases in ethanol self-administration induced by

Breese, G.R., Criswell, H.E., Carta, M., Dodson, P.D., Hanchar, H.J., Khisti, R.T., alcohol dependence in normal mice. These comple- Mameli, M., Ming, Z., Morrow, A.L., Olsen, R.W., Otis, T.S., Parsons, L.H., Pen- mentary studies support a role for the brain CRF sys- land, S.N., Roberto, M., Siggins G.R., Valenzuela, C.F., Wallner, M. Basis of the GABAmimetic profile of ethanol. Alcohol. Clin. Exp. Res. 30:731, 2006. tem in alcohol addiction. Another focus of our group is studies of self-admin- O’Dell, L.E., Purdy, R.H., Covey, D.F., Richardson, H.N., Roberto, M., Koob, G.F. Epipregnanolone and a novel synthetic neuroactive steroid reduce alcohol self- istration of intravenous cocaine, morphine, and meth- administration in rats. Pharmacol. Biochem. Behav. 81:543, 2005. amphetamine in mice. We developed a model of relapse 314 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE to drug-seeking behavior in which mice are trained to Sanchis-Segura, C., Grisel, J.E., Olive, M.F., Ghozland, S., Koob, G.F., Roberts, A.J., Cowen, M.S. Role of the endogenous opioid system on the neuropsychophar- press a lever for cocaine and then this behavior is macological effects of ethanol: new insights about an old question. Alcohol. Clin extinguished by removing the cocaine. Then the mice Exp. Res. 29:1522, 2005. are subjected to various manipulations, such as expo- Tabarin, A., Chaves, Y.D., Carmona, M.C., Catargi, B., Zorrilla, E.P., Roberts, A.J., sure to stressors or administration of neuroactive com- Coscina, D.V., Rousset, S., Redonnet, A., Parker, G.C., Inoue, K., Ricquier, D., Penicaud, L., Kieffer, B.L., Koob, G.F. Resistance to diet-induced obesity in µ-opioid pounds, and how the animals respond to the lever receptor-deficient mice: evidence for a “thrifty gene.” Diabetes 54:3510, 2005. previously associated with cocaine is recorded. We Tallent, M.K., Fabre V., Qiu, C., Calbet, M., Lamp, T., Baratta, M.V., Suzuki, C., found that treatment with the newly discovered neu- Levy, C.L., Siggins, G.R., Henriksen, S.J., Criado, J.R., Roberts, A.J., de Lecea, L. Cortistatin overexpression in transgenic mice produces deficits in synaptic plas- roactive peptide neuropeptide S results in reinstatement ticity and learning. Mol. Cell. Neurosci. 30:465, 2005. of lever pressing in this model, suggesting a novel relapse mechanism. Understanding the underlying neural mechanisms of relapse can enhance the ability to treat and Molecular Mechanisms of prevent addictive disorders. In the past year, we started a mouse behavioral Adaptive and Maladaptive assessment core facility at Scripps Research. The purpose of the facility is to provide high-quality mouse Neuronal Plasticity behavioral assessments to neuroscientists located near Scripps Research. A primary focus is to provide tests that P.P. Sanna, F. Berton, V. Canonigo, D. Lekic, K. Hagihara, allow investigators to make transitions from laboratory M. Mendez-Diaz, V. Mendoza-Fernandez, D. Thurbon, findings to clinical applications by enabling the model- L. van der Stap, W. Francesconi ing of human diseases and the development of medica- s part of our effort to apply microarray-based tions and other treatment strategies. For example, test strategies to studies of the neurobiology of drug batteries have been developed for several neuropsychi- A abuse, we profiled gene expression in reward- atric disorders, including anxiety disorders, depressive related regions of the brains in rats that had escalated disorders, disorders of learning and memory, disorders cocaine intake after extended access to cocaine. We of motor functioning, drug and alcohol abuse and compared 4 methods of analysis used to generate dependence, eating disorders, and other compulsive gene expression values: 2 that use perfect-match- and impulsive disorders. minus-mismatch models and 2 that use perfect- Our services include specific behavioral tests, gen- match-only models. Results were validated by using eral test batteries, surgical procedures, drug adminis- reverse transcriptase–polymerase chain reaction of tration protocols, and training in all of these areas. RNA from individual animals from an independent Included in these services is advice on experimental replication of the experiment. design, assistance in submitting protocols for studies We found that a small number of genes are selec- in animals, data analysis, interpretation of results, and tively associated with escalated cocaine intake. Unex- assistance in writing descriptions of these tests, results, pectedly, of the brain regions examined (prefrontal and interpretations in grant proposals and manuscripts. cortex, nucleus accumbens, septum, lateral part of the In the past few months, we have done work for inves- hypothalamus, amygdala, and ventral tegmental area), tigators in the Departments of Molecular Biology, Molec- the lateral part of the hypothalamus was the most ular and Experimental Medicine, Cell Biology, and transcriptionally responsive in escalation of cocaine Chemistry and in the Molecular and Integrative Neuro- sciences Department. intake. Most of the genes associated with escalated cocaine intake are also expressed during synaptogene- PUBLICATIONS sis and synaptic plasticity and include genes that code Carrera, M.R., Trigo, J.M., Wirsching, P., Roberts, A.J., Janda, K.D. Evaluation of the anticocaine monoclonal antibody GNC92H2 as an immunotherapy for cocaine for several presynaptic and postsynaptic proteins involved overdose. Pharmacol. Biochem. Behav. 81:709, 2005. in neurotransmission. Chen, A., Zorrilla, E., Smith, S., Rousso, D., Levy, C., Donaldson, C., Roberts, A., These results suggest that the intrinsic circuitry of Lee, K-F., Vale, W. Urocortin 2-deficient mice exhibit gender-specific alterations in circadian hypothalamus-pituitary-adrenal axis and depressive-like behavior. J. Neu- the lateral part of the hypothalamus undergoes a struc- rosci. 26:5500, 2006. tural reorganization during escalation of cocaine use. Ghozland, S., Chu, K., Kieffer, B.L., Roberts, A.J. Lack of stimulant and anxi- This remodeling could contribute to the chronic deficit olytic-like effects of ethanol and accelerated development of ethanol dependence in µ-opioid receptor knockout mice. Neuropharmacology 49:493, 2005. in reward function hypothesized to drive the transition MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 315 to drug addiction. The results also support the value of the paraventricular nucleus of the hypothalamus and using multiple analysis strategies to detect the strongest the lateral part of the hypothalamus that are thought to changes in gene expression and to compensate for the be key in the expression of “emotional memories” by biases that affect each strategy. providing an interface between the central stress and In other studies, we discovered a novel form of neu- autonomic systems. Thus, the impaired plasticity of the ronal plasticity in the juxtacapsular bed nucleus of the juxtacapsular BNST could contribute to the emotional stria terminalis (BNST) characterized by a decrease in dysregulation associated with abstinence in humans the firing threshold and an increase in the temporal with drug dependence. fidelity of firing. The plasticity was impaired during PUBLICATIONS protracted withdrawal from self-administration of vari- Ahmed, S.H., Lutjens, R., van der Stap, L.D., Lekic, D., Romano-Spica, V., ous drugs of abuse, including alcohol, cocaine, and Morales, M., Koob, G.F., Repunte-Canonigo, V., Sanna, P.P. Gene expression evidence for remodeling of lateral hypothalamic circuitry in cocaine addiction. Proc. heroin. This impairment was more pronounced in rats Natl. Acad. Sci. U. S. A. 102:11533, 2005. with a history of drug dependence. The BNST has been Izzo, E., Sanna, P.P., Koob, G.F. Impairment of dopaminergic system function after implicated in stress responses and in the motivational chronic treatment with corticotropin-releasing factor. Pharmacol. Biochem. Behav. 81:701, 2005. dysregulation associated with drug dependence. Interestingly, the long-term potentiation of the Karpova, A., Sanna, P.P., Behnisch, T. Involvement of multiple phosphatidylinositol 3-kinase-dependent pathways in the persistence of late-phase long term potentia- intrinsic excitability in the juxtacapsular BNST that was tion expression. Neuroscience 137:833, 2006. impaired in animals with a history of dependence on Lu, X., Mazarati, A., Sanna, P., Shinmei, S., Bartfai, T. Distribution and differen- alcohol, cocaine, or heroin was a potentiated population tial regulation of galanin receptor subtypes in rat brain: effects of seizure activity. Neuropeptides 39:147, 2005. spike due to enhanced intrinsic excitability and temporal fidelity of firing. Intracellular recordings revealed Sanna, P.P., King, A.R., van der Stap, L.D., Repunte-Canonigo, V. Gene profiling of laser-microdissected brain regions and sub-regions. Brain Res. Brain Res. Pro- that high-frequency stimulation of the stria terminalis toc. 15:66, 2005. induced a long-lasting shift toward hyperpolarization of the threshold for the generation of action potentials in neurons in the juxtacapsular BNST in normal rats. Cellular Physiology of Brain This shift was accompanied by a protracted increase in firing probability that was not observed in rats with Cannabinoids and Peptides histories of dependence on alcohol, cocaine, or heroin, consistent with the impaired long-term potentiation of P. Schweitzer, M. Roberto, G.R. Siggins, B. Lambolez,* D. field potentials in these rats. Piomelli** The activity-dependent decrease in the firing thresh- * Ecole Superiéure de Physique et de Chimie Industrielles, Paris, France old of juxtacapsular BNST neurons was mediated by ** University of California, Irvine, California changes in the D-type potassium current. Using DNA NEUROBIOLOGY OF CANNABINOID SUBSTANCES microarrays and reverse transcriptase–polymerase chain annabinoid substances contained in marijuana reaction, we found that the expression of the Kv1.2 have powerful psychoactive properties and alter channel, the main contributor to the D-type potassium C cognitive processes via activation of cannabi- current, was significantly increased in the juxtacapsular noid-1 (CB1) receptors, but in order to function prop- BNST in rats dependent on alcohol, cocaine, or heroin, erly, the brain produces its own cannabinoid ligands. suggesting that increased density of Kv1.2 subsequent Our objectives are to uncover the cellular mechanisms to its increased gene expression may contribute to the that underlie the central effects of cannabinoid ligands refractoriness to long-term potentiation of population and to determine the role played by endogenously formed spike in animals with histories of drug dependence. cannabinoids. Reduced capacity for plasticity of intrinsic excita- Using a physiologic approach, we are investigating bility and temporal fidelity of firing of juxtacapsular BNST the modulation of synaptic transmission and plasticity. neurons would result in inadequate feedback inhibi- In this approach, we record from neurons in brain tis- tion of the central nucleus of the amygdala. Unrestrained sue from the hippocampus and neocortex, 2 structures activation of the central nucleus of the amygdala is that have high levels of CB1 receptors and are involved expected to result in increased emotional arousal through in learning and memory processes. In collaboration with the various projection areas of the structure, including D. Piomelli, University of California, Irvine, we are using 316 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE various pharmacologic tools to study the routes of degra- Neurophysiology of Alcohol dation of endogenous cannabinoids. In our collaboration with B. Lambolez, Ecole Superiéure de Physique Exposure During Adolescence et de Chimie Industrielles, we are using single-cell reverse transcriptase–polymerase chain reaction after and Adulthood: Role of whole-cell recording to characterize the neuronal pop- Neuropeptides ulations that express transcripts for CB1 receptors. The role played by endogenous cannabinoids in C.J. Slawecki, J. Roth, D. Wills, C.L. Ehlers hippocampal and neocortical excitatory synaptic transmission and plasticity remains controversial. We found CONSEQUENCES OF EXPOSURE TO DRUGS DURING that endogenous cannabinoids acting at CB1 receptors ADOLESCENCE in the hippocampus selectively decrease excitatory trans- eurobiological, genetic, and environmental fac- mission and restrict synaptic plasticity. Consistent with tors influence alcoholism. Exposure to a drug such a role for endogenous cannabinoids, our research N of abuse during adolescence greatly increases in collaboration with Dr. Lambolez indicates that more the likelihood that a person will become dependent on than half of pyramidal neurons express CB1 receptors that drug. We have developed animal models to assess in the neocortex. The expression of the receptors also the environmental and genetic consequences of expo- did not appear to be restricted to large cholecystokinin sure to alcohol and nicotine during adolescence and interneurons as previously reported, because a majority adulthood. In these models, rats are exposed to alco- of neurons containing somatostatin and vasoactive hol or nicotine and then brain function and behavior intestinal peptide also expressed CB receptors. 1 are examined. Overall, these studies have shown the Using a physiologic approach, we confirmed that unique effects of exposure to alcohol or nicotine dur- functional CB receptors modulate neocortical networks. 1 ing adolescence. We also discovered that cyclooxygenase-2 has a pre- We examined the effects of exposure to alcohol dur- dominant role in controlling the tonic level of endoge- ing adolescence during acute withdrawal and protracted nous cannabinoids that modulate synaptic activity and abstinence. We found that when exposure abruptly ended, plasticity. Thus, the endogenous cannabinoid system interacts with the cyclooxygenase-2 pathway, and inhibi- several signs of withdrawal were more pronounced in tors of this enzyme may raise the levels of endogenous adolescents than in adults. Specifically, brain hyper- cannabinoids. arousal and behavioral hypoactivity developed more NEUROPEPTIDES AND ALCOHOL rapidly in adolescents. After more protracted periods of Neuropeptides are found throughout the brain and abstinence from alcohol, function in multiple brain regions strongly influence neuronal activity. Peptides such as known to mediate cognition was altered. Comparable corticotropin-releasing factor (CRF) and nociceptin are levels of exposure to alcohol affected behavioral indices involved in the CNS effects of ethanol. In other studies, of cognitive function more profoundly in adults. we are examining the actions of ethanol in the central Distinct changes in behavior and brain function also amygdala, a brain region prominently involved in alco- occur in adolescents after exposure to nicotine. After hol dependence and reinforcement. Our results indicate nicotine exposure ends, pronounced increases in anxi- that CRF1 receptors mediate the ethanol enhancement ety occur in several animal models. Alterations in selected of inhibitory transmission, providing a cellular mecha- brain peptides could account for the behavioral changes nism for the involvement of CRF in the effects of ethanol in rats exposed to nicotine during adolescence. Brain and supporting a role for the peptide in the motivational peptides, such as corticotropin-releasing factor and neu- effects of ethanol. CRF1 receptors could be an impor- ropeptide Y, are altered in critical brain sites known to tant therapeutic target for the treatment of stress-induced mediate anxiety. alcohol drinking. NEUROBIOLOGICAL MEDIATORS OF ALCOHOL

CONSUMPTION PUBLICATIONS Slanina, K.A., Roberto, M., Schweitzer, P. Endocannabinoids restrict hippocampal We also investigate how environmental and genetic long-term potentiation via CB1. Neuropharmacology 49:660, 2005. factors mediate alcohol drinking in rats. Using newly Slanina, K.A., Schweitzer, P. Inhibition of cyclooxygenase-2 elicits a CB1-mediated developed models, we found that exposure to alcohol decrease of excitatory transmission in rat CA1 hippocampus. Neuropharmacology 49:653, 2005. for 7–8 weeks during adulthood produced a persistent MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 317

20% increase in alcohol drinking. Neurochemical and function. We use extracellular, intracellular, and patch behavioral studies revealed that changes in neuropep- recording of brain neurons in vitro. We administer trans- tide Y could be partially responsible for the neuroad- mitters, peptides, drugs, cytokines, and neurotoxins aptive changes that increase alcohol drinking after by micropipettes and by superfusion, and we activate prolonged alcohol exposure. More recently, we used synaptic transmission via stimulating electrodes. We also this model to assess the sensitivity of adolescent rats use molecular methods to assess drug-induced alter- to this effect of alcohol exposure. We found that 2 weeks ations of transmitter receptors. of alcohol exposure was sufficient to increase ethanol We investigate synaptic mechanisms and drug effects intake in adolescent rats. This same 2-week period of in the hippocampus, nucleus accumbens, and central exposure was not sufficient to affect alcohol drinking amygdala, brain regions involved in memory, learning, in adult rats. These data further indicate that age of stress, and drug abuse. In previous studies, we discov- exposure is a critical factor in alcohol-drinking behav- ered inhibitory roles in the hippocampus for the neuro- ior. This finding is consistent with the increased risk peptides somatostatin, cortistatin, and the opioid-like of alcohol dependence associated with early onset of peptide nociceptin. All 3 peptides often depressed hip- alcohol drinking. pocampal epileptiform events. Patch-clamp studies of PUBLICATIONS neurons in the central amygdala indicated that noci- Barron, S., White, A., Swartzwelder, H.S., Bell, R.L., Ross, Z.A., Slawecki, C.J., Ehlers, C.L., Levin, E.D., Rezvani, A.H., Spear, L.P. Adolescent vulnerabilities to ceptin decreases presynaptic vesicular release of the chronic alcohol or nicotine exposure: findings from rodent models. Alcohol. Clin. inhibitory transmitter γ-aminobutyric acid (GABA) Exp. Res. 29:1720, 2005. and reverses the effect of ethanol in enhancing GABA Slawecki, C.J. Comparison of anxiety-like behavior in adolescent and adult Sprague-Dawley rats. Behav. Neurosci. 119:1477, 2005. release. A δ opioid receptor agonist also appears to reduce transmitter release in neurons in the central Slawecki, C.J., Ehlers, C.L. Enhanced prepulse inhibition following adolescent ethanol exposure in Sprague-Dawley rats. Alcohol. Clin. Exp. Res. 29:1829, 2005. amygdala in rats, with little effect on the properties of postsynaptic membranes. Slawecki, C.J., Roth, J. Assessment of sustained attention in ad libitum fed Wistar rats: effects of MK-801. Physiol. Behav. 85:346, 2005. Our previous findings suggested that glutamatergic

Slawecki, C.J., Roth, J., Gilder, A. Neurobehavioral profiles during the acute phase synapses, particularly receptors for N-methyl-D-aspar- of ethanol withdrawal in adolescent and adult Sprague-Dawley rats. Behav. Brain tate (NMDA), play a role in opiate and ethanol depen- Res. 170:41, 2006. dence. In patch-clamp recording and pharmacologic Slawecki C.J., Thorsell, A., Ehlers, C.L. Antagonism of neuropeptide Y Y1 recep- studies, chronic morphine treatment altered several tors does not inhibit ethanol’s effects on cortical EEG and ERPs in Wistar rats. J. Stud. Alcohol 66:559, 2005. pharmacologic and biophysical properties of NMDA

Thorsell, A., Slawecki, C.J., El Khoury, A., Mathé, A.A., Ehlers, C.L. The effects receptor–mediated excitatory postsynaptic potentials of social isolation on neuropeptide Y levels, exploratory and anxiety-related behav- (EPSPs) in slices and freshly isolated neurons from the iors in rats. Pharmacol. Biochem. Behav. 83:28, 2006. nucleus accumbens, suggesting changes in the function Thorsell, A., Slawecki, C.J., Khoury, A., Mathé, A.A., Ehlers, C.L. Effect of social or composition of the subunits of NMDA receptors. isolation on ethanol consumption and substance P/neurokinin expression in Wistar rats. Alcohol 36:91, 2006. Using quantitative real-time polymerase chain reaction and Western blots of NMDA receptor subunits in tissue from the nucleus accumbens, we found that RNA Neuronal Communication, for the 3 major subunits (NR1, NR2A, and NR2B) did not change in morphine-dependent rats, but the pro- Neuropeptides, and Drugs tein levels of NR1 and NR2B increased significantly, of Abuse suggesting a posttranscriptional effect of chronic morphine treatment. However, in the central amygdala, G.R. Siggins, P. Schweitzer, M. Roberto, T. Bartfai, chronic treatment with morphine significantly increased S. Madamba, Z. Nie, M. Bajo, L. Sharkey, R. Vlkolinsky, RNA levels for the NR1 subunit but had no effect on L.H.Parsons, A.J. Roberts, L. de Lecea,* S. Moore** the protein levels of any of the 3 subunits. These data * Department of Molecular Biology, Scripps Research suggest that morphine dependence causes regional- ** Duke University,Raleigh-Durham, North Carolina and subunit-specific changes in NMDA receptors. e study the effects of neuropeptides and In previous studies, in slices of the central amyg- abused drugs on electrophysiologic and molec- dala from rats never exposed to ethanol and rats chroni- W ular mechanisms of neuronal and synaptic cally treated with ethanol, acute exposure to ethanol 318 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE increased the amplitude of GABAergic inhibitory post- Finally, we are studying the effects of the neuropep- synaptic potentials (IPSPs) and decreased glutamatergic tide galanin on neurons of the dorsal raphe nucleus, EPSPs, indicating a reciprocal alteration of GABAergic known to contain both galanin and its receptors. Our and glutamatergic systems with no tolerance. Quantal findings to date indicate that galanin decreases the synaptic analysis and microdialysis studies indicated size of evoked IPSPs in dorsal raphe, probably via a that the action of ethanol on IPSPs is predominantly presynaptic decrease in GABA release. presynaptic, enhancing vesicular GABA release. PUBLICATIONS Corticotropin-releasing factor (CRF), a neuropeptide Bajo, M., Crawford, E.F., Roberto, M., Madamba, S.G., Siggins, G.R. Chronic most likely involved in stress-induced alcohol drinking, morphine treatment alters expression of N-methyl-D-aspartate receptor subunits in the extended amygdala. J. Neurosci. Res. 83:532, 2006. also presynaptically augmented IPSPs in the central Breese, G.R., Criswell, H.E., Carta, M., Dodson, P.D., Hanchar, H.J., Khisti, R.T., amygdala in both mice and rats. CRF1 receptor antag- Mameli, M., Ming, Z., Morrow, A.L., Olsen, R.W., Otis, T.S., Parsons, L.H., Pen- onists and a mutation that deleted the gene for the land, S.N., Roberto, M., Siggins, G.R., Valenzuela, C.F., Wallner, M. Basis of the GABAmimetic profile of ethanol. Alcohol. Clin. Exp. Res. 30:731, 2006. CRF1 receptor abolished the effects of both CRF and ethanol, whereas CRF antagonists and agonists had no Roberto, M., Bajo, M., Crawford, E., Madamba, S.G., Siggins, G.R. Chronic 2 ethanol exposure and protracted abstinence alter NMDA receptors in central amyg- effect, indicating that the effects of ethanol are mediated dala. Neuropsychopharmacology 31:988, 2006. by activation of endogenous CRF1 receptors. Notably, Roberto, M., Siggins, G.R. Nociceptin/orphanin FQ presynaptically decreases the CRF augmentation of IPSPs increased after chronic GABAergic transmission and blocks the ethanol-induced increase of GABA release in central amygdala. Proc. Natl. Acad. Sci. U. S. A. 103:9715, 2006. ethanol treatment, suggesting that this neuropeptide- Roberto, M., Treistman, S.N., Pietrzykowski, A.Z., Weiner, J., Galindo, R., ethanol interaction represents a novel synaptic neuroad- Mameli, M., Valenzuela, F., Zhu, P.J., Lovinger, D., Zhang, T.A., Hendricson, aptation underlying ethanol dependence. In addition, A.H., Morrisett, R., Siggins, G.R. Actions of acute and chronic ethanol on presynaptic terminals. Alcohol. Clin. Exp. Res. 30:222, 2006. in recent studies with S. Moore, Duke University, we found that ethanol increases vesicular GABA release in Tallent, M.K., Fabre, V., Qiu, C., Calbet, M., Lamp, T., Baratta, M.V., Suzuki, C., Levy, C.L., Siggins, G.R., Henriksen, S.J., Criado, J.R., Roberts, A., de Lecea, L. neurons in the central amygdala in mice with null muta- Cortistatin overexpression in transgenic mice produces deficits in synaptic plasticity tions in either δ or µ opioid receptors significantly more and learning. Mol. Cell. Neurosci. 30:465, 2005. than in neurons from control mice. Furthermore, quantal analysis indicated that ethanol augments presynaptic GABA release more after δ opioid receptors are pharma- Primate Neurobehavioral cologically blocked and that activation of δ opioid receptors diminishes IPSPs, suggesting that endogenous opioid Laboratory peptides, such as enkephalin, act opposite to the actions M.A. Taffe, S.N. Katner, R.D. Crean, S.A. Davis, C.C. Lay, of CRF, presynaptically dampening the effects of ethanol S.N. Von Huben on the GABAergic system. As we reported previously, acute ethanol also e continue to focus on the behavioral and reduced glutamatergic transmission in the central amyg- physiologic effects of exposure to drugs of dala mediated by both non-NMDA and NMDA receptors, W abuse. Currently, we are examining the in part postsynaptically. However, the depressant effect of effects of 3,4-methylenedioxymethamphetamine (MDMA), ethanol on NMDA-EPSPs was enhanced, and glutamate known as “Ecstasy,” and alcohol. Persons who use release was increased, after chronic ethanol treatment MDMA recreationally report cognitive, mood, and sleep and withdrawal, suggesting both presynaptic and post- disturbances even after prolonged abstinence from the synaptic mechanisms of sensitization to ethanol. The drug. Many laboratory studies have indicated that MDMA postsynaptic effect of chronic treatment with ethanol may can produce a selective reduction of serotonergic function involve recomposition of NMDA receptors in the central in the brain in most species, including in nonhuman amygdala to a preponderance of NR2B subunits; this primates. However, whether such changes in the brain possibility is supported by the finding that the sensitivity produce the cognitive or mood disruptions observed in of NMDA receptors to an NR2B-selective antagonist human users is unclear. We are determining how MDMA- increases and that NR2B mRNA and protein levels induced brain changes may impair cognition, mood, cir- increase after chronic exposure to ethanol. This change cadian patterns of temperature and activity, and brain in the NMDA receptor subunit associated with long-term electrophysiologic characteristics. ethanol treatment may indicate another cellular neuroad- This past year, we focused on the impact of the aptation that underlies ethanol dependence. disruption in body temperature produced by MDMA. MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 319 An elevation in temperature is a critical factor in Neurobiology of Addiction MDMA-induced neurotoxic effects in rodents, but the thermoregulatory response differs in larger bodied pri- F. Weiss, R. Ciccocioppo,* M. Heilig,** R. Martin-Fardon, mates. We found that the temperature of rhesus mon- E.P. Zorrilla, C.V. Dayas, H. Aujila, N. Sidhpura, Y. Zhao, keys was consistently elevated by MDMA even under M.A. Baptista, T.M. Kerr, N.D. Stuempfig, J.R. Lewis conditions in which body temperature in rats was * University of Camerino, Camerino, Italy decreased. In monkeys, but not in rodents, MDMA ** National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland produced temperature elevations independently of sig- e study neuroadaptive changes in signaling nificant increases in locomotor activity. mechanisms and neurocircuitries responsible Thus, our data suggest that the thermoregulatory for the development of chronic vulnerability responses to MDMA in primates, including humans, W to relapse, a defining feature of substance dependence may be even more important than would be predicted and a central issue for the successful treatment of drug on the basis of studies in rodents. Our ongoing research addiction. We recently discovered that group II metab- will determine if such sensitivity is also associated with otropic glutamate receptors (mGluRs) and the noci- increased risk for the neurotoxic effects of MDMA. ceptin/orphanin FQ (N/OFQ) opioid peptide system are In other studies, we are addressing medical emer- novel regulatory mechanisms for hyperresponsiveness gencies (and fatalities) in humans who use MDMA. to stress and drug craving, conditions implicated clini- Patients for whom MDMA use is confirmed often have cally as major risk factors for relapse to drug use. malignant hyperthermia and seizures. Using our models, ROLE OF GROUP II mGluRs IN BEHAVIORAL CHANGES we found that seizure can precede and cause hyper- This past year we began investigating the role of thermia after MDMA exposure in monkeys. Further- group II mGluRs in behavioral changes such as increased more, repeated intermittent exposure to MDMA can anxiety and sensitivity to stress that emerge in animals lower the threshold for seizure. These results provide after chronic self-administration of cocaine. In contrast new insight into the etiology of medical emergencies to the steady-state, nonescalating drug self-adminis- observed in recreational users of MDMA. tration that occurs in rats maintained on limited access We are also investigating the cognitive domains that to cocaine, in rats given extended daily access to cocaine, are disrupted by chronic alcohol drinking in periado- cocaine intake escalates. This escalation model provides lescent monkeys. We found that monkeys who drank an opportunity to study neurobiological consequences alcohol 5 days per week were impaired in learning the of cocaine addiction in rats under conditions that mimic spatial location of objects. These effects became more high-dose “bingelike” cocaine abuse in humans. pronounced when the monkeys were required to remem- We found that a history of escalated cocaine intake ber the location over several seconds, indicating a spe- was associated with profound and long-lasting increases cific impairment of spatial short-term working memory. in susceptibility to anxiogenic and stressful stimuli during withdrawal that was still unabated after 3 months of PUBLICATIONS Taffe, M.A., Lay, C.C., Von Huben, S.N., Davis, S.A., Crean, R.D., Katner, S.N. abstinence. The magnitude and persistence of these Hyperthermia induced by 3,4-methylenedioxymethamphetamine in unrestrained pathologic changes most likely contribute to the chronic rhesus monkeys. Drug Alcohol Depend. 82:276, 2006. relapsing associated with cocaine addiction. Von Huben, S.N., Davis, S.A., Lay, C.C., Katner, S.N., Crean, R.R., Taffe, M.A. Increased glutamate neurotransmission plays a major Differential contributions of dopaminergic D1-like and D2-like receptors to cognitive function in rhesus monkeys. Psychopharmacology (Berl.), in press. role in hyperresponsiveness to stress or anxiogenic stimuli. Recent advances in understanding the functional Von Huben, S.N., Lay, C.C., Crean, R.D., Davis, S.A., Katner, S.N., Taffe M.A. Impact of ambient temperature on hyperthermia induced by (±)3,4-methylene- role of mGluRs have implicated group II mGluRs dioxymethamphetamine in rhesus macaques. Neuropsychopharmacology, in press. (mGluR2/3) in modulating responses to anxiety and stress. Group II mGluRs act as autoregulatory receptors and dampen neural excitability by reducing the presynaptic release of glutamate. We therefore hypothesized that activation of group II mGluRs attenuates heightened anxiety-like behavior in rats with cocaine escalation.

As predicted, a selective mGluR2/3 agonist (LY379268) effectively reversed exacerbated anxiety- 320 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE like responses during protracted cocaine withdrawal. 2–24 hours after removal from a chronic ethanol liq- Moreover, this agent had greater anxiolytic potency in uid diet, treatment with N/OFQ significantly lowered 3 rats with cocaine escalation than in rats never exposed of 4 major behavioral indications of ethanol withdrawal. to cocaine, suggesting altered mGluR2/3 function in rats The mechanism of action for this effect is unclear. How- subjected to the cocaine escalation regimen. Thus, acti- ever, on the basis of our earlier findings that N/OFQ vation of group II mGluRs not only attenuates drug acts as a functional antagonist of the stress peptide seeking induced by environmental challenges such as corticotropin-releasing factor, most likely reduction of drug cues or exposure to stress but also ameliorates transmission of this factor is a key element in this abnormal responses to stress and anxiety. Group II pharmacologic action. Overall, these findings implicate mGluRs therefore most likely will be a highly promising N/OFQ and its receptor as an important regulatory pharmacologic treatment target for preventing relapse. system as well as a promising treatment target for N/OFQ AND ITS RECEPTOR AS TARGETS FOR alcohol abuse and addiction. PREVENTING RELAPSE TO ALCOHOL ABUSE Our previous findings implicated the opioid-like pep- PUBLICATIONS Dayas, C.V., Liu, X., Simms, J.A., Weiss, F. Distinct patterns of neural activation tide N/OFQ and its receptor, NOP, as promising targets associated with ethanol seeking: effects of naltrexone. Biol. Psychiatry, in press. for preventing relapse to alcohol abuse. We have Economidou, D., Fedeli, A., Martin-Fardin, F., Weiss, F., Massi, M., Cioccioppo, R. extended our studies to the role of the N/OFQ system in Effect of novel FQ-NOP receptor ligands on ethanol drinking in alcohol-preferring the rewarding and dependence-inducing actions of alco- msP rats. Peptides, in press. hol. In contrast to our finding that central administration Martin-Fardon, R., Lorentz, C.U., Stuempfig, N.D., Weiss, F. Priming with BTCP, a dopamine uptake blocker, reinstates cocaine-seeking and enhances cocaine cue- of N/OFQ suppresses alcohol seeking in animal models induced reinstatement. Pharmacol. Biochem. Behav. 82:46, 2005. of relapse both in genetically heterogenous Wistar rats Zhao, Y., Dayas, C.V., Aujla, H., Baptista, M.A.S., Martin-Fardon, R. Weiss, F. and genetically selected Marchigian sP rats, N/OFQ Activation of group II metabotropic glutamate receptors attenuates both stress and attenuated actual ethanol intake in Marchigian sP rats cue-induced ethanol-seeking and modulates c-fos expression in the hippocampus and amygdala. J. Neurosci. 26:9967, 2006. only. Genetically determined dysregulation of the N/OFQ system in Marchigian sP rats therefore may contribute to both increased spontaneous ethanol intake and sensitivity to modification of ethanol consumption by N/OFQ in Neurobiology of Feeding this line of rats. Consistent with this possibility, we and Stress found that Marchigian sP rats had overexpression of the genes for N/OFQ and NOP within regions of the central E.P. Zorrilla, L. Steardo,* K. Inoue,** A. Tabarin,*** amygdala and bed nucleus of the stria terminalis. K. Rice,**** S. Iwasaki,** A. Chen,***** E. Fekete, Y. Zhao, These abnormalities in N/OFQ and NOP gene expres- V. Sabino, P. Cottone, M. Brennan, M. Mattock, Y. Grant sion in brain regions linked to the regulation of behavioral responses to stress may contribute to increased * University of Palermo, Palermo, Italy spontaneous ethanol intake in animal genetic models ** Osaka City University Medical School, Osaka, Japan of alcoholism. Tests of this hypothesis are in progress. *** Université Victor Ségalen Bordeaux 2, Hôpital du Haut-Lévêque, Pessac, We next examined whether chronic ethanol intoxi- France cation can produce neuroadaptive changes in N/OFQ- **** National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland NOP function in Wistar rats and thereby contribute to ***** Weizmann Institute of Science, Rehovot, Israel the high ethanol intake that occurs in these animals after intoxication and withdrawal. Central administra- e study motivated behavior, with emphasis tion of N/OFQ reduced ethanol self-administration in on brain reward and stress neurocircuits that rats previously dependent on alcohol but not in nonde- W control food intake. Understanding food intake pendent control rats. The differential pharmacologic requires understanding how the brain organizes units effect of N/OFQ on ethanol intake as a function of of ingestive behavior, or the “feeding microstructure.” ethanol history tentatively suggests that ethanol intox- This past year we pioneered a new way to study feed- ication dysregulates the N/OFQ system. Related to this ing microstructure in rats; the method is based on the hypothesis, we identified a role of the N/OFQ system recognition that eating and drinking are behaviorally in ethanol withdrawal in Wistar rats. In rats tested integrated. We developed further tools to study dynamic MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 321 changes in the rate and regularity of eating within a ferred food is not available. Treatment with opioid meal. We generalized the methods so we could study receptor antagonists reduced the finickiness and bin- control of food intake in mutant transgenic mice. Stud- gelike eating of rats given intermittent, limited access ies with these models will help define the biological to highly preferred foods. bases for specific dysfunctional feeding patterns of obesity and eating disorders and target therapies for PUBLICATIONS Chen, A., Zorrilla, E., Smith, S., Rousso, D., Levy, C., Vaughn, J., Donaldson, C., these abnormalities appropriately. Roberts, A., Lee, K.-F., Vale, W. Urocortin 2-deficient mice exhibit gender-specific alterations in circadian hypothalamic-pituitary-adrenal axis and depressive-like For example, we showed that type 2 urocortins pro- behavior. J. Neurosci. 26:5500, 2006. duced a leptinlike facilitation of satiety, acting through Chen, S.A., O’Dell, L.E., Hoefer, M.E., Greenwell, T.N., Zorrilla, E.P., Koob, G.F. hypothalamic receptors for corticotropin-releasing fac- Unlimited access to heroin self-administration: independent motivational markers tor 2. Studies with female mice deficient in urocortin of opiate dependence. Neuropsychopharmacology, in press. 2 further revealed a regulatory role for this urocortin Funk, C.K., Zorrilla, E.P., Lee, M.-J., Rice, K.C., Koob, G.F. CRF1 antagonists in the expression of vasopressin in hypothalamic mag- selectively reduce ethanol self-administration in ethanol-dependent rats. Biol. Psy- chiatry, in press. nocellular neurons associated with a phenotype of altered circadian regulation of stress hormones by the Richardson, H.N., Zorrilla, E.P., Mandyam, C.D., Rivier, C.L. Exposure to repetitive versus varied stress during prenatal development generates two distinct anxio- hypothalamic-pituitary-adrenal axis, homeostasis of genic and neuroendocrine profiles in adulthood. Endocrinology 147:2506, 2006. body fluids, and resistance to depressive-like behavior. Tabarin, A., Chaves, Y.D., Carmona, M.C., Catargi, B., Zorrilla, E.P., Roberts, A.J., We also study ghrelin, a 28-residue stomach hor- Coscina, D.V., Rousset, S., Redonnet, A., Parker, G.C., Inoue, K., Ricquier, D., mone hypothesized to signal “energy insufficiency” to Penicaud, L., Kieffer, B.L., Koob, G.F. Resistance to diet-induced obesity in µ-opioid receptor-deficient mice: evidence for a “thrifty gene.” Diabetes 54:3510, 2005. the brain. Ghrelin may hinder consolidation of weight loss through its anabolic properties. In collaboration Valdez, G.R., Zorrilla, E.P., Koob, G.F. Homeostasis within the corticotropin-releasing factor system via CRF2 receptor activation: a novel approach for the treatment of anxi- with K.D. Janda, Department of Chemistry, we found ety and depression. Drug Dev.Res., in press. that n-octanoylation and the N-terminal third residue Zorrilla, E.P., Koob, G.F. The roles of urocortins 1, 2 and 3 in the brain. In: Hand- in the amino acid sequence of ghrelin are critical for book of Stress and the Brain, Part 1: The Neurobiology of Stress. Steckler, T., the ability of the hormone to induce feeding. Accord- Kalin, N.H., Reul, J.M.H.M. (Eds.). Elsevier Science, New York, 2005, p. 179. Techniques in the Behavioral and Neural Sciences; Vol. 15. ingly, active immunization with haptens that included the N-terminal residues of ghrelin slowed the accrual of body weight and fat in rats in proportion to the acquired capacity of the rats’ plasma to bind ghrelin. Galanin, a Neuropeptide We now are using passive immunization with transfer Involved in Depression of antibodies (whole immunoglobulin G or single-chain variable fragments) to the acylated ghrelin N terminus T. Bartfai, X. Lu, H. Badie-Mahdavi, A. Barr, J. Kinney, (residues 1–10). L. Sharkey,* G.R. Siggins* Finally, we developed models of the hedonic (rather than homeostatic) control of food intake. Rats with * Molecular and Integrative Neurosciences Department, Scripps Research intermittent, limited access to highly preferred foods alanin is a neuropeptide involved in regulation will eat large quantities of these foods when the foods of cognition, mood, seizure, and pain threshold. are available, even when fed to satiation before given G After treating rats with selective serotonin reup- access to the preferred foods. Conversely, with increas- take inhibitors such as fluoxetine, electroconvulsive ing experience with palatable foods, rats will reject less shock, and sleep deprivation, measures used to treat palatable rat chow for 5 days or longer, despite result- depression, we used transcriptional profiling to examine ing weight loss. Thus, because of hedonic factors, rats different areas of the brain. We found that galanin mRNA in this model violate regulators of short-term homeostasis was upregulated in cells of the dorsal raphe nucleus in both positive (“binge”) and negative (“finickiness”) and the locus coeruleus that are the major monoamin- directions. Despite taking in less energy (calories) over- ergic nuclei. We also found that levels of the type 2 all than chow-maintained rats do, the rats in the model galanin receptor were elevated in the dorsal raphe ultimately become heavier and fatter, have elevated nucleus, suggesting that galanin acting at this depolariz- levels of adipokines associated with metabolic compli- ing galanin receptor subtype may promote release of cations of obesity, and appear anxious when their pre- serotonin and contribute to antidepressant actions. 322 MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE

Electrophysiologic studies on serotonergic cells in the tant blisslike state.” We are doing genetic and chemi- dorsal raphe nucleus revealed that galanin has strong cal experiments to examine the mechanisms in which synaptic effects. In rodent models of the efficacy of anti- ligands for the receptor are involved. depressant agents, galnon, a galanin receptor agonist, produced antidepressant-like effects similar to those of the antidepressants fluoxetine and imipramine. Many antide- Mechanisms of Thermoregulation: pressants also promote neurogenesis, and we found that the type 2 galanin receptor mediates effects that promote Thermosensitivity in the Brain neuroprotection or neurogenesis in the hippocampus. On the basis of these data, we have made several I. Tabarean, B. Conti, M. Sánchez-Alavez, C. Davis, H. Korn, important steps toward defining the galanin receptor 2 T. Bartfai as a putative drug target for a new class of antidepres- he thermosensitivity of some peripheral sensory sant drugs. We have also made progress toward a chem- neurons with nerve endings in the skin underlies ical proof of principle of galanin receptor 2 ligands as T the ability to sense cold and heat as painful stim- antidepressant agents. uli. Using cell cultures of primary neurons and slice preparations, we are examining neurons in the anterior part of the hypothalamus that sense temperature and Search for Fast-Acting regulate core body temperature. We showed that individual neurons without the pres- Antidepressants ence of a neuronal network can sense cold and warm temperatures and can change firing rate in response to B. Conti, J.D. Hoyer, G. Bilbe, T. Bartfai these temperature changes. These neurons express recep- urrent antidepressants such as the selective sero- tors for several agents that cause fever, such as prosta-

tonin reuptake inhibitors fluoxetine (Prozac) and glandin E2, IL-1, and calcitonin gene–regulated peptide, C paroxetine (Paxil) must be taken for 14–21 days which are involved in mediating fever in response to before clinically significant improvement occurs. This inflammation and infection and in hot flashes. delay is a large problem, particularly in the treatment We also identified substances that reduce temper- of patients with depression who are at high risk for ature sensitivity, such as adenosine and histamine, and suicide. In collaboration with scientists at Novartis we are defining the receptor subtypes through which Pharma, Basel, Switzerland, we are trying to develop these effects are exerted. A molecular and cellular under- a fast-acting antidepressant. standing of the central temperature set point will be We are using 2 techniques that produce rapid, albeit helpful in the treatment of feeding and sleep disorders short lived, antidepressant effects in rats: sleep depri- because these phenomena are closely coordinated with vation and electroconvulsive therapy. We are comparing and depend mutually on changes in the temperature the transcriptional changes that occur in different brain set point. areas in response to these treatments with the changes PUBLICATIONS produced by 14 days of treatment with fluoxetine.We Conti, B., Davis, C.N., Behrens, M.M., Rebek, J., Bartfai, T. Toll-like receptors as have identified several putative new drug targets, includ- pharmacological targets. In: Toll-Like Receptors in Inflammation. O’Neill, L.A.J., Brint, E. (Eds.) Birkhäuser, Boston, 2005, p. 223. Progress in Inflammation ing sulfotransferase 1, serum- and glucocorticoid-induc- Research. Parnham, M.J. (Series Ed.). ible kinases, and G protein–coupled receptor 88, that Davis, C.N., Mann, E., Behrens, M.M., Gaidarova, S., Rebek, M., Rebek, J., Jr., if validated in behavioral experiments such as learned Bartfai, T. MyD88-dependent and -independent signaling by IL-1 in neurons probed by bifunctional Toll/IL-1 receptor domain/BB-loop mimetics. Proc. Natl. helplessness may form the basis of efforts to develop Acad. Sci. U. S. A. 103:2953, 2006. a fast-acting antidepressant. Davis, C.N., Tabarean, I.V., Gaidarova, S., Behrens, M.M., Bartfai, T. IL-1β Transcription of G protein–coupled receptor 88 is induces a MyD88-dependent and ceramide-mediated activation of Src in anterior affected by lithium, the most common therapy for manic hypothalamic neurons. J. Neurochem. 98:1379, 2006. depression/bipolar disorder, and most surprisingly, the Florén, A., Sollenberg, U., Lundström, L., Zorko, M., Stojan, J., Budihna, M., levels of this receptor are also altered in lactating Wheatley, M., Martin, N.P., Kilk, K., Mazarati, A., Bartfai, T., Lindgren, M., Lan- gel, Ü. Multiple interaction sites of galnon trigger its biological effects. Neuropep- females, who are considered to be in a “stress resis- tides 39:547, 2005. MOLECULAR AND INTEGRATIVE NEUROSCIENCES 2006 THE SCRIPPS RESEARCH INSTITUTE 323

Kinney, J.W., Davis, C.N., Tabarean, I., Conti, B., Bartfai, T., Behrens, M.M. A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons. J. Neurosci. 26:1604, 2006.

Kovacs, Z., Kekesi, K.A., Szilagyi, N., Abraham, I., Szekacs, D., Kiraly, N., Papp, E., Csaszar, I., Szego, E., Barabas, K., Peterfy, H., Erdei, A., Bartfai, T., Juhasz, G. Facilitation of spike-wave discharge activity by lipopolysaccharides in Wistar Albino Glaxo/Rijswijk rats. Neuroscience 140:731, 2006.

Lundström, L., Elmquist, A., Bartfai, T., Langel, Ü. Galanin and its receptors in neurological disorders. Neuromolecular Med. 7:157, 2005.

Ostenson, C.G., Gaisano, H., Sheu, L., Tibell, A., Bartfai, T. Impaired gene and protein expression of exocytotic soluble N-ethylmaleimide attachment protein receptor complex proteins in pancreatic islets of type 2 diabetic patients. Diabetes 55:435, 2006.

Sánchez-Alavez, M., Tabarean, I.V., Behrens, M.M., Bartfai, T. Ceramide mediates the rapid phase of febrile response to IL-1β. Proc. Natl. Acad. Sci. U. S. A. 103:2904, 2006.

Shi, T.J., Hua, X.Y., Lu, X., Malkmus, S., Kinney, J., Holmberg, K., Wirz, S., Cec- catelli, S., Yaksh, T., Bartfai, T., Hokfelt, T. Sensory neuronal phenotype in galanin receptor 2 knockout mice: focus on dorsal root ganglion neurone development and pain behaviour. Eur. J. Neurosci. 23:627, 2006.

Tabarean, I.V., Conti, B., Behrens, M., Korn, H., Bartfai, T. Electrophysiological properties and thermosensitivity of mouse preoptic and anterior hypothalamic neurons in culture. Neuroscience 135:433, 2005.

Wirz, S.A., Davis, C.N., Lu, X., Zal, T., Bartfai, T. Homodimerization and internalization of galanin type 1 receptor in living CHO cells. Neuropeptides 39:535, 2005.

Wirz, S.A., Tobias, P.S., Ulevitch, R.J., Aribibe, L., Bartfai, T. TLR2 is required for the altered transcription of p75NGF receptors in gram positive infection. Neu- rochem. Res. 31:297, 2006.

Neurobiology

Rbm3 is highly expressed in the cerebellum. Immunostaining

(top left panel, in green) and in situ hybridization (top right panel, in red) for Rbm3 were performed on a sagittal section of 12-day-old rat brain. The bottom left panel (in cyan) shows nuclei stained with 4',6-diamidino-2-phenylindole; the bottom right panel is an overlay of the three panels.

Note the high expression of both Rbm3 protein and mRNA in purkinje cell bodies and dendrites. Image provided by

Julie Pilotte, Ph.D., and Peter W. Vanderklish, Ph.D. Robyn Meech, Ph.D.

Assistant Professor and

Olivier Harismendy, Ph.D.

Senior Research Associate

Department of Neurobiology NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 327

DEPARTMENT OF NEUROBIOLOGY

STAFF STAFF SCIENTIST VISITING * Joint appointment in The Skaggs INVESTIGATORS Institute for Chemical Biology Gerald M. Edelman, M.D., Wei Zhou, Ph.D. ** Appointment completed; new Ph.D.* Sigeng Chen, Ph.D. Professor and Chairman Neurosciences Institute location shown SENIOR RESEARCH San Diego, California Kathryn L. Crossin, Ph.D. ASSOCIATES Associate Professor David Edelman, Ph.D. Annette R. Atkins, Ph.D. Neurosciences Institute Bruce A. Cunningham, Ph.D. San Diego, California Professor Stephen A. Chappell, Ph.D. Helen Makarenkova, Ph.D. Ralph Greenspan, Ph.D. Neurosciences Institute San Diego, California Adjunct Professor RESEARCH ASSOCIATES

Geoffrey Owens Vincent P. Mauro, Ph.D. S. Armaz Aschrafi, Ph.D.** Neurosciences Institute Associate Professor Massey University San Diego, California Auckland, New Zealand Robyn Meech, Ph.D. Assistant Professor John Dresios, Ph.D.

Peter W. Vanderklish, Ph.D. Katie N. Gonzalez, Ph.D. Assistant Professor Olivier Harismendy, Ph.D.

Dora Chin Yen Koh, Ph.D.

Panagiotis Panopoulos, Ph.D.

Julie Pilotte, Ph.D.

Marina Tsatmali, Ph.D.** Department of Immunology, Scripps Research 328 NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

produced from neural progenitors, which in turn will enhance their potential use in stem cell therapies. Consolidation of the mechanisms that underlie learning and memory involves an elaborate set of molecular events that alters the shape and function of synapses. Some of these changes require protein synthesis, and recent work has revealed a new set of events that link the activity of the synapse to changes in synaptic strength. It appears that granules containing mRNAs and parts of the translation machinery can be transported to the vicinity of dendritic spines. Synaptic activity can trigger local translation by the elements in the granules, providing specific synaptic proteins at sites of activity. Bruce Cunningham and Peter Vanderklish, along with their colleagues, have extended their studies of the proteins involved in the organization of granules and their transport into dendrites. To these efforts they have added high-throughput proteomics studies in collaboration with Lujian Liao and John Yates. One project focuses on Dr. Gerald M. Edelman, M.D., Ph.D. Vanderklish’s analysis of the changes in synaptic components in response to brain-derived neurotrophic factor, which influences a variety of processes in neural devel- Chairman’s Report opment and synaptic plasticity. In another study, these investigators are examining the proteins that interact vast amount of new information about cellular and with the RNA-binding protein RBM3, which is involved neurobiological events has become available as a in regulating mRNA transport at multiple levels. In earlier result of new methodologies, particularly in molecu- A studies, they found that RBM3 is present in a subset of lar biology. Integrating these new data into our under- dendritic granules and showed that its overexpression standing of central problems of biology continues to be a can enhance protein synthesis as much as 3-fold. major challenge. In the past year, members of the Depart- Dr. Vanderklish has also been studying the biochemi- ment of Neurobiology have made extensive progress in cal and synaptic events altered in the fragile X mental their studies of the fundamental processes of develop- retardation syndrome. This year he was honored by the ment, particularly those involved in development of the Fragile X Research Foundation for his analysis of the nervous system and those that regulate synaptic plasticity. molecular and cellular changes in the mouse model for Much can be gained by understanding these mech- this syndrome. The results of these studies promise to pro- anisms, particularly those that generate synaptic change to regulate higher-order processes such as learning and vide new assays for testing drugs to treat the condition. memory. The vertebrate nervous system derives from Although much is to be learned about translation of multipotent stem or progenitor cells in the neural tube, mRNA at the synapse, there are still considerable gaps which divide and differentiate into mature neurons and in our knowledge of the fundamental events in mRNA glial cells. Kathryn Crossin and her colleagues have pro- translation itself. Translation in eukaryotes is initiated vided convincing evidence that reactive oxygen species, via 2 mechanisms, cap dependent and internal ribosome molecules usually associated with cell death, provide entry site (IRES) dependent, which differ in how ribo- positive signals to neural stem cells. While not altering somes are recruited to the mRNA. In the first mecha- the proportion of neurons and glia that arise from the nism, ribosomes are recruited at the cap structure, a stem cells, these highly reactive molecules regulate the modified nucleotide found at the 5′ ends of mRNAs. In proportion of different neuronal subtypes in development. the second mechanism, which has been the focus of many The results of these studies may provide new mecha- of our studies, ribosomes are recruited by sequence ele- nisms for regulating the types of neurons that can be ments (IRESs) contained within the mRNA. Differential NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 329 use of these 2 mechanisms appears to be important in processes such as synaptic plasticity in the brain. Vince Mauro and his colleagues have made major inroads into understanding the events that regulate translation initiation. They have shown convincingly that base pairing between messenger RNA and ribosomal RNA is a critical step in selective initiation of translation of many mRNAs. Moreover, they have found that similar processes are involved in the ability of the translation machinery to “shunt” past structural obstacles, such as hairpin structures in mRNAs, and past upstream start codons (AUGs) that are present in many mRNAs. As these studies gain in breadth, they are beginning to challenge established concepts and are likely to modify current textbook views on the factors that regulate translation initiation. Transcription of selected genes has long been noted as a key regulator of development. Robyn Meech and her colleagues have developed a method for exhaustive identification of the targets of transcription factors in the genome. Initial experiments are being tested on the transcription factor REST, which plays critical roles in regulating neural-specific genes. Homeobox genes are particularly important because they control cascades of genes that regulate entire developmental patterns. Dr. Meech and her colleagues are defining the ability of the homeobox protein Barx2 to regulate development in a variety of tissues. Their recent work shows that Barx2 plays multiple roles in muscle development and repair. It also has an intricate pattern of coregulation with the estrogen receptor, a finding that may lead to new insights into the factors that cause breast cancer. All of these studies open exciting new areas. More- over, they are gradually merging into what promises to be a series of synergistic interactions among our investigators that will allow them to attack these and other fundamental problems in new ways. 330 NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

INVESTIGATORS’REPORTS Type I neurons were large pyramidal-like neurons that fired no action potentials or only a single action potential and expressed low levels of the calcium-bind- Cell-Surface and Metabolic ing protein calretinin in their processes. These cells accounted for about 70% of the neurons. Type II neurons Influences on Differentiation were smaller bipolar-like neurons with rounded cell bodies that fired repeated action potentials and contained of Neural Stem Cells high levels of calretinin in their nuclei. These neurons accounted for about 30% of the neurons in the cultures. K.L. Crossin, M. Tsatmali, G.C. Owens, D.B. Edelman, Decreasing the levels of ROS with cell-permeant S. Chen, G.M. Edelman antioxidants strongly influenced the numbers of the 2 he ability to control the differentiation of neural neuronal cell types, although the total numbers of neu- stem cells into neurons is critical for therapeutic rons remained the same. Under these conditions, the T use of the stem cells in neurodegenerative disease type II neurons accounted for 80% of the neurons and and neural trauma. During earlier studies on the influ- type I neurons for 20%. ence of signaling by cell adhesion molecules on neural Changes in ROS levels therefore modulate several stem/progenitor cells, we found another molecular mech- aspects of neuronal differentiation and could be used anism that appears to be important for the differentia- in development to bias a population of cells toward a tion and maturation of neural progenitors into newborn particular phenotype. The results of these and ongoing neurons. This mechanism involves the production of studies should provide a broad molecular and cellular reactive oxygen species (ROS) by mitochondria and the foundation that may aid in the design of strategies for influence of ROS in controlling neuronal development. the expansion and manipulation of progenitors for use Recently, we showed that newborn neurons produce in clinical applications. higher levels of ROS than do the progenitor cells from which the neurons are derived. ROS have been associated with cellular stress and cell death by apoptosis. Structure and Function of However, evidence is accumulating for a role for ROS signaling in normal developmental processes in several RNA-Binding Proteins diverse systems. We previously hypothesized that ROS B.A. Cunningham, P.W. Vanderklish, A. Atkins, J. Pilotte, might promote neuronal differentiation or maturation. G.M. Edelman We have shown that cells with high levels of ROS occur in embryonic and early postnatal stages of brain or some time, we focused on the structure and development in rats. The cells have normal physiologic function of proteins in the nervous system, includ- properties, indicating that they are healthy neurons. The F ing cell-surface glycoproteins that mediate cell-cell pattern of expression of ROS is consistent with that of interactions and proteins involved in synaptic plasticity. young migratory neurons in the developing cortex. By Recently, we turned our attention to intracellular proteins postnatal day 20, few cells with high levels of ROS that mediate protein-protein and protein–nucleic acid remain except in neurogenic regions such as the olfac- interactions. We have been characterizing RNA-binding tory bulb and the dentate gyrus of the hippocampus. motif protein 3 (RBM3), which is induced by mild cold Thus, it appears that high levels of ROS are a property shock and other forms of cellular stress. The protein is of newborn neurons. part of a small family of proteins characterized by having We explored this possibility further with clonal cul- a single RNA-binding domain and an arginine/glycine- tures of cells from the brains of embryonic rats. Cells rich domain that is thought to mediate protein-protein with low levels of ROS were isolated by using fluores- interactions. Our structural studies indicate that RBM3 cence-activated cell sorting. At embryonic day 15, such is expressed in multiple forms. At least one of the forms cells from the cortex were multipotent progenitors that arises by alternative splicing; the others most likely are differentiated into neurons, astrocytes, and oligodendro- generated by posttranslational modifications. cytes when growth factors were removed from the cul- In earlier studies, we found that RBM3 is present ture. Two types of neurons were produced. in granules that are transported from the cell body to NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 331 dendrites and that include mRNAs, a variety of proteins, nucleotides long and revealed that many were comple- ribosomes, and other parts of the translation machin- mentary to segments of the 18S rRNA, the RNA com- ery. The granules are translationally silent, but their ponent of the small (40S) ribosomal subunit. contents can be used for translation at the dendrites These findings suggested that some mRNA elements after synaptic stimulation. In the past year, in collabo- recruit 40S ribosomal subunits directly by base pairing ration with V. Mauro, Department of Neurobiology, we to the subunits. Although mRNA-rRNA base pairing is found that overexpression of RBM3 can enhance transla- used by prokaryotic mRNAs to initiate translation, no tion as much as 3-fold. The protein was associated with direct evidence exists for this mechanism in eukaryotes. the large ribosomal subunit, but the amounts associated Our most recent analysis of a short mRNA element with the ribosome were too small to account for the from the 5′ leader of the mouse Gtx homeodomain influence of RBM3 on translation. A more likely possi- mRNA now provides this evidence. bility is that overexpression of the protein can signifi- To show base pairing between the Gtx element and cantly decrease the size of a fraction that contains 18S rRNA, we used yeast, taking advantage of the fact micro-RNAs, which influence translation. that yeast 18S rRNA, which differs from mouse 18S To help define the range of functions for RBM3, we rRNA by approximately 20%, lacks a complementary examined its expression throughout the brain during match to the Gtx element. When the Gtx element was development. We found that the levels of expression tested in yeast, in the 5′ leader of a reporter mRNA, were highest in early development and decreased with it did not enhance translation. However, the element age except in areas of active cell proliferation. The high- did enhance translation in yeast when mouse rRNA est levels were in the cerebellum both in embryos and in sequences containing the complementary match were adults. These results and earlier studies indicated that introduced into yeast ribosomal subunits or when the in addition to being expressed in the cytoplasm, RBM3 yeast 18S rRNA was mutated to introduce a comple- is strongly expressed in the nucleus, but the levels of mentary match to the Gtx element. nuclear vs cytoplasmic concentration vary with cell type We confirmed mRNA-rRNA base pairing by show- and the stages of development. Results were similar in ing that the activity of the Gtx element was disrupted studies in cell lines; different cell lines expressed differ- by mutations of the 18S rRNA that disrupted comple- ent ratios of nuclear to cytoplasmic levels of RBM3. mentarity. That activity was restored when the Gtx ele- RBM3 appears to be present in discrete regions of ment was mutated to restore complementarity to the the nucleus, but its role in the nucleus is at this stage 18S rRNA. unclear. Currently, we are assessing the role of the vari- In other studies, we showed that the base-pairing ous forms of this unusual protein in RNA processing, interaction between the Gtx element and 18S rRNA also transport, and translation. facilitates ribosomal shunting, enabling ribosomal subunits to bypass obstacles in the 5′ leader of an mRNA, including an upstream initiation codon that resides in Translational Regulation of a favorable context and a stable RNA hairpin structure. A low level of shunting occurred when multiple Gtx Gene Expression elements were present upstream of the obstacles, but shunting was highly efficient when a single Gtx element V.P. Mauro, S.A. Chappell, W. Zhou, J. Dresios, D.C.Y. Koh, was also present downstream of the obstacle. Control P.Panopoulos, G.M. Edelman experiments indicated that the high level of activity e focus on understanding the mechanisms observed when the Gtx elements flanked the obstacle that underlie the initiation of translation. In could not be accounted for by the activity of the single W eukaryotic cells, the first step in translation downstream element alone. Studies in yeast revealed is recruiting the translation machinery to the mRNA. that the ability of the ribosomal subunits to shunt This recruitment can be mediated by the cap structure, required base pairing to 18S rRNA. On the basis of a modified nucleotide found at the 5′ ends of mRNAs, these studies, we propose that clustering and/or teth- or by specific sequences within some mRNAs that act ering of the translation machinery in the vicinity of the as internal ribosome entry sites. Our earlier studies indi- mRNA increases the likelihood that ribosomes will inter- cated that such recruitment sites could be less than 10 act with other accessible recruitment sites in the mRNA, 332 NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE enhancing the likelihood of direct recognition of the sis, we are examining muscle repair in mice that lack initiation codon via the initiator methionine-tRNA. the gene for Barx2. If the notion of clustering and/or tethering is correct, Interestingly, although Barx2 initially promotes the initiation of translation should be less efficient as the differentiation of myoblasts into myofibers, its continued distance between ribosomal recruitment sites and the expression in myofibers appears to be incompatible with initiation codon increases. A second prediction is that the differentiated state. Thus, ectopic expression of translation should begin efficiently regardless of whether Barx2 in myofibers in culture induces dedifferentiation ribosomal subunits are recruited from sites located of the fibers. Dedifferentiation is an important pathway upstream or downstream of the initiation codon. Using for muscle repair in amphibians. In these animals, mus- model mRNAs with either the 5′ cap structure or mul- cle damage induces myofibers to dedifferentiate into tiple Gtx elements as ribosomal recruitment sites, we myoblasts that proliferate and then redifferentiate into recently tested both of these predictions. The results new myofibers. However, the importance of this pro- confirmed both predictions. These studies with model cess in mammals remains unclear. We are investigat- mRNAs indicate that initiation of translation can occur ing whether Barx2-induced dedifferentiation is involved efficiently through nonlinear mechanisms, and we sug- in the repair of muscle in mice. gest that similar mechanisms underlie movement of To better understand how transcription factors such ribosome subunits during the initiation of translation as Barx2 control cellular behaviors, we have been devel- of natural mRNAs. oping a method to comprehensively identify transcription factor binding sites by using chromatin immunoprecipitation. After completion of proof-of-principle studies, Transcriptional Control of we will use this method to examine the targets that are bound and regulated by Barx2 at different stages of Vertebrate Development muscle development and during repair. Overall, these studies may provide new avenues R. Meech, O. Harismendy, K.N. Gonzalez, G.M. Edelman for treatment of muscle degenerative disease and mus- e focus on the basic mechanisms of tran- cle injury. scriptional control in mammalian develop- W ment. One factor of particular interest is the homeodomain transcription factor Barx2, which was Interrelationships Between mRNA discovered in this department. Barx2 is expressed in embryonic cartilage, muscle, and branching tissues such Translation and Synaptic Structure as mammary, prostate, and lacrimal glands. Recently, we showed important roles for Barx2 in the develop- in Consolidation of Plasticity and ment of several of these tissues. In muscle cells, Barx2 interacts with the serum Fragile X Syndrome response factor and the myogenic regulatory factor MyoD P.W. Vanderklish, J. Pilotte, G.M. Edelman to regulate muscle-specific gene expression. Early in the development of muscle, Barx2 increases cell adhesion he strength and reliability of synaptic communica- and upregulates expression of smooth muscle actin. This tion between neurons (synaptic efficacy) are not change leads to more rapid fusion of the myoblasts into T fixed properties. Rather, the ability of synapses multinucleated myofibers. After birth, Barx2 is down- to undergo long-term changes in efficacy in response regulated in myofibers but persists in a quiescent stem to particular patterns of synaptic activity (synaptic cell–like population called satellite cells. Satellite cells plasticity) is an essential property of neural circuits are responsible for the repair of muscles in adults. After involved in learning, memory, and other higher order injury, these cells activate, proliferate, and then differ- brain functions. The goal of our research is to define entiate to produce new myofibers. the mechanisms by which changes in efficacy are con- Our working hypothesis is that Barx2 is involved in solidated and how these mechanisms are altered in muscle repair by promoting the differentiation of myo- fragile X syndrome (FXS), the most common inherited fibers derived from satellite cells. To test this hypothe- form of mental retardation. NEUROBIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 333

Three basic observations guide our hypotheses. ticity, these changes are of interest because they are First, translation of dendritically localized mRNAs is important in cortical development and function. We required to stabilize changes in efficacy in at least 3 discovered that potentiation induced by spike timing– forms of synaptic plasticity: long-term potentiation, dependent plasticity is absent in the cortex of mice long-term depression (LTD), and synaptic enhancement lacking Fmr1. Depression elicited by spike timing was induced by brain-derived neurotrophic factor. Second, normal, as was a form of mGluR-dependent plasticity changes in efficacy can outlast the half-lives of new of intrinsic neuronal properties. However, neither of proteins synthesized during induction of the changes. these forms of plasticity required translation, and the Third, each form of plasticity can be associated with results are thus consistent with the mGluR theory. unique changes in the morphology of dendritic spines. To identify protein changes that produce synaptic We propose that local translation plays a role in abnormalities in FXS, in collaboration with L. Liao and transforming synaptic shape and that molecular deter- J. Yates, Department of Cell Biology, we are examining minants of synaptic shape in turn regulate the synthesis the “synaptic proteome” of mice lacking Fmr1. This and placement of new proteins that determine synaptic analysis has revealed a large set of proteins that are efficacy. Such regulatory interrelationships are predicted increased or decreased relative to those in wild-type to be unique for each form of plasticity, and defects in mice. Many of the proteins may represent novel mech- specific aspects of these consolidation mechanisms are anisms whereby lack of FMRP alters synapses. responsible for synaptic abnormalities in FXS. We are also conducting proteomic analyses of syn- Previously, we found that stimulation of metabo- apses to determine if translation is regulated differently tropic glutamate receptors (mGluRs), which induces a in distinct forms of plasticity. We observed that brain- translation-dependent form of LTD, led to a translation- derived neurotrophic factor upregulates protein synthe- dependent elongation of dendritic spines. The new sis broadly, and we are comparing these effects with spines resembled the abnormally long and thin spines those stimulated by receptors that induce long-term that occur in FXS and suggested that this hallmark potentiation and LTD. In addition, we are comparing abnormality of FXS might be the result of an altered the activities of FMRP and a protein that we recently LTD consolidation process. showed enhances protein synthesis, RNA-binding motif FXS is caused by the silencing of a single gene, protein 3. In collaborative studies with B. Cunningham, Fmr1, which encodes a protein (FMRP) that can act Department of Neurobiology, we are studying how the as a suppressor of translation in dendrites. In wild-type protein affects translation and how it is regulated and animals, stimulation of mGluRs leads to synthesis of expressed in brain. FMRP, presumably to limit translation by negative feed- PUBLICATIONS back. In mice lacking Fmr1, mGluR-induced LTD is Chappell, S.A., Dresios, J., Edelman, G.M., Mauro, V.P. Ribosomal shunting medi- enhanced. Because longer, thinner spines have fewer ated by a translational enhancer element that base pairs to 18S rRNA. Proc. Natl. Acad. Sci. U. S. A. 103:9488, 2006. glutamate receptors, our data suggest that mGluR- induced translation leads to changes in the shape of Desai, N., Casmiro, T., Gruber, S.M., Vanderklish, P.W. Altered developmental plasticity in neocortex of FMR1 knockout mice. J. Neurophysiol., in press. spines that express LTD and that this process is exaggerated in FXS. Dresios, J., Chappell, S.A., Zhou, W., Mauro, V.P. An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes. Nat. Struct. Mol. Biol. 13:30, 2006. The preceding observations have led to the proposal that exaggerated mGluR-induced translation leads Greenspan, R.J. No critter left behind: an invertebrate renaissance. Curr. Biol. 15:R671, 2005. to changes in synaptic plasticity and shape that are Meech, R., Edelman, D.B., Jones, F.S., Makarenkova, H.P. The homeobox tran- the proximal causes of the signs and symptoms of FXS. scription factor Barx2 regulates chondrogenesis during limb development. Develop- This theory, known as the “mGluR theory,” has found ment 132:2135, 2005. broad support, including our observation that trans- Stevens, T.A., Meech, R. BARX2 and estrogen receptor-α (ESR1) coordinately reg- lational activation of mRNA granules by mGluRs is ulate the production of alternatively spliced ESR1 isoforms and control breast cancer cell growth and invasion. Oncogene, in press. enhanced in mice lacking Fmr1. Our current research on FXS includes studies of Vanderklish, P.W., Edelman, G.M. Differential translation and fragile X syndrome. Genes Brain Behav. 4:360, 2005. forms of synaptic plasticity that are elicited according to the relative timing of presynaptic and postsynaptic action potentials. Termed spike timing–dependent plas-

Biochemistry

High-throughput cell-based screening in the laboratory of M. D. Conkright, Ph.D., is used to uncover novel components of the cyclic AMP signaling pathway. Over 15,000 proteins were assayed for the ability to activate cAMP- responsive genes. Three out of 25 putative activators of the pathway are shown in green along with positive and negative controls in the bottom right 4 X 4 quadrant. Michael D. Conkright, Ph.D.

Assistant Professor

Department of Biochemistry BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 339

DEPARTMENT OF STAFF SCIENTISTS C-Y Liu, Ph.D.*** BIOCHEMISTRY Michael J. Chalmers, Ph.D. Julien Mamet, Ph.D.***

Takato Imaizumi, Ph.D.*** Brooke H. Miller, Ph.D.

STAFF Ling Li, Ph.D. William Mwangi, Ph.D.

Yi An Lu, Ph.D. Steve A. Kay* Hiroshi Nakase, Ph.D. Professor and Chairman Qitao Yu, Ph.D. Michael H. Ober, Ph.D.

Alessandra Cervino, Ph.D.** Jiu Xiang Zhao, Ph.D. Alessia Para, Ph.D.*** Assistant Professor Quansheng Zhou, Ph.D. Jeffery Pitman, Ph.D.*** Nicholas Gekakis, Ph.D. *** Assistant Professor Swati Prasad, Ph.D.

RESEARCH ASSOCIATES Jose Pruneda-Paz*** Patrick R. Griffin, Ph.D.** Professor Troy D. Ryba, Ph.D. Hedieh Badie, Ph.D.*** Tomothy J. Jegla, Ph.D. *** Achim M. Sorg, Ph.D. Antoine Baudry, Ph.D.*** Assistant Professor Sahba Tabrizifard, Ph.D. Mario Bengston, Ph.D.*** Claudio A.P. Joazeiro, Mariola Tortosa, Ph.D. Ph.D.*** Ghislain Breton, Ph.D.*** Assistant Professor Hein Tran, Ph.D.*** Darren Bykowski, Ph.D. Paul J. Kenny, Ph.D. Axel Ulbrich, Ph.D.*** Assistant Professor Ning Chen, Ph.D.*** Oliver Umland, Ph.D. Jessie Chu, Ph.D.*** Malcolm A. Leissring, Ph.D. Matthew Wheeler, Ph.D.*** Assistant Professor Sonead Clancy, Ph.D.*** Jason, Willkes, Ph.D.*** Phillip LoGrasso, Ph.D.** Kristin Clarke, Ph.D. Associate Professor Amie L. Williams, Ph.D. Etzer Darout, Ph.D. Howard T. Petrie, Ph.D. Eric Zhang, Ph.D.*** Amy C. DeBaillie, Ph.D. Professor Joshua R. Dunetz, Ph.D. Mathew T. Pletcher, Ph.D.** Assistant Professor Eva Farré, Ph.D.*** VISITING INVESTIGATOR

Kerim Gattas-Asfura, Ph.D. William R.Roush, Ph.D.**** David Welsh, M.D., Ph.D.*** Assistant Professor Professor Elizabeth Hamilton, Ph.D.*** Associate Dean,Kellogg Department of Psychiatry School of Science and Masaki Handa, Ph.D. University of California, Technology, Florida San Diego Samuel Hazen, Ph.D.*** Thomas Schultz, Ph.D.*** Anne Helfer, Ph.D.*** Assistant Professor of * Joint appointments in the Biochemistry Department of Cell Biology and Tamara Hopkins, Ph.D. the Institute for Childhood and Neglected Diseases James Tam, Ph.D. Rebecca J. Kocerha, Ph.D. ** Joint appointment in the Professor Translational Research Institute Warren Lewis, Ph.D.*** *** California campus Claes Wahlestedt, M.D., Fangzheng Li, Ph.D. **** Joint appointments in the Ph.D.** Translational Research Institute Professor Wei Li, Ph.D.*** and the Department of Chemistry 340 BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

INVESTIGATORS’REPORTS

Genetics and Genomics of Circadian Clocks

S.A. Kay, A. Baudry, G. Breton, N. Chen, J. Chu, A. deSchöpke, E. Farré, E. Hamilton, S.P. Hazen, A. Helfer, T. Imaizumi, B. Jenkins, W. Lewis, C.Y. Liu, A. Para, J. Pruneda, A.L. Quiroz, T.F. Schultz, H.G. Tran,* D.K. Welsh,** E. Zhang * Genomics Institute of the Novartis Research Foundation, La Jolla, California ** University of California, San Diego, California

vast array of cellular processes fluctuate with a 24-hour periodicity, and an endogenous circadian A clock is responsible for generating these biological rhythms. Circadian rhythms are found in all kingdoms of life and control diverse events ranging from sleep-wake cycles in mammals to the overall rate of photosynthesis

Steve A. Kay, Ph.D. in plants. Many pathologic changes in humans, such as sleep disorders, most likely are due to a defect in circadian rhythms, so understanding how the circadian clock Chairman’s Overview operates within the cell will have significance for human health. To study how circadian clocks are built inside of he Department of Biochemistry at Scripps Research cells, we use molecular, genetic, and genomic approaches was recently created to recruit faculty members in 2 model systems: mouse and Arabidopsis. T to both the California and Florida campuses. The In mammals, the circadian clock plays an integral overall theme of the department centers on the need role in timing many physiologic rhythms, such as blood to understand physiologic processes from the molecular pressure, body temperature, and liver metabolism, in level to the whole organism. Our faculty members are anticipation of dusk and dawn. The master circadian generally multidisciplinary biologists who wield cutting- clock resides within a region of the brain that receives edge tools of structural biology, protein dynamics, biolog- light information from the eyes. However, this brain region ical chemistry, genetics and genomics, pathway analysis, can still keep time even in the absence of light, as occurs and computational approaches to understand how organ- in some who are visually blind. Mutations in the genes isms maintain homeostasis and respond to stress. that encode components of the circadian clock are mani- We have broad interests and seek to answer contem- fested as abnormal activity rhythms in rodents and as porary questions in neurobiology, metabolic control, sleeping disorders in humans, although which photore- immunology, and cancer biology. By taking integrative ceptors set the clock is unclear. Thus, although marked approaches to substantial problems in modern biology, advances have been made in understanding how the mam- we will affect the understanding of a wide variety of dis- malian clock itself runs, little is known about the how eases such as diabetes, cancer, and CNS disorders. light transduces synchronizing signals to the clock. To address this major question, we are using genetic and genomic approaches to identify new gene functions in circadian biology. We are producing a number of mouse strains with mutations in known and potential photoreceptors and are testing the mice for defects in circadian rhythm. Thus far, we have determined that one photoreceptor, melanopsin, is an important contributor in maintaining synchrony between the clock and environmental BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 341 light conditions. With the completed sequencing of the long days and short days to identify other components human and mouse genomes, we now know the sequences involved in perception of day length. of more than 30,000 genes that can be investigated By combining molecular, genetic, and genomic for potential roles in circadian function. We developed approaches, we are beginning to define a number of large-scale, in vitro, cell-based assays that can be used molecular links between the circadian clock and rhyth- to rapidly determine if genes control clock activity. Com- mic regulation of behavior and development. Analysis bining this approach with genetic analysis will enable of circadian rhythms in multiple organisms provides a us to further dissect the connection between environmen- unique opportunity to define molecular controls for the tal stimuli, in the form of light, and the behavioral and behavior of whole organisms. These results will provide physiologic events regulated by the circadian clock. targets for clinical and agricultural applications to improve In recent years, researchers have found that intrin- the quality of life. sic circadian clocks exist in various peripheral tissues PUBLICATIONS and cell types, directly controlling local physiology and Farré, E.M., Harmer, S.L., Harmon, F.G., Yanovsky, M.J., Kay, S.A. Overlapping behavior. We are studying the circadian oscillators in and distinct roles of PPR7 and PPR9 in the Arabidopsis circadian clock. Curr. Biol. 15:47, 2005. the liver and in the vasculature. As the first step, we are investigating the heterogeneity and distinct functions of Harmer, S.L., Kay, S.A. Positive and negative factors confer phase-specific circadian regulation of transcription in Arabidopsis. Plant Cell 17:1926, 2005. the central and peripheral oscillators. In particular, we are examining the distinct roles of the retinoid-related Hazen, S.P., Borevitz, J.O., Harmon, F.G., Pruneda-Paz, J.L., Schultz, T.F., Yanovsky, M.J., Liljegren, S.J., Ecker, J.R., Kay, S.A. Rapid array mapping of circadian clock orphan nuclear receptors in the clock mechanism. These and developmental mutations in Arabidopsis. Plant Physiol. 138:990, 2005. orphan nuclear receptors were recently identified in Hazen, S.P., Schultz, T.F., Pruneda-Paz, J.L., Borevitz, J.O., Ecker, J.R., Kay, S.A. our functional genomics studies. LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Second, we are asking how environmental cues, Proc. Natl. Acad. Sci. U. S. A. 102:10387, 2005. mainly light-dark cycles and feeding time, entrain or Imaizumi, T., Schultz, T.F., Harmon, F.G., Ho, L.A., Kay, S.A. FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science synchronize peripheral oscillators. Peripheral oscillators 309:293, 2005. most likely are synchronized by hormonal outputs of Welsh, D.K., Imaizumi, T., Kay, S.A. Real-time reporting of circadian-regulated the suprachiasmatic nucleus or by physiologic inputs such gene expression by luciferase imaging in plants and mammalian cells. Methods as feeding-mediated metabolic changes. We are using Enzymol. 393:269, 2005. transgenic mice, mice lacking certain genes, and immor- Welsh, D.K., Kay, S.A. Bioluminescence imaging in living organisms. Curr. Opin. talized hepatocytes and vascular smooth muscle cells Biotechnol. 16:73, 2005. in these studies along with real-time bioluminescence imaging and biochemical and genetic approaches. Fur- thermore, we are using high-resolution bioluminescence Mechanism of Activation of imaging to examine whether single neurons in the suprachiasmatic nucleus and peripheral cells are autono- Nuclear Receptors mous circadian clocks and to characterize the precise P.R. Griffin, S.A. Busby, M.J. Chalmers, S. Prasad, S.Y. Dai nature of synchronization of the molecular clockwork of individual cells. eroxisome proliferator-activated receptor γ (PPARγ), Flowering is a major event in the life cycle of higher a ligand-dependent transcription factor and mem- plants. Many plants use seasonal changes in the length P ber of the nuclear receptor superfamily, is the of days as a signal to flower, and higher plants use their molecular target of the drug class known as the glita- circadian clocks to perceive these changes. Recently, zones. These compounds are used to improve muscle we defined a molecular link between the circadian clock insulin resistance, a sign or cause of type 2 diabetes, and day length–dependent regulation of flowering. A and are referred to as insulin sensitizers. The glitazones flowering time gene known as CONSTANS was identified are widely prescribed for treatment of type 2 diabetes. a number of years ago and is regulated by the circadian However, they have limited usefulness in patients clock. We showed that clock regulation of CONSTANS with mild insulin resistance or a history of cardiovas- expression is the key to seasonal control of flowering in cular disease because the drugs are associated with Arabidopsis.We are extending these studies by com- specific receptor-mediated side effects, such as weight paring gene expression profiles under conditions of gain, fluid retention, and plasma volume expansion. 342 BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

Unfortunately, the incidence of cardiovascular disease exchange. We used fluorescence assays to determine is high in patients with type 2 diabetes. In addition, recruitment of coactivators and to measure the affinity a clear link exists between body mass index and the of the receptor heterodimer composed of PPARg and incidence of insulin resistance and type 2 diabetes. retinoic X receptor a. A genetic screen was used to probe Thus, the search continues for a PPARγ modulator that the molecular determinants of ligand-dependent coac- increases muscle insulin sensitivity without causing tivator selectivity. The functional relevance of the screen weight gain and plasma volume expansion. was further confirmed in a 3T3-L1 preadipocyte differen- In addition, recent data indicate that thiazolidine- tiation assay as an in vitro model of adipogenesis. dione-based PPARγ agonists may be carcinogenic in In other studies, we are using coactivators as chemi- rodents. Because of this finding, the Food and Drug cal tools to generate desired functional responses and Administration requires the completion of 2-year car- distinguish beneficial functions from adverse functions, cinogenicity studies in rodents before any new PPARγ a novel therapeutic avenue for treating insulin resistance modulator can be tested in phase 2 studies in humans. that has not yet been exploited. Our goals are to deter- Studies with animal models of insulin resistance have mine the structure-activity relationships between PPARγ shown that weight gain and plasma volume expansion ligands and their coactivator recruitment selectivity and can be minimized without loss of insulin sensitization to obtain PPARγ ligands with preferences for specific by using partial agonists of PPARγ, although the mech- coactivators. To this end we have developed a validated anism of this dissociation of efficacy from unwanted fluorescence assay for ligand-dependent recruitment of events is unclear. Studies with preadipocytes revealed the coactivator to PPARγ for a large-scale high-through- that these partial agonists do not induce adipogenesis put screen to identify coactivator-selective agonists of the as the full agonists do, and expression profiling has receptor. The results obtained from this research will shown that the gene expression patterns differ for the provide molecular insight into how recruitment of coacti- different functional classes of activators. vators modulates activation of PPARγ and shed light Currently, we are studying the mechanism of action the role of specific coactivators in the pharmaco- vation of PPARγ by partial agonists and the role of logic behavior of PPARγ modulators. conformation-mediated recruitment of coactivators in Research is also under way to improve the techni- adipogenesis. Activation of PPARγ is regulated by bind- cal aspects of the amide hydrogen-deuterium exchange ing of ligands to the receptor’s ligand-binding domain, method. We have developed a fully automated system which induces a change in the conformational dynam- for hydrogen-deuterium exchange experiments. The ics of the domain-mediated dissociation of corepressor automation of the method created an absolute require- molecules and forms suitable neoepitopes for binding ment for an integrated data management and analysis coactivator molecules. Ligands of PPARγ are charac- system. In collaboration with B. Pascal, Scripps Florida, terized as full or partial agonists on the basis of their we are developing software to control workflow, stor- ability to transactivate PPARγ target genes. Full agonists age, and analysis of data obtained in hydrogen-deuterium have maximal transcriptional activity, whereas partial exchange experiments. When the software solution, agonists have moderate activity. Although the structural named HD Desktop, is completed, it will be offered to and molecular determinants of full-agonist regulation academic laboratories as an open source program. of PPARγ have been studied in detail, the determinants PUBLICATIONS for partial agonists have not been completely character- Chalmers, M.J., Busby, S.A., Pascal, B.D., He Y., Hendrickson, C.L., Marshall ized. We are using structural, biochemical, and cell-based A.G., Griffin, P.R. Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry. Anal. Chem. 78:1005, 2006. techniques to examine the mechanism of regulation of PPARγ transcriptional activity by partial agonists. Hamuro, Y., Coales, S.J., Morrow, J.A., Molnar, K.S., Tuske, S.J., Southern, M.R., Griffin, P.R. Hydrogen/deuterium-exchange (H/D-Ex) of PPARγ LBD in the presence Hydrogen-deuterium exchange coupled with mass of various modulators. Protein Sci. 15:1883, 2006. spectrometry was used to characterize ligand-dependent Holt, T.G., Flick, R.B., Rohde, E., Griffin, P., Rohrer, S.P. Biomarker discovery in structure and dynamic changes in PPARg. Ligand- rat plasma for estrogen receptor-α action. Electrophoresis 26:4486, 2005. induced protection of amide exchange in the transcrip- Quint, P., Ayala, I., Busby, S.A., Chalmers, M.J., Griffin, P.R., Rocca, J., Nick, tional complex composed of PPARg, retinoic X receptor H.S., Silverman, D.N. Structural mobility in human manganese superoxide dismu- a, and steroid receptor coactivator-1 was probed by tase. Biochemistry, in press. using automated solution-phase hydrogen-deuterium BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 343 Insulin-Degrading Enzyme to yield new insights into the function of IDE and its homologs in vivo and could pave the way to a novel M.A. Leissring, J. Zhao, L. Li, Q. Lu class of therapeutic agents based on slowing insulin clearance. Characterization of these compounds in cells AGING AND NEURODEGENERATION and animal models is under way. e study diseases of the nervous system that occur during aging and the fundamental mech- PUBLICATIONS anisms that regulate aging. Currently, we are Leissring, M.A. Proteolytic degradation of the amyloid β-protein: the forgotten side of W Alzheimer’s disease. Curr. Alzheimer Res., in press. examining a fascinating but poorly understood zinc-metal- Leissring, M.A., Saido, T.C. Aβ degradation. In: Alzheimer’s Disease: Advances in loprotease known as insulin-degrading enzyme (IDE). Genetics, Molecular, and Cellular Biology. Sisodia, S.S., Tanzi, R.E. (Eds.). Springer, IDE is responsible for degradation of insulin and amy- New York, 2006, p. 139. loid β-protein, peptide substrates central to the pathogenesis of diabetes and Alzheimer’s disease, respectively. We have shown that enhancing the proteolysis of amy- Inhibition of Jun N-Terminal loid β-protein by IDE or other proteases can completely prevent Alzheimer-type pathologic changes in a mouse Kinase 2/3 for the Treatment model of Alzheimer’s disease. We are using techniques ranging from in vitro enzy- of Parkinson’s Disease matic assays to transgenic and gene-targeted rodent P. LoGrasso, M. Cameron, D. Duckett, R. Jiang, T. Kamenecka, models to explore the normal biology and therapeutic L. Lin potential of IDE and other proteases. In parallel, we are using high-throughput screening of compounds, solid- poptosis or programmed cell death plays a vital phase peptide synthesis, and medicinal chemistry to role in the normal development of the nervous discover and rationally design pharmacologic modulators A system and is also thought to contribute to the of IDE. These pharmacologic tools promise to deepen aberrant neuronal cell death that characterizes many neurodegenerative diseases. Therefore, blocking of neu- our understanding of IDE biology and could lead to ronal apoptosis could be an approach for treating neu- novel therapeutic interventions for Alzheimer’s disease rodegenerative diseases. A major pathway implicated in or diabetes. neuronal cell death and survival is the MAP kinase path- RATIONAL DESIGN OF POTENT AND SELECTIVE INHIBITORS OF IDE way, which controls cell proliferation and cell death in Despite the potentially great importance of IDE in response to many extracellular stimuli. Recent studies health and disease, no useful pharmacologic tools have have linked Jun N-terminal kinase (JNK) activity with been available for manipulating the activity of this pro- the cell death associated with Parkinson’s disease and tease in in vivo models. To meet this important need, we Alzheimer’s disease. deduced the cleavage-site specificity of IDE by analyz- JNK is linked to many of the hallmark pathophysio- ing combinatorial mixtures of peptides. On the basis of logic components of Parkinson’s disease, such as oxidative this knowledge, we designed and synthesized peptide stress, programmed cell death, and microglial activation. hydroxamates, which are potent IDE inhibitors. We next Many pieces of evidence support JNK as a target for used solid-phase peptide synthesis to generate a series treatment of the pathologic changes that underlie Parkin- of retro-inverso peptide hydroxamates, which led to the son’s disease. One attractive feature of JNK3 as a selec- identification of unnatural amino acid substitutions that tive drug target is that this kinase is almost exclusively improved potency by several 100-fold. The simplicity expressed in the brain; levels expressed in the kidney and and versatility of this approach have enabled us to gen- testis are extremely low. In contrast, JNK1 and JNK2 erate fluorescently labeled and affinity-tagged probes and are ubiquitously expressed. Despite the ubiquitous expres- cyclic peptide hydroxamates with exquisite selectivity sion of JNK2, we are developing a therapy to prevent for IDE. degeneration of dopaminergic neurons and halt the pro- The best compounds inhibit in the low nanomolar to gression of Parkinson’s disease by targeting JNK2/3. high picomolar range, making them greater than 10,000 This strategy is based on the results of experiments times more potent than any previously described small- with mice in which the gene for JNK3 or JNK2 was molecule inhibitors for IDE. These new reagents promise deleted and mice in which the genes for both JNK2 and 344 BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE JNK3 or both JNK1 and JNK2 were deleted. In contrast Genetic Determinants of the to mice lacking the gene for JNK1 alone, which had defective T-cell differentiation, mice lacking the gene for JNK2 Efficacy of Selective Serotonin alone had normal T- and B-cell development and normal T-cell proliferation. Moreover, mice lacking the gene for Reuptake Inhibitors JNK2 alone and mice lacking the gene for JNK3 alone were protected against the effects of 1-methyl-4-phenyl- M.T. Pletcher, A. Gulati, B.H. Miller, B.M. Young, 1,2,3,6-tetrahydropyridine (MPTP), a compound used to G.M. Zastrow induce parkinsonian signs in animal models of Parkin- linical depression is a mood disorder with high son’s disease, whereas both wild-type mice and mice morbidity and mortality that is estimated to occur lacking the gene for JNK1 were not. In other research, C in more than 15% of the adult population in the compared with wild-type mice, mice lacking the genes United States. Depression has a wide range of symptoms, for both JNK2 and JNK3 were dramatically protected including loss of energy, changes in weight, diminished against acute MPTP-induced injury of the nigrostriatal interest or pleasure in daily activities, insomnia or exces- pathway. This protective effect resulted in a 3-fold sive sleep, anxiety, slowness of movement, feelings of increase in the number of neurons positive for tyrosine worthlessness, difficulty concentrating, and thoughts hydroxylase, an indication of the increase in survival of death. of dopaminergic neurons. Depression can be a one-time occurrence but is often On the basis of these data, we are synthesizing an ongoing problem throughout a person’s lifetime. A potent, selective JNK 2/3 inhibitors that may be effica- total of 15% of patients with major depressive disorders cious in MPTP animal models of Parkinson’s disease. We die of suicide. The onset of depression is often linked have established homogeneous time-resolved fluorescence to environmental factors such as life events that greatly biochemical assays for JNK3 and counterscreens for JNK1 increase stress. Despite this association, depression and p38. We have generated nanomolar inhibitors of has a strong genetic component. Genetics accounts for JNK3 from 2 structural classes of compounds. We have 40%–50% of the risk for depression in a person’s life- tested compounds in vivo in rats and mice for drug metab- time, and the risk for members of a family does not olism and pharmacokinetic properties. Many of the JNK3 change if they are raised separately. inhibitors have had good oral absorption, good brain pen- We are using cell-based screening technology and a etration, and good pharmacokinetic properties that enable mouse model to identify the genes and pathways that efficacy studies. Moreover, we have screened 400,000 contribute to depressive behavior. Currently, we are con- compounds in an effort to discover new structural classes ducting a survey in 30 strains of mice for differences in that may be optimized as described. Current efforts are molecular, biochemical, and behavioral traits that repre- centered on (1) development of cell-based assays to mon- sent endophenotypes (i.e., single well-defined symptoms itor inhibition of c-Jun phosphorylation and survival of or traits of a much more complex disorder) associated dopaminergic neurons and (2) structure-based drug design with depression. In cataloging the variation in quantities to help guide medicinal chemistry studies and optimize of neurotransmitters and stress hormones in the blood, compounds for potency, selectivity, brain penetration, gene expression in regions of the brain such as the hippo- oral absorption, half-life, clearance, and efficacy. campus and hypothalamus, and performance in behavioral models such as the tail suspension test and open PUBLICATIONS Fricker, M., LoGrasso, P., Ellis, S., Wilkie, N., Hunt, P., Pollack, S.T. Substituting field tests, we are producing the data necessary to iden- c-Jun N-terminal kinase-3 (JNK3) ATP-binding site amino acid residues with their tify the genetic controls for each of these traits. Using p38 counterparts affects binding of JNK- and p38-selective inhibitors. Arch. Biochem. Biophys. 438:195, 2005. the in silico genetic mapping method, we can correlate the natural variation for these measurements in the inbred Zhang, W.X., Wang, R., Wisniewski, D., Marcy, A.I., LoGrasso, P., Lisnock, JM., Cummings, R.T., Thompson, J.E. Time-resolved Forster resonance energy transfer lines of mice with the underlying haplotype structure of assays for the binding of nucleotide and protein substrates to p38α protein kinase. the mice, thereby pinpointing biologically important genes. Anal. Biochem. 343:76, 2005. In parallel, we are developing a cell-based assay to monitor the function of the serotonin transporter, the gene target of the most common pharmaceutical treatment for depression: selective serotonin reuptake inhibi- BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 345 tors. Using this assay, we can screen the 18,000 full-length clone cDNA library of Scripps Florida for genes that modify the function of the transporter. Addi- tionally, introducing a selective serotonin reuptake inhibitor into the screen will enable us to identify genes that modulate the efficacy of this important class of drugs. Cumulatively, we hope to provide a better understanding of the molecular basis of depression and its treatment to allow improved diagnosis and therapies.

PUBLICATIONS Fries, S., Grosser, T., Price, T.S., Lawson, J.A., Kapoor, S., DeMarco, S., Pletcher, M.T., Wiltshire, T., FitzGerald, G.A. Marked interindividual variability in the response to selective inhibitors of cyclooxygenase-2. Gastroenterology 130:55, 2006.

Ishimori, N., Li, R., Walsh, K.A., Korstanje, R., Rollins, J.A., Petkov, P., Pletcher, M.T., Wiltshire, T., Donahue, L.R., Rosen, C.J., Beamer, W.G., Churchill, G.A., Paigen, B. Quantitative trait loci that determine BMD in C57BL/6J and 129S1/SvImJ inbred mice. J. Bone Miner. Res. 21:105, 2006.

Reijmers, L.G., Coats, J.K., Pletcher, M.T., Wiltshire, T., Tarantino, L.M., May- ford, M. A mutant mouse with a highly specific contextual fear-conditioning deficit found in an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Learn. Mem. 13:143, 2006.

Synthesis of Natural Products, Development of Synthetic Methods, and Medicinal Chemistry

W.R.Roush, R. Bates, Y.-T. Chen, E. Darout, A. DeBaillie, J. Dunetz, G. Halvorsen, M. Handa, J. Hicks, T. Hopkins, C.-W. Huh, W. Lambert,* R. Lira, C. Nguyen, M. Ober, R. Owen,* P. Orahovats,* R. Pragani, J. Qi,* T. Ryba, A. Sorg, K. Takao,** R. Thalji,* M. Tortosa, P. Va, A. Williams, S. Winbush * University of Michigan, Ann Arbor, Michigan Fig. 1. ** Keio University, Yokohama, Japan Structures of recently synthesized natural products.

ur research has 2 major themes. One is the syn- (Z)-dienes, double allylboration reactions of aldehydes thesis of structurally complex, biologically active with γ-boryl–substituted allylboranes for stereocontrolled O natural products (Fig. 1). These efforts in total synthesis of 1,5-endiols, synthesis of highly substituted synthesis are pursued in parallel with stereochemical tetrahydrofurans via [3 + 2]-annulation reactions of studies and the development of new synthetic methods. highly functionalized allylsilanes, and use of 2-deoxy- We have been particularly interested in stereochemical 2-iodoglycosyl imidates and 2-deoxy-2-iodoglycosyl aspects of intramolecular and transannular Diels-Alder fluorides for the highly stereocontrolled synthesis of reactions, in the development of methods for the dia- 2-deoxy-β-glycosides. stereoselective and enantioselective reactions of allyl- Natural products of current interest to us include metal compounds with carbonyl compounds, and in the amphidinolides C and F, amphidinol 3, angelmicin B, development of highly stereoselective methods for syn- annonaceous acetogenin analogs, durhamycin A and thesis of 2-deoxyglycosides. analogs, integramicin, lomaiviticin A, peloruside A, Recent studies to develop new synthetic methods psymberin, quartromicin D1, reidispongiolide A, spinosyn included studies on the Diels-Alder reactions of A biosynthetic intermediates, scytophycin C, superstolide 346 BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE

A, tedanolide, and tetrafibricin. We selected these addition to drug discovery efforts, currently we are molecules as targets because of their biological prop- focusing on basic aspects of mammalian genomics erties and their interesting and complex structures. We and transcriptomics. place a significant emphasis on the discovery, develop- IDENTIFICATION AND FUNCTIONAL ANALYSIS OF ment, and/or illustration of new reactions and synthetic NONCODING AND ANTISENSE TRANSCRIPTS methods for achieving high levels of stereochemical Conventional protein-coding genes account for a control in each of these synthesis efforts. minority of human RNA transcripts. A substantial com- Our second major area of interest focuses on prob- ponent of the full-length mouse and human cDNA sets lems in bioorganic chemistry and medicinal chemistry. that we and others have analyzed does not contain an One long-term project involves the design and synthesis annotated protein-coding sequence and most likely cor- of inhibitors of cysteine proteases isolated from tropical responds to noncoding RNA. Many of the noncoding parasites, such as Trypanosoma cruzi, the causative sequences constitute natural antisense RNA transcripts. agent of Chagas disease, and Plasmodium falciparum, We have shown that the majority of noncoding RNAs the most virulent of the malaria parasites. This research identified to date have substantial conservation across is performed in collaboration with colleagues at the species. We have also shown that many noncoding RNA University of California, San Francisco. We have also and antisense transcripts have differential expression participated in the development of proapoptotic benzodi- under various conditions and can affect conventional gene expression. azepines, in collaboration with G.D. Glick at the Univer- Antisense transcription, yielding both coding and sity of Michigan, and the design of novel heterocycles noncoding RNA, is a widespread phenomenon in mam- targeting HIV. New projects at Scripps Florida involve mals. The mechanisms by which natural antisense discovery of small molecules that affect cancer and transcripts may regulate gene expression are largely other validated targets, studies of the structure-activity unknown. We recently provided direct evidence against relationship of certain natural products, and optimiza- the formation of sense-antisense RNA duplexes in the tion of the pharmacologic profiles of natural products. cytoplasm of human cells and subsequent activation of

PUBLICATIONS RNA interference associated with the nuclease Dicer. Dias, L.C., Aguilar, A.M., Salles, A.G., Jr., Steil, W.J., Roush, W.R. Concerning the Interestingly, however, a number of antisense tran- application of the 1H NMR ABX analysis for assignment of stereochemistry to aldols deriving from aldehydes lacking β-branches. J. Org. Chem. 70:10461, 2005. scripts regulate the expression of their sense partners, either discordantly (silencing of antisense transcripts Hicks, J.D., Flamme, E.M., Roush, W.R. Synthesis of the C(43)-C(67) fragment of amphidinol 3. Org. Lett. 7:5509, 2005. results in increases in sense transcripts) or concordantly (silencing of antisense transcripts results in concomi- Lambert, W.T., Roush, W.R. Synthesis of the A-B subunit of angelmicin B. Org. Lett. 7:5501. 2005. tant decreases in sense transcripts). We have proposed

Thalji, R.K., Roush, W.R. Remarkable phosphine-effect on the intramolecular aldol 2 new pharmacologic strategies based on the suppres- reactions of unsaturated 1,5-diketones: highly regioselective synthesis of cross-con- sion of antisense RNA transcripts by small interfering jugated dienones. J. Am. Chem. Soc. 127:16778, 2005. RNA (siRNA). First, in discordant regulation, silencing of antisense transcripts increases the expression of the conventional (sense) gene, thereby conceivably mimic- Neuroscience Discovery and king agonist/activator action. Second, in concordant regulation, silencing of antisense transcripts alone or a Pharmacogenomics synergistic concomitant silencing of antisense as well as sense transcripts results in reduction of the conven- C. Wahlestedt, P. Kenny, P. McDonald, M.A. Faghihi, tional (sense) gene expression. J. Kocerha, M. Przydzial, H. Thonberg,* H.-Y. Zhang,* RNA INTERFERENCE AND DEVELOPMENT OF HIGH- T. Andersson,* O. Larsson,* L. Huminiecki,* C. Scheele,* THROUGHPUT GENOMICS TECHNOLOGY P. Engstrom,* B. Lenhard,* C. Dahlgren,* K. Wennmalm,* RNA interference has become one of the most impor- Z. Liang, J.A. Timmons* tant gene manipulation technologies. siRNA, the inducer * Karolinska Institutet, Stockholm, Sweden of RNA interference in mammals, can be used to elu- ur research involves aspects of Alzheimer’s dis- cidate gene functions by rapidly silencing expression of ease, Parkinson’s disease, depression, addic- a target gene. Today, siRNAs are widely used as research O tion, fragile X syndrome, autism, and aging. In tools and have potential for becoming therapeutic agents. BIOCHEMISTRY 2006 THE SCRIPPS RESEARCH INSTITUTE 347

We have built up a portfolio of siRNA technology. In this package we have a powerful siRNA vector system, a validation system, and a design system, all of which are unique. Combining these technologies with the high-throughput chemistry for on-chip DNA synthesis, we have set up a system for constructing siRNAs. Finally, we have introduced the use of locked nucleic acids in siRNAs and have shown beneficial properties of these agents. G PROTEIN–COUPLED RECEPTORS AS DRUG TARGETS More than half of known drugs bind to G protein– coupled receptors. We have continued our long-standing work on these receptors, particularly certain neuropeptide receptors. These efforts are now forming part of our drug discovery program. HUMAN GENETICS AND PHARMACOGENOMICS We are also pursuing drug discovery related to several human disorders that affect the brain. Our goal is to identify markers in the human genome that are associated with such common disorders. We wish to understand what makes certain individuals susceptible and how their responses to drug treatment may differ (pharmacogenomics).

PUBLICATIONS Carninci, P., Sandelin, A., Lenhard, B., et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38:626, 2006.

Dahlgren, C., Wahlestedt, C., Thonberg, H. No induction of anti-viral responses in human cell lines HeLa and MCF-7 when transfecting with siRNA or siLNA. Biochem. Biophys. Res. Commun. 341:1211, 2006.

Engstrom, P.G., Suzuki, H., Ninomiya, N., Akalin, A., Sessa, L., Lavorgna, G., Brozzi, A., Luzi, L., Tan, S.L., Yang, L., Kunarso, G., Ng, E.L., Batalov, S., Wahlestedt, C., Kai, C., Kawai, J., Carninci, P., Hayashizaki, Y., Wells, C., Bajic, V.B., Orlando, V., Reid, J.F., Lenhard, B., Lipovich, L. Complex loci in human and mouse genomes. PLoS Genet. 2:e47, 2006.

Faghihi, M.A., Wahlestedt, C. RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals. Genome Biol. 7:R38, 2006.

Fredman, D., Sawyer, S.L., Stromqvist, L., Mottagui-Tabar, S., Kidd, K.K., Wahlestedt, C., Chanock, S.J., Brookes, A.J. Nonsynonymous SNPs: validation characteristics, derived allele frequency patterns, and suggestive evidence for natural selection. Hum. Mutat. 27:173, 2006.

Frith, M.C., Wilming, L.G., Forrest, A., Kawaji, H., Tan, S.L., Wahlestedt, C., Bajic, V.B., Kai, C., Kawai, J., Carninci, P., Hayashizaki, Y., Bailey, T.L., Huminiecki, L. Pseudo-messenger RNA: phantoms of the transcriptome. PLoS Genet. 2:e23, 2006.

Timmons, J.A., Norrbom, J., Scheele, C., Thonberg, H., Wahlestedt, C., Tesch, P. Expression profiling following local muscle inactivity in humans provides new perspective on diabetes-related genes. Genomics 87:165, 2006.

Wahlestedt, C. Natural antisense and noncoding RNA transcripts as potential drug targets. Drug Discov. Today 11:503, 2006.

Wennmalm, K., Wahlestedt, C., Larsson, O. The expression signature of in vitro senescence resembles mouse but not human aging. Genome Biol. 6:R109, 2005.

Cancer Biology

Activation of the estrogen receptor-a (ER-α). Ribbon diagram shows ER-α (yellow) bound to a peptide of the Grip1 coactivator (red) and to the ER-α agonist tetrahydrocrysene (blue). ER-α has well-known roles in the progression and treatment of breast and uterine cancer, whereas ER-β contributes to resistance to prostate and colon cancer. This structure defines features that are required for tetrahydrocrysene to act as an

ER-α agonist, but as an antagonist of ER-β, and it reveals the mechanism of ligand-selective signaling.

Work and image done in the laboratory of Kendall W.

Nettles, Ph.D. 350 CANCER BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE

DEPARTMENT OF CANCER BIOLOGY

STAFF

John L. Cleveland, Ph.D. Professor and Chairman

Nagi G. Ayad, Ph.D. Assistant Professor

Michael D. Conkright, Ph.D. Assistant Professor

Kendall W. Nettles, Ph.D * Assistant Professor

RESEARCH ASSOCIATES

Antoino Amelio, Ph.D John L. Cleveland, Ph.D. John Bruning, Ph.D.

Frank C. Dorsey, Ph.D. Chairman’s Overview

Jeffrey E. Habel, Ph.D. he Department of Cancer Biology was launched this year to recruit faculty to the Florida campus who Dympna Harmey, Ph.D. T have close ties with cancer researchers at the Cali- fornia campus. Broadly, the goals of our research pro- Jelena Janjic, Ph.D. grams are to understand the molecular pathogenesis of Becky Mercer, Ph.D. cancer. Our focus is on signaling pathways directed by oncogenes and tumor suppressors that control cell division, Anthony D. Smith, Ph.D. growth, survival, and differentiation, as well as those that modify the response to therapeutics. Members in our department use a battery of state-of-the-art technologies * Joint appointment in the for target discovery and validation, and have developed Translational Research Institute preclinical models to evaluate the efficacy of new leads in cancer prevention and therapeutics. Our investigators are primarily interested in understanding the molecular underpinnings of all the major human cancers, but also have an interest in pediatric oncology, the interplay between cancer and the immune system, and the relationships between aging and cancer. One of the many strengths of Scripps Research is its access to high-throughput technologies, which enable investigators to explore potential leads very quickly using both genetic and small-molecule screens. Collaborations with the major cancer centers in the state of Florida, along with cancer researchers at the California campus, further enable us to convert leads into translational and clinical studies. CANCER BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 351

INVESTIGATORS’REPORTS into mitosis in both frogs and humans. We are determining the intracellular location of wee1 degradation in humans and the role of phosphorylation on wee1 Role of Ubiquitin-Mediated degradation. In collaboration with J. Busby, Scripps Florida, we are using mass spectrometry to identify Proteolysis in Irreversible wee1 phosphorylation sites. This multidisciplinary approach will provide greater insight into proteolysis, Transitions During the Cell Cycle the cell cycle, and neurogenesis and may be useful in cancer research and nerve regeneration studies. N.G. Ayad, A. Smith, D. Harmey, S. Simanski

e are interested in the cell biological basis of PUBLICATIONS Ayad, N.G., Rankin, S., Ooi, D., Rape, M., Kirschner, M.W. Identification of ubiquitin irreversible transitions that occur during the ligase substrates by in vitro expression cloning. Methods Enzymol. 399:404, 2005. W eukaryotic cell cycle. Ubiquitin-mediated proteolytic pathways ensure that irreversibility is achieved by targeting specific inhibitors of these transitions for Molecular Mechanisms of proteasomal degradation. One of the most important ubiquitin E3 ligases is the anaphase-promoting com- cAMP-Mediated Transcription plex (APC). The APC is active during G1 and in fully differentiated cells. Furthermore, our recent studies M.D. Conkright, B.A. Mercer, A.L. Amelio, M.A. Morris indicate that the APC is required to initiate differentia- variety of biological functions depend on the tion of neuronal precursors. This finding is especially cAMP signaling cascade, including long-term important because it suggests that the APC is controlling A memory, survival of beta cells, glucose metabo- an essential step in cell-cycle exit or differentiation, a lism, cardiomyopathy, and proliferation of chondrocytes. control that is both biologically and medically relevant. We study the molecular mechanisms involved in the Although we understand a great deal about the role conversion of these signals into transcriptional events. of the APC during the cell cycle, its role in initiating exit Increases in cellular levels of cAMP stimulate the expres- from the cycle is virtually unknown. We are uncovering sion of numerous genes by liberating the catalytic sub- the mechanism of APC activation and the proteins turned units of protein kinase A, which phosphorylates the over via the APC during differentiation. For these studies, transcription factor cAMP-responsive element binding we are using both PC12 cells and the primary cere- protein (CREB). Phosphorylation of CREB promotes the bellar granule cell system to probe the role of the APC recruitment of the coactivators CREB-binding protein/ during differentiation. In collaboration with J. Hogenesch, p300 and the initiation of transcription. Scripps Florida, we are using cell-based screening The diversity of biological functions associated with technologies to identify novel activators and inhibitors CREB and the cAMP pathway will be impossible to of the APC. For this research, we have developed a fully understand until all of the components involved high-throughput luciferase-based measure of APC activ- in the pathway have been identified and characterized. ity. In this method the N terminus of the APC substrate Currently, we are using high-throughput cell-based cyclin B is fused with luciferase, so an increase in screening technologies, including cDNA expression luciferase corresponds to increased levels of cyclin B, libraries, small interfering RNA libraries, and small- a finding that indicates lower levels of APC activity. We molecule libraries, to identify all of the proteins that are continuing our screening of 15,000 human cDNAs make up and regulate the cAMP pathway. Using this to identify novel APC regulators. Identification of these technology, we identified proteins called transducers of regulators most likely will illuminate both temporal and regulated CREB activity, a novel family of CREB coac- spatial control of APC activity during development. tivators. Ascertaining the factors involved in the cAMP In addition to identifying novel regulators and sub- signaling pathway will be paramount in delineating strates of the APC, we have concentrated on one of why the biological function of CREB differs so drasti- the known APC substrates, the cytosolic protein trigger cally between tissues. of mitotic entry 1. This protein is required for degradation of the mitosis-inhibitory kinase wee1 and entry 352 CANCER BIOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE Suppression of NF-κB by the Estrogen Receptor

K.W. Nettles, J. Bruning, J. Janjic

ur overall goal is to improve the treatment of inflammatory diseases through a novel path- O way by which the estrogen receptor reduces the action of a key transcription factor, NF-κB. Sup- pression of NF-κB by the estrogen receptor is critical for maintaining the protective effects of estrogens in inflammatory bowel disease, sepsis, arthritis, atherosclerosis, and lipid metabolism, highlighting the broad importance of this pathway. Several mechanisms have been proposed to explain cross talk between the estrogen receptor and NF-κB, including inhibition of DNA binding by NF-κB, competition for limiting transcriptional coactivators, and the formation of a direct inhibitory complex. Our preliminary data indicate that the estrogen receptor is recruited to the NF-κB response element of the gene for monocyte chemoattractant protein 1 in the cell line MCF7, which is positive for the estrogen receptor, and that the coactivator CREB-binding protein is a bridging factor between the estrogen receptor and NF-κB. We have also developed a technique that greatly speeds our ability to obtain x-ray crystal structures of the estrogen receptor, allowing us to obtain structural information on entire classes of receptor-ligand complexes in a short time. We are using this approach to define the chemical and structural features that determine NF-κB–selective signaling through the estrogen receptor.

PUBLICATIONS Nettles, K.W., Greene, G.L. Ligand control of coregulator recruitment to nuclear receptors. Annu. Rev. Physiol. 67:309, 2005. Infectology

Colored transparent surface rendering of the dimeric amino-terminal domain of the hepatitis C virus NS5A protein overlaid on a background of hepatitis C virus–infected cells stained to detect NS5A using a monoclonal antibody. Work and image done in the laboratory of Tim Tellinghuisen, Ph.D., Assistant

Professor, Department of Infectology, Scripps

Research, Florida. Sukhvir Mahal, Ph.D. Staff Scientist Department of Infectology INFECTOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 355

DEPARTMENT OF INFECTOLOGY

STAFF

Charles Weissmann, M.D., Ph.D. Professor and Chairman

Joaquin Castilla, Ph.D. Assistant Professor

Corinne Lasmézas, Ph.D. Professor

A. Donny Strosberg, Ph.D. Professor

Tim Tellinghuisen, Ph.D. Assistant Professor

SENIOR STAFF SCIENTIST Charles Weissmann, M.D., Ph.D. Nicole Salès, Ph.D. Chairman’s Overview

STAFF SCIENTISTS esearchers in the Department of Infectology cur- Sukhvir Mahal, Ph.D. rently focus on 3 areas: prion diseases, hepatitis R C, and leishmaniasis. Two groups, those of Corinne Lasmézas and my own, study the molecular biology of SENIOR RESEARCH prions and the pathogenesis of the disease. A further prion ASSOCIATES group was established when Joaquin Castilla joined us as an assistant professor in September. Chris Baker, Ph.D. Prion diseases are of interest not only because of the Carlos Coito, Ph.D. emergence of bovine spongiform encephalopathy (mad cow disease) in the United Kingdom and chronic wast- Vittorio Verzillo, Ph.D. ing disease in the United States but also because of the unusual properties of the infectious agent, which consists mainly or entirely of a protein, PrPSc, a conformer of the RESEARCH ASSOCIATES normal host protein PrPC. Interestingly, many strains of prions are associated with the same protein sequence, Shawn Browning, Ph.D. raising the question as to how strain-specific properties are encoded. An important resource we have established Paula Saá Prieto, Ph.D. is a semiautomated, cell-based assay for prions (scrapie cell assay), which replaces in most instances the slow, Prem Subramaniam, Ph.D. expensive, and less accurate mouse-based bioassay. The scrapie cell assay is being used to analyze the proper- Minghai Zhou, Ph.D. ties of prions and prion propagation, both in cell culture and in cell-free systems. Sukhvir Mahal has isolated cell lines that will replicate some prion strains but not other strains and has 356 INFECTOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE extended the scrapie cell assay to differentiate between INVESTIGATORS’REPORTS prion strains. A major effort at elucidating the mechanism by which cells distinguish between different prion strains involves, in addition to Dr. Mahal and Chris Baker, Generation and Transmission Shawn Browning and Prem Subramaniam, Dr. Lasmézas and her group are collaborating on this effort but also of Prions are investigating the therapeutic possibilities of using C. Weissmann, C.A. Baker, S.P. Mahal, S. Browning, antibodies to PrP and PrP-binding aptamers to cure C. Demczyk, A. Sherman prion diseases. A further research effort is directed toward screening he “protein only” hypothesis proposes that prions, for drugs against leishmaniasis, specifically against J-bind- the agents that cause transmissible spongiform ing protein, a putative target, and involves Scripps scien- T encephalopathies, consist mainly or entirely of tists in both California and Florida. Dr. Subramaniam, in PrPSc, an abnormal conformer of a normal host protein, collaboration with D.P. Millar, Department of Molecular PrPC, and that the agents propagate by a PrPSc-catalyzed Biology, and P. Wentworth, Department of Chemistry, has conversion of PrPC.A specific mechanism is suggested developed a high-throughput assay for the binding of an by the “seeding hypothesis.” Intriguingly, distinct prion oligonucleotide containing the modified DNA base J. In strains, which generate different disease phenotypes, collaborative studies with P.R. Griffin and his colleagues, may be associated with the same PrP sequence, sug- Department of Biochemistry, hydrogen-deuterium exchange gesting that the phenotypes are encoded by different was used to identify residues involved in the binding of PrP conformations. We are elucidating the mechanism the oligonucleotide to J-binding protein. of prion replication, the structural basis of strain specific- A third field of endeavor is hepatitis C. Donny Stros- ity, and the mechanism of strain recognition by cells. berg and members of his group are studying interactions We have improved the sensitivity and accuracy of the between hepatitis C virus proteins to find drug candi- cell-based prion assay (scrapie cell assay), which can now dates that disrupt such interactions. Tim Tellinghuisen, be carried out in 10 days and allows the simultaneous, who joined the department as an assistant professor in semiautomated processing of hundreds of samples per November 2005, is elucidating the role of the hepatitis week. This assay largely obviates the mouse bioassay, C virus protein NS5A in viral replication. As further space which requires large numbers of animals and many months becomes available, the studies on hepatitis C virus will to complete. We have isolated cell lines susceptible to be extended. infection by certain prion strains but not by other strains A histopathology service is being set up by and have established a cell panel (strain discrimination Nicole Salès. panel) that allows us to distinguish various prion strains, a task that otherwise requires many mouse bioassays and might take years to complete. We have also isolated neuroblastoma sublines that are susceptible to the RML prion strain, the 22L strain, both strains, or neither strain. This finding suggests that PrP conformation alone most likely is insufficient to allow discrimination by cells and that prions contain other components that codetermine strain specificity.We are puri- fying prions to identify such components. In other research, using the scrapie cell assay, we studied the kinetics of infectivity amplification in a cell-free system containing PrPC and primed with prion-infected brain homogenate. Using the strain discrimination panel, we are determining whether prions are replicated in a strain-specific manner in the cell-free system. Even after repeated cloning, cell populations persistently infected with prions are heterogeneous, composed INFECTOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 357 of infected and uninfected cells. We have identified a detrimental to growth or survival of Leishmania organ- nondividing subpopulation that is the most prolific in isms. Because J and JBP do not occur in the host, such producing and secreting prions and a dividing subpop- a compound might lead to a therapeutic drug for treat- ulation that generates low levels of prions. Our hypothe- ment of leishmaniasis. sis is that the doubling rate of dividing cells is greater In collaboration with P.R. Griffin, Department of than the doubling rate of the prions within the cells, Biochemistry, using hydrogen-deuterium exchange tech- so that infectivity is diluted out of the cells. This pro- nology, we compared JBP with the JBP-oligonucleotide cess would ultimately result in curing of the cells if the complex. We identified 4 regions in JBP that had sig- cells were not continuously reinfected by prions secreted nificant reduction in hydrogen-deuterium exchange by the nondividing population. The hypothesis assumes upon binding of the oligonucleotide and that may that the dividing population continuously throws off define a binding site. nondividing producer cells and predicts that seques- We will search for compounds that interfere with tration of secreted prions will cause “curing” of the the binding of J to JBP, seek proof of principle in the cell culture; this prediction has been confirmed in pre- mouse leishmaniasis model, and, if we are successful, liminary experiments. The switch between productive endeavor to develop a therapeutic drug. The fluores- and nonproductive states is an epigenetic phenomenon cence polarization assay for the binding of a J-contain- that may also underlie susceptibility to infection by ing, fluorescently labeled oligonucleotide to recombinant prion strains. JBP is adequate for high-throughput screening. A trial run with 360 randomly selected compounds yielded 1 PUBLICATIONS significant hit. The compound, reactive blue 2, a poly- Heikenwälder, M., Zeller, N., Seeger, H., Prinz, M., Klöhn, P.C., Schwarz, P., Ruddle, N.H., Weissmann, C., Aguzzi, A. Chronic lymphocytic inflammation speci- sulfonated anthraquinone dye, is not an attractive can- fies the organ tropism of prions. Science 307:1107, 2005. didate for a lead compound. We have also set up a

Jackson, G.S., McKintosh, E., Flechsig, E., Prodromidou, K., Hirsch, P., Linehan, J., Leishmania growth assay and used it to screen the Brandner, S., Clarke, A.R., Weissmann, C., Collinge, J. An enzyme-detergent method 360 compounds for ones that inhibit growth of the for effective prion decontamination of surgical steel. J. Gen. Virol. 86:869, 2005. organism. We found 2 inhibitors, ketoconazole and Weissmann, C. Birth of a prion: spontaneous generation revisited. Cell 122:165, ribavirin, the first of which is a recognized second-line 2005. treatment for leishmaniasis. This finding validates the Weissmann, C., Aguzzi, A. Approaches to therapy of prion diseases. Annu. Rev. screening approach. Med. 56:321, 2005.

J-Binding Protein of Leishmania Pathogenesis of Transmissible as a Potential Drug Target Spongiform Encephalopathies C.I. Lasmézas, N. Salès, P. Saá Prieto, M. Zhou, C. Weissmann, P. Subramaniam, P. Wentworth,* D.P. Millar,** M. Lefebvre-Roque, A. Sturny, G. Sferrazza R. Sabatini,*** P.R. Griffin,**** P. Borst***** rions, the infectious agents responsible for prion * Department of Chemistry, Scripps Research diseases, are thought to consist mainly or entirely ** Department of Molecular Biology, Scripps Research of an abnormally folded isoform, PrPSc, of the *** Marine Biological Laboratory, Woods Hole, Massachusetts P prion protein PrP. Prions are thought to replicate by an **** Department of Biochemistry, Scripps Research autocatalytic process of template-induced conforma- ***** Netherlands Cancer Institute, Amsterdam, the Netherlands tional change. he DNA of Leishmania organisms contains a mod- Most approaches for treatment of prion diseases ified base, J (β-D-glucosyl-hydroxymethyluracil), have focused on PrP and are difficult to implement T that does not occur in higher eukaryotes. Leish- because PrP is a normal host protein. We are exploring mania species contain a protein, J-binding protein (JBP), the possibility of using PrP-specific antibodies to impede that binds J-containing DNA sequences and is involved prion replication in the brain. Our findings are reveal- in the conversion of thymine to J. Deletion of JBP is ing the limitations of such an approach. lethal for the organisms. Therefore, most likely com- Using osmotic pumps, we delivered antibodies pounds that interfere with the binding of JBP to J are against PrP into the brains of prion-infected mice. We 358 INFECTOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE found that PrP-specific Fab′2 fragments were more widely Moreover, because Ap9 can be used to distinguish distributed in the brain than were immunoglobulins. PrPSc from PrP without the need for a protease diges- The potential efficacy of such a treatment was offset tion step, we are exploring use of the aptamer as a by the toxicity of the antibodies, as indicated by glial fluorescent probe for detecting PrPSc on a variety of reactions and neuronal apoptosis. Overall, these results samples, including tissue sections and living cells. We call for caution in the use of therapies based on anti- are also developing lentiviral vectors to optimize in vitro bodies to PrP that target the CNS. In order to determine and in vivo delivery of the PrPSc-specific aptamer or new targets of intervention, our current goals are to bet- other PrP ligands. ter understand the replication of prions and the mecha- A functional histology core facility for standard tis- nisms of the neurotoxic effects and to identify factors sue processing and immunolabeling and classical or other than PrP that are involved in these processes. confocal microscopy analysis is being set up as a service Although the central role of PrP in the pathogene- for the Department of Infectology and Scripps Florida. sis of transmissible spongiform encephalopathies is Another goal of the facility is to develop innovative firmly established, one or more other molecules might molecular labeling and imaging techniques. play an important role, particularly in regard to the different kinetics of replication and different distributions in the body and brain of different prion stains. In col- Protein-Protein Interactions in laboration with C. Weissmann, Department of Infectol- ogy, we are searching for such putative factors. Hepatitis C As part of our effort to devise approaches for treatment of prion diseases, we are studying the route of A.D. Strosberg, C. Coito, S. Kota, N. Ayad,* C. Nahmias** entry of prions after oral infection of mice. It is known * Department of Biochemistry, Scripps Research that prions accumulate in follicular dendritic cells and ** CNRS, Paris, France macrophages of the germinal centers of the intestinal INTERACTIONS BETWEEN HEPATITIS C VIRUS Peyer’s patches. However, the mechanism of transport PROTEINS from the gut lumen to the lamina propria and the cells e are interested in interactions between the that take up prions and transport the proteins to the proteins of hepatitis C virus (HCV). Our goals lymphoid follicles are not known. Our immunohisto- W are to better understand the respective roles chemical studies revealed accumulation of PrP in 2 of the proteins and to identify small-molecule inhibitors distinct cell populations of the mucosa and in nerve that might actually have an effect on HCV replication. endings. In further studies, we hope to identify the Previously, we identified 5 pairs of interacting HCV pro- relevant cells and determine their role during the early tein domains and tagged the domains with glutathione- phases of oral infection. S-transferase or the octapeptide FLAG. We have now We are also studying an RNA aptamer that binds synthesized and cloned 4 of the 10 cDNA constructs that specifically to PrPSc. We plan to use this aptamer as a encode these HCV protein domains. The corresponding tool to prevent PrPSc from acting as a template for the proteins were expressed in Escherichia coli and were conversion of PrP into PrPSc and to detect PrPSc. affinity purified by using nickel-containing agarose, Aptamers are small nucleic acids (DNA or RNA) which recognizes the 6-histidine–containing epitope selected from a pool of random DNA or RNA sequences attached at the C terminus of the interacting domains. for their ability to bind to a given molecular target. The Interactions within one of these pairs of domains, in vitro procedure involves several rounds of selection composed of the first 106 residues of the HCV core and amplification. We focused on RNA aptamers because labeled with FLAG and with glutathione-S-transferase, RNAs adopt specific, sometimes complex, 3-dimensional have been confirmed. Attempts to inhibit this core-core structures. Using PrPSc purified from hamster brains, interaction by using 10 core-derived 18-residue pep- in collaboration with the Gene Center, Munich, Germany, tides or a mixture thereof have been unsuccessful. A we selected the aptamer Ap9, which binds selectively more sensitive and high-throughput homogenous time- to PrPSc. We are testing the ability of the aptamer to resolved fluorescence assay will soon be implemented interfere with prion replication and formation of PrPSc in to further extend screening of peptide and small-mole- cells infected with the prion agent that causes scrapie. cule inhibitors of the protein-protein interactions. Once INFECTOLOGY 2006 THE SCRIPPS RESEARCH INSTITUTE 359 this assay is operational for the core protein domains, cifically interacts with the C-terminal region of the AT2 it will used for studies of the other pairs of domains. receptor for angiotensin II. This putative tumor suppres- INTERACTIONS BETWEEN HCV AND HOST sor gene is also underexpressed in other forms of can- PROTEINS IN HUMANS cer. We are expanding these studies to investigate how To identify the human host proteins that interact the mutations affect the function of ATIP, in particular with individual HCV proteins, in collaboration with its capacity to activate the tyrosine phosphatase regu- N. Ayad, Department of Biochemistry, we are using lated by binding of angiotensin II to the AT2 receptor. In S35 translation and a collection of more than 7000 addition, using the system implemented with Dr. Ayad individually sequenced human cDNA clones available for the HCV NS5A protein, we are evaluating protein at Scripps Florida. Using this method, we searched for partners of ATIP tagged with glutathione-S-transferase. human protein partners of the NS5A domain I of HCV PUBLICATIONS labeled with glutathione-S-transferase. The proteins that Di Benedetto, M., Bieche, I., Deshayes, F., Vacher, S., Nouet, S., Collura, V., Seitz, bound to this HCV polypeptide were recovered and I., Louis, S., Pineau, P., Amsellem-Ouazana, D., Couraud, P.O., Strosberg, A.D., Stoppa-Lyonnet, D., Lidereau, R., Nahmias, C. Structural organization and expres- identified (Fig. 1). After several independent experi- sion of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptor-interacting proteins, ATIP. Gene 380:127, 2006.

Di Benedetto, M., Pineau, P., Nouet, S., Berhouet, S., Seitz, I., Louis, S., Dejean, A., Couraud, P.-O., Strosberg, A.D., Stoppa-Lyonnet, D., Nahmias, C. Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/MTUS1 in hepatocellular carcinoma. Mol. Cell. Endocrinol. 252:207, 2006.

Hepatitis C Virus RNA Replication

Fig. 1. Autoradiographs of in vitro translated pools interacting T.L. Tellinghuisen, J.C. Treadaway, K.L. Foss with NS5A domain I of HCV tagged with glutathione-S-transferase epatitis C virus (HCV) is a human pathogen of and captured on glutathione-covered beads. global importance; according to some estimates, ments, we found that 7 proteins reproducibly bound to H nearly 3% of the world’s population is chroni- the NS5A protein domain I. We are further identifying cally infected with HCV. Long-term viral replication in and characterizing these proteins. these individuals leads to severe liver disease, includ- HEPA TOMA CELL CULTURE SYSTEMS FOR ing cirrhosis and often hepatocellular carcinoma. The HCV SUBTYPES current treatment regimen with agents nonspecific for To further evaluate potential inhibitors of interactions HCV is poorly tolerated and is ineffective in about half between HCV proteins or interactions between HCV of the patients, emphasizing the need for effective antivi- proteins and human host proteins, we have assembled ral drugs specific for HCV. the components necessary to re-create a culture system Extensive structural and functional characterization in which HCV subtype 1b replicates at low levels in of the HCV proteins protease/helicase (NS3) and RNA hepatoma cells. Corresponding replicons have been pro- polymerase (NS5B) has led to the development of potent duced, and transfected cells have been subcloned to small-molecule inhibitors of HCV replication. Despite isolate producers of HCV particles. these advances, the high mutation rate inherent in HCV PUTA TIVE TUMOR SUPPRESSOR GENE IN RNA replication and the subsequent evolution of resis- HEPATOCELLULAR CARCINOMA tance dictate the need for new antiviral agents. Study The end stage of HCV infection is hepatocellular of the protein NS5A, another replicase component, has carcinoma. Despite its rare occurrence, HCV infection lagged behind research on NS3 and NS5B. NS5A remains is nevertheless the leading cause of liver cancer in the enigmatic, with no known function in HCV replication, United States. Recently, in collaboration with C. Nahmias, except its requirement in this process. CNRS, Paris, France, we found that more than 10% of We have been characterizing NS5A. Our goal is to patients with hepatocellular carcinoma have mutant understand the role of this protein in replication and, forms of the gene that encodes ATIP, a protein that spe- more generally, the replicase itself. Using biochemical 360 TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE and genetic methods, we have defined NS5A as an absolutely required 3-domain metalloprotein component of the replicase. Our recent crystal structure of the amino-terminal domain of NS5A (domain I) has provided a glimpse of the potential membrane, protein, and RNA interactions of NS5A in the viral replicase and has suggested potential antiviral targets. In addition, using genetic screens, we recently discovered an interaction between the membrane anchor of NS5A and the HCV protein NS4B, another component of the replicase. This interaction appears to localize NS5A to the replicase and is essential for RNA replication. We are conducting biochemical, genetic, and structural experiments to evaluate the potential interaction surfaces of the NS5A observed in the NS5A domain I structure and our genetic screens. These experiments include using the HCV replicon system, the HCV infectious cell culture system, mass spectrometry, nuclear magnetic resonance, molecular modeling, and crystallography. A parallel focus of our research is the development of small-molecule inhibitors that disrupt these putative interactions. Our ultimate goal is to understand, at the molecular level, the HCV RNA replication machinery. Greater insight into the poorly understood replicase components, such as the NS5A protein, will provide a more complete view of the replicase complex and will fuel new drug design.

PUBLICATIONS Lindenbach, B.D., Evans, M.J., Syder, A.J., Wolk, B., Tellinghuisen, T.L., Liu, C.C., Maruyama, T., Hynes, R.O., Burton, D.R., McKeating, J.A., Rice, C.M. Complete replication of hepatitis C virus in cell culture. Science 309:623, 2005.

Tellinghuisen, T.L., Marcotrigiano, J., Rice, C.M. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435:374, 2005.

Tellinghuisen, T.L., Paulson, M.S., Rice, C.M. The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein. J. Virol. 80:7450, 2006. Translational Research Institute

Inhibition of stress fiber formation in Swiss 3T3 cells by rho kinase

Inhibitor for potential treatment of spinal cord Injury. Work performed by

Thomas Schroeter, Ph.D., Evelyn Griffin, and Philip LoGrasso, Ph.D. Jennifer C. Busby, Ph.D.

Associate Scientific Director

Protein Sciences and Proteomics

Translational Research Institute TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE 363

TRANSLATIONAL Nicholas F. Tsinoremas, Ph.D. Ahmad Khalil, Ph.D. RESEARCH INSTITUTE Senior Director, Informatics Magdalena Przydzial, Ph.D. Claes Wahlestedt, M.D., STAFF Ph.D.* Michael Smolinski, Ph.D. Director, CNS Disorders Patrick R. Griffin, Ph.D.* Jeremiah D. Tipton, Ph.D. Head, Drug Discovery SENIOR SCIENTISTS Jennifer C. Busby, Ph.D. Associate Scientific Director, Thomas D. Bannister, Ph.D. SCIENTIFIC ASSOCIATE Protein Sciences and Dmitriy Minond, Ph.D. Proteomics Derek R. Duckett, Ph.D.

Michael Cameron, Ph.D. Marcel Koenig, Ph.D. Laboratory Head, Drug INFORMATICS STAFF Metabolism and Louis D. Scampavia, Ph.D. Pharmacokinetics Mohammad Fallahi-Sichani

Alessandra Cervino, Ph.D. Mark M. Gosink, Ph.D. Informatics SENIOR STAFF Assistant Professor SCIENTISTS Christopher C. Mader

Yangbo Feng, Ph.D. Jiu-Xiang Ni, Ph.D. Bruce D. Pascal Associate Director, Medicinal Chemistry Tomas Vojkovsky, Ph.D. Stephan Schuerer, Ph.D.

Peter S. Hodder, Ph.D. Mark R. Southern Associate Director, Lead Identification STAFF SCIENTISTS

Ted Kamenecka, Ph.D. Scott A. Busby, Ph.D. Associate Director, Medicinal * Joint appointment in the Chemistry Patricia H. McDonald, Ph.D. Department of Biochemistry Thomas Schroeter, Ph.D. ** Joint appointment in the Chris Liang, Ph.D. Department of Cancer Biology Director, Medicinal Chemistry *** Joint appointments in the Department of Biochemistry and Phillip LoGrasso, Ph.D.* RESEARCH ASSOCIATES the Department of Chemistry Director, Discovery Biology Yen Ting Chen, Ph.D.

Kendall W. Nettles, Ph.D.** Julie Conkright, Ph.D. Discovery Biology Yuan Dai, Ph.D. Mathew T. Pletcher, Ph.D.* Chinh Dao, Ph.D. Genome Technologies Brian Ember, Ph.D. William R.Roush, Ph.D.*** Executive Director, Medicinal Bozena Frackowiak, Ph.D. Chemistry, Yuanjun He, Ph.D. Associate Dean,Kellogg School of Science and Jia Huang, Ph.D. Technology, Florida Rong Jiang, Ph.D. Layton H. Smith, Ph.D. Associate Scientific Director, Discovery Biology 364 TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE

Metabolism and Pharmacokinetics, headed by Mike Cameron, associate director. The Lead Identification department enables high- throughput screening–related drug discovery research. Using state-of-the-art automation and instrumentation, the department is responsible for developing and executing biochemical or cell-based high-throughput screening assays in a miniaturized 1536-well microtiter plate format. In addition to its support of internal Scripps Research objectives, the group participates in the National Insti- tutes of Health’s Molecular Library Screening Center Network, where qualified assays are screened against the network’s high-throughput screening compound library. Several internal and external investigators have accessed the department’s expertise via collaborative or core-charge mechanisms. The Genomics platform is headed by Mathew Pletcher, assistant professor of biochemistry. The Genomics core oversees genotyping and gene expression profiling technology platforms. The services provided by this core allow Patrick R. Griffin, Ph.D. Scripps Research investigators to query the genome at Head, Drug Discovery both the genetic and transcriptional levels for the genes that underlie common diseases. In collaboration with Chairman’s Overview Scripps Florida colleagues, the core has been involved in projects seeking to identify the genes responsible he Translational Research Institute, headed by Pat for pathologies such as addiction and alcoholism, lupus, Griffin, professor of biochemistry, merges drug dis- autism, obsessive-compulsive disorder, obesity, and T covery efforts at Scripps Florida with advanced prion pathogenesis. technology platforms to rapidly identify and validate The Cell-Based Screening platform is headed by biological pathways that can be targeted for therapeutic Mike Conkright, assistant professor of Cancer Biology. intervention. The technology platforms are grouped into The Cell-Based Screening platform leverages high- genomics, cell-based screening, and proteomics cores. throughput technologies toward a systematic descrip- The goal of the drug discovery operation is to discover tion of the function of genes encoded by the human and develop small-molecule therapeutic agents for unmet genome and a more comprehensive understanding of medical needs in neurodegeneration, Parkinson’s disease, the genetic basis for human disease. The Cell-Based acute respiratory distress, spinal cord injury, cardiovas- Screening group provides investigators with access to cular disease, cancer, and metabolic disorders such as genome-wide collections of cDNAs and siRNAs that insulin resistance and type 2 diabetes. Therapeutic areas can be used to interrogate cellular models of signal and targets, which include G protein–coupled receptors, transduction pathways and phenotypes. proteases, channels, and kinases, are selected on the The Proteomics platform is headed by Jennifer Cald- basis of unmet needs and the ability to attract funding. well-Busby, associate director. The core focuses on the The drug discovery operation is fully integrated with the application of liquid chromatography and state-of-the- following groups: Lead Identification, headed by Peter art mass spectrometry technology to the identification, Hodder, director and associate professor; Medicinal Chem- quantitation, and characterization of proteins and their istry, headed by William Roush, executive director and modifications. The laboratory is involved in scientific professor of biochemistry; Discovery Biology, headed by collaborations that use novel technologies to identify Phil LoGrasso, senior director and associate professor of biologically important proteins and modifications. Large- biochemistry; CNS Disorders, headed by Claes Wahlest- scale differential analysis is being used to map the path- edt, director and professor of biochemistry; and Drug ways related to insulin sensitization and adipogenisis. TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE 365

Another project uses targeted peptide scanning to deter- sites of phosphorylation and other post-translational mine the rate of degradation of key proteins involved modifications. The Proteomics Lab works with collabo- in cellular regulation. Still other projects are using rating scientists to create experiments that will provide chromatographic enrichment techniques to identify meaningful mass spectrometric results.

INVESTIGATORS’REPORTS Data analysis is performed primarily via an automated workflow on a cluster maintained by the bioinformatics group. Automation of the front-end processing Proteomics Laboratory allows for a more thorough review of the resultant data and more time for development of innovative software J.A. Caldwell-Busby, V. Cavett, J.D. Tipton in collaboration with information technology groups at he Proteomics Laboratory provides proteomics Scripps Research and beyond. services and expertise to scientific collaborators at all Scripps Research facilities, universities PUBLICATIONS T Kirkland, P.A., Busby, J., Stevens, S., Jr., Maupin-Furlow, J.A. Trizol-based within the state of Florida, and other educational insti- method for sample preparation and isoelectric focusing of halophilic proteins. Anal. Biochem. 351:254, 2006. tutions. We use cutting-edge mass spectrometry technology to identify proteins, map modifications that occur after translation, and do relative quantitation experiments with a variety of samples. Drug Discovery: The Lead In its lifetime, a protein can have several locations and functions within a cell. Location, function, and Identification Department 3-dimensional structures of proteins are all influenced P. Hodder,P. Baillargeon, P. Chase, C. Chung, F. Madoux, by static and dynamic chemical modifications that occur D. Minond, K. Ryan, L. Scampavia, T. Spicer after translation. These modifications vary from small methyl and acetyl groups, which are a part of the his- he Lead Identification Department is responsible tone codes, to large lipid and glycosylation modifications, for developing and executing biological and bio- which act as cellular markers and signaling molecules. T chemical high-throughput screening assays and for With mass spectrometry, we can detect both the small supporting downstream medicinal chemistry efforts. The and the large changes in mass that occur in proteins anchors of the department are 2 fully automated robotic because of these modifications, and we can identify platforms (Fig. 1). One supports screening of 384- and the specific amino acids modified. 1536-well microtiter plates in a variety of assay formats. Relative changes in protein levels between multiple The other is used to manage the library of compounds samples provide biologically relevant information about used for drug discovery at Scripps Research. cellular pathways and proteins of interest. Large-scale This past year, we completed implementation of the studies of this type require rigorous sample preparation ultra-high-throughput screening (uHTS) operation and methods and highly tuned algorithms for comparing successfully completed several uHTS-related collabora- different mass spectrometric analyses. We are currently tions. Table 1 provides a list of the assay formats we validating methods for both sample fractionation and can use in screening. data analysis for these types of large-scale differential INFRASTRUCTURE ENHANCEMENTS protein experiments. The library of compounds for drug discovery con- Mass spectrometers at the facility include an ion-trap tains more than 600,000 unique small molecules. Most spectrometer, which is used mostly to identify proteins of the compounds have been acquired from commercial and peptides, and a triple quadrupole mass spectrom- suppliers. This past year, we augmented our collection eter, which is used for relative quantitation experiments. with compounds derived from internal research efforts. A new addition is a mass spectrometer that can be used Our newest additions include collections of kinase-spe- to perform accurate mass and high-resolution experi- cific and click chemistry compounds; the click chem- ments. Each mass spectrometer is interfaced to nanoflow istry compounds were generously donated by V. Fokin electrospray ionization sources and capillary high-per- and K.B. Sharpless, Department of Chemistry. Through formance liquid chromatography columns. our involvement in the National Institutes of Health 366 TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE

T able 1. Examples of uHTS-compatible assay formats for some popular drug discovery targets and indications.

Desired target or indication Measured species Assay formats G protein–coupled receptors Reporter gene β-Lactamase Luciferase Melanophore Second messenger Homogeneous fluorescence resonance energy transfer (cAMP) Fluorescence imaging plate reader (intracellular calcium)

Nuclear hormone receptor Reporter gene β-Lactamase Luciferase

Protease Labeled substrate Fluorescence dequenching Homogeneous fluorescence resonance energy transfer Fluorescence polarization

Kinase Labeled substrate Homogeneous fluorescence resonance energy transfer Fluorescence polarization

Antifungal or antibiotic Cell viability Luminescence (ATP activity)

Apoptosis Cell viability Luminescence (ATP activity)

Protein-protein interactions Labeled protein Homogeneous fluorescence resonance energy transfer Enzyme-linked immunosorbent assay

Protein folding Labeled protein Fluorescence dequenching

Roadmap Initiative, we have also acquired approxi- folds that might be useful chemical probes for relevant mately 100,000 compounds to support HTS collabo- biological or biochemical targets. rations associated with the Molecular Library Screening This past year we acquired specialized equipment Center Network (MLSCN). This collection contains scaf- and software to further automate the selection and

Fig. 1. The Scripps Research uHTS platform. A, The entire robot occupies a footprint of 220 square feet. B, A 1536-pin tool is used to transfer test compounds rapidly from compound plates to assay plates. C, Two liquid handlers capable of dispensing up to 32 different reagents and washing 1536-well plates are integrated onto the platform. D, Three incubators, each capable of holding approximately 700,000 samples in 1536-well format are used to store assay and compound plates. E, An imaging plate reader measures absorbance, luminescence, and fluorescence (with time resolution) on 1536-well plates. Not shown is a compound management robot capable of storing approximately 700,000 samples in a 384-well format for selecting the best or most desirable compounds; it occupies a footprint of 198 square feet. TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE 367 reformatting of medicinal chemistry compounds. We also subdivided our collection of compounds into smaller collections on the basis of known scaffold templates and target classes. This subdivision has enabled us to screen subsets of the Scripps collection of compounds. Additionally, we acquired a specialized liquid chromatography–mass spectrometry platform that allows us to rapidly determine the structure and purity of collections of small-molecule compounds, important for quality control and structure-activity efforts. Through collaborations with the Bioinformatics Department, we have installed a database to manage Fig. 2. Results of a uHTS campaign to determine the effects of the data produced by uHTS activities. We also devel- the MLSCN collection of compounds on the proliferation and viabil- oped or helped develop several software tools that ity of Jurkat cells. Representative structure-activity relationships interface with our uHTS and compound management derived from in silico analysis of active compounds are shown. A cluster of 4 antiparasitic agents, 2-carbamoyl-benzimidazoles, is platforms. Most important among these tools are those shown, along with percentages of inhibition and IC values. Also that enable us to automatically create lists of compounds 50 found in the screen were known apoptosis inducers (vincristine of interest, track sample locations, and generate uHTS and vinblastine); chemotherapeutic agents (paclitaxel and docetaxel, summary reports. doxorubicin and daunorubicin); and adenosine, pyrimidoindole, RESEARCH COLLABORATIONS phenylthiazidine, and benzimidazole derivatives. In collaboration with various internal and external We have also been actively screening the Scripps partners, we have successfully developed and executed several biological and biochemical uHTS assays. Our collection of compounds against drug discovery targets assays chiefly come from scientists at Scripps Research internal to Scripps Research. For example, in one uHTS involved in drug discovery, the MLSCN, and the Florida campaign, we used homogeneous time-resolved fluo- Access to Technologies program. rescence resonance energy transfer to discover inhibi- The first assay completed on the uHTS platform was tors of a serine-threonine kinase. The primary screen under the auspices of the MLSCN. In this campaign, was conducted in a miniaturized assay volume of 10 a collection of compounds from the National Institutes µL/well. More than 400,000 discrete compounds were of Health was tested for the effects of the compounds tested for activity, with a throughput of approximately on the proliferation and viability of a Jurkat cell line. 8000 tests/hour (Fig. 3). Because the collection contains several probes with From the primary uHTS campaign, 4220 compounds known cytotoxic activity, it was expected that the assay were identified as compounds of interest and were would unambiguously indicate the cytotoxic compounds. retested to confirm activity and selectivity to the kinase The uHTS campaign was conducted in 3 phases. of interest. After inspection of the resulting data, 1177 In the first phase, the primary screen, we determined of the compounds were selected for titration assays in if any compounds in the collection were active. In the order to determine IC50 values. From this set of com- second phase, we selected active compounds, sorted pounds, several novel scaffolds were identified that them into a new compound plate, and then tested them will complement future medicinal chemistry efforts. in triplicate to confirm activity. In the third phase, we As part of our commitment to the State of Florida, reformatted compounds with confirmed activity so that we participate in the Access to Technologies program, potency could be determined. In the potency assay, an initiative to help Florida academicians develop uHTS- 63 compounds had IC50 values less than 10 µM. As compatible assays. Currently, we are collaborating with expected, clustering these compounds by structural B. Dunn, University of Florida in Gainesville, and H. similarity revealed several chemotherapeutic agents and Weissbach, Florida Atlantic University in Boca Raton, other compounds with structural similarity to known to develop assays for discovering novel antimalarials cytotoxic agents (Fig. 2). These findings and all other and activators of antioxidant enzymes. MLSCN-generated assay data are available for public viewing at the PubChem Web site: http://pubchem PUBLICATIONS Hodder,P.S. Building a uHTS laboratory. Drug Discov.World 31, 2005/2006, .ncbi.nlm.nih.gov/assay/assay.cgi?aid=364. Winter Issue. 368 TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE

Hodder, P.S. Integration of miniaturisation technologies into the high throughput arthritis and asthma. For these reasons, protein kinases screening laboratory. Eur. Pharm. Rev. 2:47, 2005. are being investigated as valuable therapeutic targets by virtually every pharmaceutical company, and according to estimates, about 25% of all current pharmaceutical research is devoted to these targets. We successfully completed 2 drug discovery projects in the past year. The compounds from one project have been exclusively licensed to a large pharmaceutical company for further development; those from the other are being considered by another company. We also made significant progress in our collaboration with the NeoRx Corporation, Seattle, Washington, to discover novel multitargeted protein kinase inhibitors for the treatment of cancer. We have filed 2 invention disclo- sures covering 2 novel classes of compounds as inhibitors of protein kinases of great interest to NeoRx. Further optimizations of these compounds are under way.

Identification of Biomarkers in Tissues of Different Cell Types

N.F. Tsinoremas, M. Gosink

Fig. 3. A, Summary data from a uHTS campaign done with the iomarkers are increasingly important in the devel- Scripps Research robotic screening platform. Each data point repre- opment of personalized medicine. However, sents the results from one 1536-well plate. Z′ measures the assay identification of suitable biomarkers between window, and S/B measures the signal-to-background ratio from B individuals and disease states is complicated by the each plate. The well-resolved traces are indicative of high-quality data. B, A graph of the individual percentage of inhibition values incredible heterogeneity within tissues affected by dis- for each of the 400,000 compounds tested in the primary uHTS ease. These underlying tissues can play a crucial role campaign for a serine-threonine kinase. in the development, regulation, and progression of disease in adjacent tissues. The identification of particular biomarkers within a single cell type in a mixture of cells currently involves extensive and laborious “wet Development of Protein biology” experimentation to purify a critical cell type Kinase Inhibitors so that potential markers can be isolated. Once this cell type has been isolated, a number of approaches, C. Liang, M. Koenig, T. Vojkovsky, Y. He including expression profiling, are used to identify genes specific to the cell type. ur goal is to discover protein kinase inhibitors We have developed an algorithmic approach to iso- that can be used as therapeutic agents for the late the expression pattern of a single set of tissues from O treatment of human diseases such as cancer a mixture of tissues (Fig. 1, left). With this approach, and arthritis. Protein kinases are a class of enzymes that catalyze the transfer of the γ-phosphate from ATP to protein substrates. These enzymes play critical roles in signal transduction for a number of cellular functions. In particular, they regulate most of the hallmarks of cancer: cell proliferation, cell survival, cell motility/metastasis, cell cycle/division, and angiogenesis. Protein kinases Fig. 1. Isolating the expression profile of a single tissue from a are also implicated in inflammatory diseases such as mixture of tissues. TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE 369 some tissue types from a mixed sample can be isolated of the major genes in the signaling process have been (Fig. 1, middle). However, we are interested in the other identified, most have not. The thymus contains a mix- tissues that cannot be purified from the mixture (Fig. 1, ture of tissues, including the stromal cells and a sig- right, red blood cells). In our approach, computational nificant number of lymphocytes/thymocytes. Currently, methods are used to identify the part of the expression the stroma cells cannot be purified from the thymic profile of a mixed sample that is due to the tissue type tissue; however, thymocytes can be isolated from the that can be purified. Once this proportion is known, a thymus, and from these cells an expression profile can simple calculation can be performed to determine the be determined. This simplification method (deconvolu- expression profile of the genes in the remaining tissues. tion) is being used to electronically subtract the expres- This approach can also be used to deconvolute or sion profile of isolated thymocytes from the expression improve the resolution of expression patterns in wild- profile of the cells that make up the whole thymus. The type vs mutant genes or normal vs diseased tissues calculated stromal cell expression profile has revealed when the gene or disease affects only a subset of tissues. not only genes known to be involved in thymocyte mat- IMPROVEMENT OVER STRAIGHT SUBTRACTION uration but also genes known to be involved in the When the interesting subset of tissues in a mixture immune response (by mutational analysis) but by makes up a significant part of the total mixture, genes unknown mechanisms. with dramatic changes in expression can be identified We are also applying this in silico subtraction method by performing a simple fold-change calculation between to understand the development of regulatory T cells mixed and purified tissues. However, this approach that suppress the immune response to self-antigens. breaks down when the interesting tissue makes up only Although these cells play a critical role in preventing a small percentage of the mixture. For example, in a autoimmune disease, they constitute only a small fraction mixture of 2 tissues in which the tissue of interest of all T cells, and they are difficult to isolate. How- accounts for 5% of the total, a gene with an expres- ever, the transcription factor Foxp3 is a master regulator sion that is 10 times higher in the tissue of interest of the development of regulatory T cells. Expression pro- than in the overall mixture would have only a 1.45- files of T cells from mice lacking the gene for Foxp3 fold change by the standard calculation. have been reported, and a limited number of genes with In addition to increased sensitivity, the method can significant differences in expression between Foxp3− and be expanded to allow algorithmic removal of multiple wild-type T cells have been identified. We reanalyzed tissues. A combined fold-change calculation between the expression data from this experiment and identi- the mixed sample and the purified tissue samples is fied many more genes that are upregulated in regula- not possible, and sequential subtraction of each tissue tory T cells (Table 1 and Fig. 2). from the mixed sample would result in oversubtraction T able 1. Number of genes identified as having 2-fold or greater for most gene signals. In order to use this method, the expression in regulatory T cells. amount each tissue is contributing to the expression profile of the mixed sample is first calculated on the basis Method No. of genes of genes that are strongly expressed in one tissue in the Standard fold-change calculation 1355 sample but not in the other tissues. Once the individual Deconvolution method 7657 contribution fractions are calculated, the expression profile of the remaining tissue of interest is calculated An analysis of these gene sets indicates that as before. although both sets have a significant number of genes APPLICATIONS OF THE IN SILICO annotated by gene ontologies as involved in immune SUBTRACTION METHOD responses, only the set identified by using the decon- In collaboration with H.Petrie, Department of Bio- volution method had a significant number of genes chemistry, we are using this approach to identify genes involved in cell-mediated immune responses. It is expressed in the stroma of the thymus. Thymic stroma thought that in order to exert their activity, regulatory cells play a critical role in the maturation of T cells T cells must interact directly with other T cells. In addi- after the T cells leave the bone marrow. The stroma tion, the genes for IL-6 signal transducer and transform- cells display numerous signaling molecules and secrete ing growth factor-β receptor 2 were induced more than many factors involved in this process. Although a few 2-fold in regulatory T cells when the new method was 370 TRANSLATIONAL RESEARCH INSTITUTE 2006 THE SCRIPPS RESEARCH INSTITUTE

Fig. 2. Signals for genes that changed by 2-fold or more in regulatory T (T-reg) cells. used but not when the standard fold-change calculations were used. Recent evidence indicates that IL-6 and transforming growth factor-β play important roles in the differentiation of regulatory T cells. Awards, Education, Centers and Institutes, and Organizations

The 3A protein of coxsackievirus B3 disrupts the Golgi complex to inhibit protein trafficking. HeLa cells were transfected with a reporter construct expressing both the 3A protein and a marker membrane-trafficking protein, eGFP(mem). Bottom, Untransfected cell showing intact Golgi (GM130 marker, red). Middle, Early transfection shows some trafficking of eGFP(mem), with some of the protein being retained in the Golgi, which is beginning to disperse.

Top, Late in transfection, there is virtual disappearance of the Golgi, and scattered distribution of eGFP(mem). Nuclei are shown in blue. Images represent isosurface renderings of a confocal z series, created with the Imaris software package (Bitplane Scientific

Solutions). Work done by J. Lindsay Whitton, M.D., Ph.D.,

Christopher T. Cornell, Ph.D., and Stephanie Harkins, M.S., in

Dr. Whitton’s laboratory; and William B. Kiosses, Ph.D., Core

Microscopy Facility, Scripps Research. Class of 2006 Kellogg School of Science and Technology THE SCRIPPS RESEARCH INSTITUTE 2006 373 Staff Awards and Activities Chairman, Hepatitis B Virus Symposium, International Congress of Virology, San Francisco, California; Editorial Boards, Journal of Virology, Viral Immunology, Virology, Baran, P.S.—Beckman Young Investigator Award, Arnold Microbial Pathogenesis, Journal of Clinical Investigation. and Mabel Beckman Foundation; Career Award, National Conkwright, M.D.—Ruth L. Kirschstein National Research Science Foundation; Young Investigator Award, Eli Lilly Service Award, National Institutes of Health. and Company; Excellence in Chemistry Award, Astra- Zeneca; Young Professor Award, E.I. du Pont de Nemours Curtiss, L.K.—Member, Atherosclerosis and Inflammation and Company; Excellence in Chemistry Award, Hoff- Cardiovascular Sciences Study Section, National Institutes mann-La Roche; Young Investigator Award, Amgen, of Health; Associate Editor, Journal of Lipid Research; Inc.; Searle Scholar Award, Kinship Foundation; Editorial Board, Arteriosclerosis, Thrombosis, and Chemistry Scholar Award, GlaxoSmithKline; Fellow, Vascular Biology. Alfred P. Sloan Foundation. Danuser, G.—Associate Editor, IEEE Transactions on Barbas, C.F. III—Fellow, American Association for Image Processing; Editorial Board, Biophysical Journal. the Advancement of Science; In-Cites Highly Cited Dawson, P.E.—Member, Faculty in Chemical Biology, Researcher, Thomson Scientific, Philadelphia, Pennsyl- Faculty 1000, Biology Reports, Ltd.; Editorial Boards, Faculty vania; Member, Faculty in Chemical Biology, International Journal of Peptide Research and Thera- 1000, Biology Reports, Ltd.; Editorial Boards, Bioor- peutics, Letters in Peptide Science. ganic and Medicinal Chemistry Letters, Bioorganic and Medicinal Chemistry. Dyson, H.J.—Editorial Boards, Journal of Magnetic Resonance, Biophysical Journal. Bartfai, T.—Fellow, American Academy of Arts and Sciences. Elder, J.H.—Editorial Boards, Journal of Virology, Virology.

Boger, D.L.—Editor-in-Chief, Bioorganic and Medicinal Fokin, V.V.—William. H. Nichols Medal Award Distin- Chemistry Letters; Editorial Boards, Tetrahedron Pub- guished Speaker, American Chemical Society New York lications, Organic Reactions, Current Opinion in Drug Section, White Plains, New York. Discovery and Development, Current Drugs. Friedlander, M.—Alcon Research Award, Alcon, Inc.; Bokoch, G.M.—Editorial Boards, Journal of Leukocyte Chairman, Special Emphasis Panel, National Eye Insti- Biology, Journal of Biological Chemistry, Molecular tute; Member, Neurosciences Blueprint Advisory Panel, Pharmacology. National Institutes of Health; Member, Nanomedicine Initiative Advisory and Review Panel, National Institutes Buchmeier, M.J.—Fellow, American Association for the of Health Roadmap Program; Member, Trans-Institute Advancement of Science; Fellow, American Academy Angiogenesis Research Program Portfolio Review, National of Microbiology; Codirector,Pacific Southwest Center Institutes of Health. for Biodefense and Emerging Infectious Disease; Member, Scientific Advisory Boards, PathoSystems Resource Gale, A.J.—Early Career Investigator Award, Bayer Integration Center (Virginia BioInformatics Institute), Hemophilia Awards Program, Bayer HealthCare L.L.C., Predictive Biology Initiative, Pacific Northwest National Research Triangle Park, North Carolina; Career Devel- Laboratories; Member, National Multiple Sclerosis opment Award, National Hemophilia Foundation. Society Study Section; Editorial Boards, Journal of Gascoigne, N.R.J.—Member, Cellular and Molecular Virology, Virology, Viral Immunology, BMC Microbiol- Immunology A Study Section, National Institutes of ogy, The Virology Journal, Journal of Neurovirology, Health; Section Editor, Journal of Immunology. Microbiology and Molecular Biology Reviews. Gerace, L.—Editorial Boards, Journal of Cell Biology, Case, D.A.—Associate Editor, Biopolymers; Editorial BMC Cell Biology. Board, Journal of Biomolecular NMR. Gottesfeld, J.M.—Associate Editor, Journal of Biological Chisari, F.V.—Member, National Academy of Sciences; Chemistry. Member, Institute of Medicine, National Academy of Sciences; Member, Board of Scientific Councillors, Gottlieb, R.A.—Member, Research Committee, American National Institute of Allergy and Infectious Diseases; Heart Association, Western Regional Affiliate; Member, 374 THE SCRIPPS RESEARCH INSTITUTE 2006

Myocardial Ischemia and Metabolism Study Section, Medication Development, American College of Neuropsy- National Institutes of Health; Editorial Boards, American chopharmacology; Member, Neurobiology of Motivated Journal of Physiology: Heart and Circulatory Physiol- Behavior Study Section, National Institutes of Health; ogy, Biochemical Journal. Field Editor, Neuropharmacology; Editorial Boards, American Journal on Addictions, Biological Psychiatry. Griffin, J.H.—Distinguished Career Award, International Society for Thrombosis and Hemostasis. Mason, B.J.—Field Editor, Neuropsychopharmacology.

Havran, W.L.—Editorial Board, Immunological Reviews. Miles, L.A.—Thrombosis Special Recognition Award, Council on Ateriosclerosis, Thrombosis, and Vascular Horwich, A.L.—Corecipient, Stein and Moore Award, the Biology, American Heart Association; President, XVIIth Protein Society. International Congress on Fibrinolysis and Proteolysis; Janda, K.D.—Section Head, Faculty in Chemical Biology, Chair, Women’s Leadership Committee, Council on Arte- Faculty 1000, Biology Reports, Ltd.; Editorial Boards, riosclerosis, Thrombosis and Vascular Biology, American Chemical Reviews, Combinatorial Chemistry Research Heart Association and Review Committee 4B, American and Applications, Bioorganic and Medicinal Chem- Heart Association Western States Affiliate Research istry Letters, Bioorganic and Medicinal Chemistry, Committee; Member, American Heart Association Combinatorial Chemistry High-Throughput Screening. Western Consortium; Council Member, International Society for Fibrinolysis and Proteolysis; Member, Sci- Johnson, E.F.—Editor-in-Chief, Drug Metabolism and entific Advisory Board Member, International Society of Disposition; Editorial Boards, Journal of Biological Thrombosis and Haemostasis; Editorial Boards, Frontiers Chemistry, Molecular Pharmacology, Archives of Bio- in Bioscience, Thrombosis and Haemostasis. chemistry and Biophysics. Mowen, K.A.—Hulda Irene Duggan Arthritis Investigator Joyce, G.F.—Member, National Academy of Sciences; Award, Arthritis Foundation. Member, Committee on International Security and Arms Control, National Academy of Sciences; Member, External Nicolaou, K.C.—G.M. Kosolapoff Award, Auburn Section, Advisory Board, Beckman Institute, California Institute American Chemical Society; Burkhardt-Helferich Prize, of Technology, Pasadena, California; Head of Faculty in Institute of Organic Chemistry, University of Leipzig, Chemical Biology, Faculty 1000, Biology Reports Ltd.; Leipzig, Germany; Co-Editor-in-Chief, Chemistry & Biol- Associate Editor, BioSystems, Evolutionary Computa- ogy; Editorial Boards, Tetrahedron Publications, Syn- tion, Origins of Life and Evolution of the Biosphere. thesis, Carbohydrate Letters, Chemistry—A European Journal, Perspectives in Drug Discovery and Design, Lerner, R.A.—Fellow, American Association for the Indian Journal of Chemistry, Section B, Combinatorial Advancement of Science; Robert A. Good Lecture in Chemistry High-Throughput Screening, Current Opin- Immunochemistry,Robert A. Good Immunology Society, ion in Bioorganic Chemistry, Current Organic Chem- St. Petersburg, Florida; Editorial Boards, Bioorganic istry, Organic Letters, ChemBioChem, Chemistry and and Medicinal Chemistry, Bioorganic and Medicinal Biodiversity, Bulletin for the Chemical Society of Japan, Chemistry Letters, Catalysis Technology, Drug Target- Chemistry—An Asian Journal. ing and Delivery, Journal of Virology, Molecular Biol- ogy and Medicine, Molecular Medicine, Journal of Oldstone, M.B.A.—Fellow, American Academy of Micro- Peptide Research, Vaccine, Angewandte Chemie. biology; Member, Institute of Medicine, National Acad- emy of Sciences; Elected Member, Scandinavian Society Lotz, M.—President, Osteoarthritis Research Society of Immunology, American Association of Physicians, International; Member, Skeletal Biology and Skeletal American Society for Clinical Investigation; Member, Regeneration Study Section, National Institutes of Health; Scientific Advisory Committee, Pew Scholars Program Member, Faculty in Medicine, Faculty 1000, Biology in the Biomedical Sciences; Editor, Virology, Current Reports, Ltd.; Associate Editor, Arthritis Research and Topics in Microbiology and Immunology; Editorial Therapy, Journal of Immunology; Editorial Boards, Boards, Immunity, Journal of Clinical Investigation. Biotherapy, Osteoarthritis and Cartilage, Journal of Polich, J.—Member, Cognitive Neuroscience Study Sec- Orthopedic Research, Modern Rheumatology. tion, National Institutes of Health; Editorial Boards, Markou, A.—Chair, Animal Models and Their Validity to Brain Topography, Brain and Cognition, Clinical Neu- the Disease Disorder Subcommittee, Task Force on rophysiology, Journal of Psychophysiology. THE SCRIPPS RESEARCH INSTITUTE 2006 375

Pollard, K.M.—Member, External Advisory Committee, York; Novartis Lecturer in Central Europe, Budapest, Center for Environmental Health Sciences, University Hungary, Bratislava, Slovakia, and Prague, Czech of Montana, Missoula, Montana. Republic; Sessler Lecture, Stanford University, Stanford, California; J.P. Freeman Lectureship, University of Notre Rebek, J., Jr.—Medal of the Academy of Sciences, Dame, Notre Dame, Indiana; Backer Lecture, University Prague, Czech Republic; Medal of the National Acad- of Groningen, Groningen, the Netherlands; Wheeler emy of Sciences, Letters and Arts, Modena, Italy; Lecture, University College Dublin, Dublin, Ireland; Member, Academia Europaea; Editorial Boards, Tetra- Closs Lecture, University of Chicago, Chicago, Illinois; hedron Publications, Bioorganic and Medicinal Chem- Masamune Memorial Lecture, Massachusetts Institute istry Letters, Bioorganic and Medicinal Chemistry, of Technology, Cambridge, Massachusetts. Editorial Chemistry and Biology, Current Opinion in Chemical Boards, Advanced Synthesis and Catalysis, Beilstein Biology, Journal of Supramolecular Chemistry. Journal of Organic Chemistry, Bulletin of the Chemical Reed, S.I.—Editorial Board, Molecular and Cellular Society of Japan, Chirality, Current Opinion in Drug Biology. Discovery and Development, Current Drug Discovery Reisfeld, R.A.—Honorary Degree in Medicine, University Technologies, Enantiomer, Organic Letters. Synlett. of Genova, Italy; Coeditor, Journal of Clinical Laboratory Stevens, R.C.—Editorial Boards, Protein Expression and Analysis; Editorial Boards, Bioconjugate Chemistry, Purification, Biodrugs, Drug Discovery Today, The Cancer Biotherapy and Radiopharmaceuticals, Cancer Protein Journal. Immunology and Immunotherapy, Cancer Research, Clinical Cancer Research, Hybridoma, International Stuhlmann, H.—Member, Cardiovascular Differentiation Journal of Oncology, Journal of Immunology, Tumor and Development Study Section, National Heart, Lung, Targeting. and Blood Institute; Editorial Board, Stem Cells.

Ruf,W.—Thrombosis Special Recognition Award, Sutcliffe, J.G.—Member, International Advisory Board, Council on Arteriosclerosis, Thrombosis, and Vascular International Institute of Molecular and Cell Biology, Biology, American Heart Association; Editorial Board, Warsaw, Poland; Member Program Committee, Inter- Journal of Thrombosis and Haemostasis. national Society for Neurochemistry; Editorial Boards, DNA and Cell Biology, Molecular Neurobiology Reviews, Salomon, D.R.—Chair, National Islet Center Resources Journal of Neuroscience Research, Journal of Molec- Program, National Institutes of Health; Member,Trans- ular Neuroscience, Advances in Neuroscience, Jour- plantation,Tolerance, and Tumor Immunology Study Section, National Institutes of Health; Associate Edi- nal of Neurochemistry. tor, American Journal of Transplantation; Editorial Tellinghuisen, T.L.—Career Development Award, National Board, Transplantation. Institute of Allergy and Infectious Disease.

Schlaepfer, D.D.—Established Investigator, American Theofilopoulos, A.N.—18th Annual Paul Klemperer Heart Association. Award, New York Academy of Science; Honorary Doc- Schmid, S.L.—MERIT Award, National Institutes of toral Degree, Medical School, Aristotle University, Health; Alex Novikoff Plenary Lecture, Lysosomes and Thessaloniki, Greece; Honorary Doctoral Degree, Dem- Endocytosis Gordon Conference, Andover, New Hamp- ocritos Medical School, University of Thrace, Alexan- shire; Fellow, American Association for the Advancement droupolis, Greece; Corresponding Member, Academy of of Science; Board Member and Treasurer, Athena, Uni- Athens; Editor, Current Directions in Autoimmune versity of California, San Diego; Member, Activities Diseases (book series); Editorial Boards, Survey of Review Panel, American Heart Association Western Immunologic Research, Journal of Clinical Immunol- Division; Member, Review Panel, Howard Hughes Med- ogy, Journal of Experimental and Clinical Research, ical Institute International Research Scholar Program; Journal of Clinical Immunology and Immunopathol- Member, Advisory Committee, Burroughs Wellcome ogy, Journal of Immunopharmacology and Immuno- Career Awards in Biomedical Research; Editor-in-Chief, toxicology, Journal of Autoimmunity, International Molecular Biology of the Cell. Journal of Oncology, Scandinavian Journal of Immu- Sharpless, K.B.—William H. Nichols Medal, American nology, Human Immunology, Japanese Journal of Chemical Society New York Section, White Plains, New Rheumatology. 376 THE SCRIPPS RESEARCH INSTITUTE 2006

Torbett, B.E.—Consultant, Center for Biologics Evalua- Acting Editor-in-Chief, Viral Immunology; Editorial tion and Research Response, Food and Drug Adminis- Boards, Journal of Virology, FEMS Medical Microbiol- tration, States as Certifiers; Member, AIDS Molecular ogy and Immunology. and Cellular Biology Study Section, National Institute Wilson, I.A.—Fellow, Royal Society of London; Fellow, of Allergy and Infectious Diseases; Reviewer, Special American Academy of Arts and Sciences; Member, Sci- Emphasis Panel, Program Project in Myeloid Biology, entific Advisory Board, Keystone Symposia; Associate National Heart, Lung, and Blood Institute. Editor, Journal of Molecular Biology, Immunity; Editor- Vogt, P.K.—Medal of Distinction, Institute of Organic ial Boards, Science, Journal of Experimental Medicine. Chemistry and Biochemistry, Academy of Sciences of Wong, C.-H.—Georges Smets Chair Award for Organic or the Czech Republic; Fellow, American Academy of Arts Polymer Chemistry, University of Leuven, Belgium; Sci- and Sciences; Chairman, Scientific Advisory Board, entific Advisor, Max-Planck-Institut, Dortmund, Germany; Oncogene Research Institute, University of Singapore; Editor-in-Chief, Bioorganic and Medicinal Chemistry; Member, Selection Committee, Robert Koch Foundation; Editorial Boards, Tetrahedron Publications, Current Member, Board of Directors, Foundation for Advanced Opinion in Chemical Biology, Biocatalysis, Advanced Cancer Studies; Editorial Boards, Virology, Journal of Synthesis and Catalysis, Journal of the American Virology, Current Topics in Microbiology and Immunol- Chemical Society, Chemistry—An Asian Journal. ogy, Cancer Research, Proceedings of the National Academy of Sciences, Blood Cells, Molecules and Wright, P.E.—Honorary Doctor of Science, University of Sydney, Sidney, Australia; Editor-in-Chief, Journal of Diseases, Cell Cycle. Molecular Biology; Editorial Boards, Biochemistry, Waterman-Storer, C.M.—Who’s Who in Science and Current Opinion in Structural Biology, Journal of Bio- Engineering, Marquis Publishing; Keith R. Porter Fellow, molecular NMR. Keith R. Porter Endowment for Cell Biology; Director’s Wüthrich, K.—Doctor of Science honoris causa, King Pioneer Award, National Institutes of Health; R.R. Bens- George’s Medical University, Lucknow, India; Doctor ley Award in Cell Biology, American Association of honoris causa, University of Pécs, Pécs Hungary; Hon- Anatomists; Established Investigator, American Heart orary Member, Indian Biophysical Society; Corresponding Association; Chair, Summer Meeting Series, American Member, Nordrhein-Westfälische Akademie der Wissen- Society for Cell Biology; Member, Cell Structure and schaften; Foreign Member, Korean Academy of Science Function Study Section, National Institute of General and Technology; Honorary Member, Korean Magnetic Medical Sciences; Faculty Member, Annual Summer Resonance Society; Sarojini Damodoran Lecture, Tata Course, Marine Biological Laboratory, Woods Hole, Institute of Fundamental Research, Mumbai, India; Massachusetts. G.N. Ramachandran Memorial Lecture, Indian Biophysi- Weissmann, C.—Distinguished Research Professor, cal Society, Pune, India; Editor-in-Chief, Journal of Department of Biological Sciences, Florida Atlantic Biomolecular NMR; Editorial Boards, Biochimie, Bio- University, Jupiter, Florida; Warren Alpert Foundation polymers, ChemBioChem, Current Opinion in Structural Prize, Harvard Medical School, Boston, Massachusetts; Biology, IUBMB Life, Journal of Magnetic Resonance, DART/NYU Biotechnology Achievement Award for Basic Journal of Membrane Biology, Journal of Structural Biotechnology, Biotechnology Study Center, NYU School and Functional Genomics, Proteins, Structure. of Medicine, New York, New York; Bernard Fields Lec- Yagi, T.—Special Reviewer, Neurodegeneration, Neuroin- ture, Scripps Research Institute, La Jolla, California; flammation, Oxidative Stress, and Mitochondria Study Henry Kunkel Lecture, Cambridge, England; Geoffrey Section, National Institutes of Health; Editorial Board, H. Bourne Memorial Lecture, St. George’s University, Journal of Bioenergetics and Biomembranes. Grenada, West Indies; Editorial Board, Journal of NeuroVirology. Yates, J.R. III—Herbert A. Sober Lectureship Award, American Society for Biochemistry and Molecular Biol- Whitton, J.L.—Chair, Special Study Section on Vaccine ogy; Christian B. Anfinsen Award, Protein Society. Development, National Institutes of Health; Ad Hoc Member, Experimental Virology and Virology study sections, National Institutes of Health; Editor, Virology; THE SCRIPPS RESEARCH INSTITUTE 2006 377

versity of Hamburg, National Taiwan University, and the University of California system. Members of this year’s entering class originally come from countries as far away as Australia, Slovenia, and China. The entering biology class of 27 is the largest in the history of the program. Shortly after the first-year students arrived at Scripps Research, they began a 12-week class called Critical Thinking and Communication in Science to sharpen their skills in assessment and communication of scientific information and ideas. For the first time in 2006, the course included an introduction to the Scripps Research Kresge Library, with an overview of the library’s resources and services, a hands-on orientation, and in-depth seminars on topics such as databases and citation management software. One of the course requirements is a research proposal suitable for submission to a variety of predoctoral fellowship competitions. Another opportunity to learn about the institute’s resources and to meet student and faculty colleagues was provided by the 2006 Faculty Student Retreat. Held at the Bahia Resort on Mission Bay, the retreat was similar to a professional scientific conference, with Jeffery W. Kelly, Ph.D. students from both Florida and California campuses presenting their research through 17 oral presentations and 150 posters that explored topics such as Micro- Kellogg School of Science and Capillary Crystallization and Adventures in Total Synthe- Technology sis: The Stephacidin Family. Mike Burkart (Class of ‘99), now a faculty member in the chemistry and biochemistry itality, innovation, interdisciplinary scientific department of the University of California, San Diego, exchange—these are some of the cornerstones also gave a talk, passing on his experiences in science V of the Kellogg School of Science and Technology and offering career advice to the Ph.D. candidates. at Scripps Research. In 2006, numerous honors and awards were This year was a significant one for graduate studies bestowed on Kellogg School students highlighting at the Florida campus with the enrollment of the first their accomplishments: recruited graduate student, John Whitaker. He joins • An unprecedented number of students (5) were eight other Ph.D. candidates who transferred to Scripps selected for National Science Foundation Fellow- Florida from the University of Michigan with Professor ships: Daniel Bachovchin, Christine Fang, Gra- and Associate Dean William Roush. The introduction of ham Johnson, Costas Lyssiotis, and Adrian Ortiz. 2-way, web-based conferencing technology now enables • David Horning, a member of the entering class, the Florida students to participate in California lectures won a highly competitive Hertz Foundation in real time, as well as opening future Florida chem- Fellowship, which aims to support the graduate istry classes on asymmetric synthesis and related top- education of “America’s most promising tech- ics to interested California students. The technology nical talent.” also facilitates the meeting of thesis committees with • Lindsey Macpherson received a National Insti- faculty on both Scripps Research campuses. tutes of Health Ruth L. Kirschstein National In 2006, we welcomed a total of 42 new students Research Service Award. to our Ph.D. program from undergraduate institutions including Dartmouth, Brown, Stanford, Cornell, Duke, • Stuart Webb won a 3-year fellowship from the Pennsylvania State, Tufts, University of Chicago, Uni- National Institute on Deafness and Other Com- 378 THE SCRIPPS RESEARCH INSTITUTE 2006

munication Disorders of the National Institutes directs the private capital management firm, The Drey- of Health. foos Group, was honored for his numerous scientific • Sherry Niessen won a 2-year Career Develop- and engineering accomplishments and for his service ment Award from the California Breast Cancer as a member of the Board of Trustees. He and his wife, Research Program. Renate, provided a generous gift of $1 million to the institute in 2004. • Katherine Marcucci won an American Heart At the ceremony, Judge Sullivan, whose remarks Association fellowship. emphasized the importance of keeping an open mind • Noah Z. Burns and Scott T. Harrison were and welcoming the unexpected, praised this year’s honored for their research accomplishments graduates. “Today you are to be congratulated on your at Roche’s 3rd annual graduate research tremendous accomplishments in the classroom and the symposium, Excellence in Chemistry. laboratory,” she said to them. “This is a day to enjoy In 2006, Kellogg School student stipends and tuition the promise of success that awaits you because of your were supported by generous donations from individuals, intellect, your dedication, and your hard work. The foundations, and corporations—including the Gustavus trustees of Scripps Research are immensely proud to and Louise Pfeiffer Research Foundation, the William know that you will carry the name and reputation of and Sharon Bauce Family Foundation, the Fletcher Jones The Scripps Research Institute wherever you go. . . Foundation, the ARCS Foundation, the Hertz Foundation, you will honor us as we honor you.” the Donald E. and Delia B. Baxter Foundation, the Koshland Foundation, the American Chemical Society, Novartis, the Gilula Memorial Fund, the Andrea Eliza- beth Vogt Memorial Fund, David and Ursula Fairchild, and Lesly Starr Shelton. The Skaggs Oxford Scholarship Program, a joint 5-year program of study at Scripps Research and Oxford University, continued in 2006 thanks to generous support by supermarket and drugstore leader L.S. Skaggs and his wife, Aline. The program’s first student has now completed training at Scripps Research and is in the process of moving to Oxford University, where she will complete work for a joint Ph.D./D.Phil. degree. In other program news, the Kellogg School has begun the 3-year process of obtaining reaccreditation. Accredi- tation is a continuous process of improvement and is divided into 3 stages: an institutional proposal, a capacity and preparatory review, and an educational effectiveness review. Six committees are providing input for the 3-year self-study process. (See http://www.scripps.edu/ library/Accreditation/i_index.html for news and updates.) In May, we celebrated the many accomplishments of our students and the Kellogg School program at the institute’s 14th commencement ceremony, which honored 31 graduating students and two honorary degree recipients. Hon. Alice Sullivan (Ret.), a former Califor- nia Superior Court judge and founder and chief executive officer of Private Judge, was honored for her role as former chair and current member of the Scripps Research Board of Trustees. Alexander Dreyfoos, a resident of West Palm Beach, Florida, who owns and THE SCRIPPS RESEARCH INSTITUTE 2006 379

STUDENTS IN CHEMISTRY Elizabeth Culyba Nadia Haq Ang Li AND CHEMICAL BIOLOGY College of William & Mary, California Institute of Peking University, B.S. B.S. Technology, B.S. PROGRAMS Weiwei Li Trevor Dale David Harris National University of Simon Fraser University, B.S. Cornell University, B.A. Singapore, M.S. Adrian Accurso Jennifer Hazen Yee Hwee Lim Dartmouth College, B.A. Stephen Dean Vanderbilt University, B.S. Franklin & Marshall College, University of Bristol, B.Sc. Robert Aversa B.A. David Lin Cornell University, B.A. Jessica DeMartino Simon Hilcove Stanford University, B.S. University of Delaware, B.S. Arizona State University, B.S. Daniel Bachovchin Ricardo Lira Harvard College, A.B. Michael DeMartino Vu Hong University of California, University of Delaware, B.S. University of California, San Diego, M.S. Catherine Barglow Berkeley, B.S. Stanford University, B.S. Damian Ekiert Ewa Lis University of Chicago, B.A. Chan-Woo Huh Cornell University, B.A. Robert Bates Yonsei University, M.S. Massachusetts Institute of Shelby Ellery Chang Liu Technology, B.S. Cedar Crest College, B.S. Wooyoung Hur Harvard University, B.A. Pohang University, B.S. John Beierle Michael Evans Jonathan Lockner Boston College, B.S. St. Mary’s College of Reshma Jagasia University of Illinois at Maryland, B.A. University of Alberta, B.S. Urbana-Champaign, B.S. Jacqueline Blankman Valer Jeso Andre Loutchnikov Northwestern University, B.A. Christine Fang Massachusetts Institute of University of Toronto, M.S. University of California, Los Grant Boldt Technology, B.S. Angeles, B.S. Colin Lowery San Diego State University, Robert Jones University of Virginia, B.S. M.A. Doug Fowler Duke University, B.S. Northwestern University, B.A. Costas Lyssiotis Diana Bowley Daisuke Kato University of Michigan, B.S. University of Northern Iowa, University of California, Davis, Michael Frederick Karen MacMillan B.S. B.S. University of Minnesota, B.S. University of California, Davis, William Brenzovich Dong-In Koo B.S. Brian Frezza College of William & Mary, Brown University, B.S. Thomas Maimone B.S. Carnegie Mellon University, B.S. Paul Krawczuk University of California, Steven Brown New York University, B.S. Berkeley, B.S. University of Wisconsin, Joie Garfunkle Tun-Hsun Kuo Dena Marrinucci Madison, B.S. Boston College, B.S. National Taiwan University, University of Vermont, B.S. Jovana Grbic Noah Burns M.S. Casey Mathison Columbia University, B.A. Northwestern University, B.A. Sen Wai Kwok Massachusetts Institute of Jason Chen Yevgeniy Grigoryev University of California, Technology, B.S. Harvard University, A.B. City University of New York, San Diego, B.S. Jeremy Mills B.A. Shuibing Chen Jolene Lau Vanderbilt University, B.S. Tsinghua University, M.S. Carlos Guerrero California Institute of Amira Moreno Vera Harvard University, B.A. Technology, B.S. Johnathan Chittuluru University of Pennsylvania, B.A. Cornell University, B.A. Benjamin Hafensteiner Aaron Leconte University of Rochester, B.A. Carleton College, B.A. Timothy Newhouse Chung-Han Chu Colby College, B.A. National Taiwan University, Geoff Halvorsen Hyun Soo Lee B.S. University of Illinois, B.S. Pohang University, M.S. Andrea Nold Indiana University, B.S. Ryan Clark Sarah Hanson Sangyeul Lee University of California, University of California, University of California, Christine Nguyen San Diego, B.S. Berkeley, B.S. Berkeley, B.S. Boston College, B.S. 380 THE SCRIPPS RESEARCH INSTITUTE 2006

Daniel O’Malley Corin Slown STUDENTS IN BIOLOGY Melissa Dix Rice University, B.S. Yale University, B.S. AND BIOPHYSICS Pennsylvania State University, B.S. Adrian Ortiz Houchao Tao PROGRAMS University of Arizona, B.A. Shanghai Institute of Organic Jonas Dorn Chemistry, M.S. Swiss Federal Institute of Paresma Patel Parinaz Aliahmad Technology, M.S. University of North Carolina Mark Tichenor McGill University, B.S. at Chapel Hill, B.S. University of California, San Phillip Aoto Bao Duong Diego, B.S. University of California, Irvine, University of California, Francis Peters Los Angeles, B.A. University of New South Theresa Tiefenbrunn B.S. Wales, B.S. California Institute of Rena Astronomo Hunter Elliot Technology, B.S. Simon Fraser University, B.S. Colorado College, B.A. Anna Polk Stevens Institute of Jennifer Treweek Ann Atwood Kelly Flanagan Technology, B.S. California Institute of Brown University, B.S. Saint Louis University, B.S. Technology, B.S. Rajan Pragani Michael Barnes Amandeep Gakhal Goucher College, B. A. George Scott Tria University of Notre Dame, B.S. Simon Fraser University, B.S. Boston University, B.A. Duane Prasuhn Gira Bhabha Anna Galkin Carnegie Mellon University, Porino Va University of Chicago, B.A. Cornell University, B.S. B.S. University of Michigan, M.S. Sara Brownell Marin Gantner University of Puget Sound, Benjamin Pratt Hillary Van Anda Cornell University, B.S. B.S. Dartmouth College, B.A. Bryn Mawr College, A.B. Eric Brustad Sulagna Ghosh Stanislav Presolski Jianhua Wang Purdue University, B.S. University of Maryland, B.S. Colby College, B.A. University of Montreal, M.S. Anne Bunner Russell Gordley Jessica Raushel Sheng-Kai Wang Iowa State University, B.S. Swarthmore College, B.A. National Tsing Hua Texas A&M University, B.S. Russell Burge University, B.S. Daniel Groff Jeremy Richter Arizona State University, B.S. Albertson College, B.S. John Whitaker Butler University, B.S. Stuart Cahalan Washington State University, Jing Guo Tucker Roberts University of California, B.S. Peking University, B.S. Vanderbilt University, B.S. San Diego, B.S. Landon Whitby Joshua Chappie Peter Hawkins William Robertson University of Utah, B.S. Brandeis University, M.S. Bringham Young University, University of Colorado, B.A. B.S. SusAnn Winbush Stephen Chen Valentin Rodionov University of California, Christine Johanna Heideker Rice University, B.S. University of Maryland, M.S. Los Angeles, B.S. Julius Maximilians Universitaet Yee-Ting Chong Wuerzburg, Diplom David Sarlah Yang Xu Cornell University, B.A. University of Ljubljana, B.S. University of California, Davis, Dawn Hill B.S. Ryan Cirz Martin Schnermann University of Maryland, B.S. Pennsylvania State University, Colby College, B.A. Yang, Yu-Ying Ronald Hills B.S. National Chiao Tung Florida State University, B.S. Ian Seiple University, M.S. Ronald Coleman University of California, David Horning California State University, Berkeley, B.S. Isaac Yonemoto Fullerton, B.S. Harvard University, A.B. University of Chicago, B.S. Ryan Shenvi Amanda Hoyt Corey Dambacher Pennsylvania State University, Travis Young University of Washington, B.S. San Diego State University, B.S. Boston College, B.S. M.S. Julie Hsu Jun Shi Yu, Wayne University of California, Neekesh Dharia Wuhan University, B.S Portland State University, B.S. Berkeley, B.A. University of California, Sarah Siegel Andrea Zuhl San Diego School of Pei-hsin Hsu University of Virginia, B.S. Northwestern University, B.A. Medicine, B.S. Stanford University, M.S. THE SCRIPPS RESEARCH INSTITUTE 2006 381

Jason Jens Tracey Lincoln Sanjeev Ranade Kathryn Weinell Michigan State University, B.S. Williams College, B.A. Northeastern University, M.S. University of Colorado, B.A.

Audra Johnson Victor Mitch Luna William Ridgeway Laura White San Francisco State Stanford University, B.S. University of California, Emory University, B.S. University, B.S. Berkeley, B.A. Lndsey Macpherson Joann Wu Graham Johnson University of California, San Christopher Roth University of California, Johns Hopkins School of Diego, B.S. University of California, San Diego, B.S. Medicine, M.A. Ranjan Mannige Santa Barbara, B.S. Fei Xu Jeffrey Johnson University of Houston, B.S. Sophie Rozenzhak University of Science and Technology of China, B.S. University of Illinois, B.S. Andrea Manuell Wayne State University, M.S. Iowa State University, M.S. Craig Yoshioka Eiton Kaltgrad April Saunders University of Florida, B.S. University of California, Katherine Marcucci University of California, San Diego, B.S. Northwestern University, B.A. Davis, B.S. Jason Young University of Wisconsin, B.S. Piotr Kazmierczak Christopher Martin Erin Scherer University of Warsaw, M.Sc. Tufts University, B.A. University of Arkansas, B.S.

Donald Kerkow Alexandre Matov Gabriel Simon University of California, Technical University Varna, University of Pittsburgh, B.S. M.Sc. San Diego, B.S. Peter Smith Christopher Kimberlin Mayako Michino Purdue University, B.S. Georgia Institute of Technology, University of California, Sevil Sofueva M.S. Santa Barbara, B.S. International University Takashi Miyamoto Breman, B.Sc. Robert Kirchdoerfer University of Tokyo, B.S. University of Wisconsin, Bogdan Tanasa Madison, B.S. Crystal Moyer “Gr.T.Popa” University of University of Pittsburgh, B.S. Medicine and Pharmacy Heather Kiyomi Komori Iasi, M.D. Albertson College, B.S. Anke Mulder Purdue University, B.S. Shishi Tang Kristopher Koudelka University of Toronto, M.S. University of Wisconsin, Amber Murray River Falls, B.S. Massachusetts Institute of Megan Thielges Technology, B.S. Arizona State University, B.S. Sherman Ku Georgia Institute of Technology, Sherry Niessen Kathryn Thompson B.S. McGill University, M.S. Centenary College, B.S.

Jinny Kwong Bryan O’Neill Anne-Marie Turner University of California, University of San Diego, B.A. Wake Forest University, B.S. San Diego, B.S. Wendelien Oswald Lisa Tuttle Gabriel Lander Cornell University, B.S. University of Minnesota, M.S. State University of New York, Katherine Petrie José Vela Binghamton, B.S. University of Pittsburgh, B.S. California State University, Pick-Wei Lau John Picuri Northridge, B.A. University of Arizona, M.S. Cornell University, B.S. Sarah Voytek Daniel Leaman William Placzek Brown University, B.S. University of Pittsburgh, B.S. Washington University, B.A. Andrew Ward Joon Youb Lee Gunner Poplawsk Duke University, B.S. Seoul National University, M.S. University of Hamburg, Diplom Stuart Webb James Lim Randor Radakovits University of California, McGill University, B.S. Stockholm University, M.Sc. Santa Barbara, B.S. 382 THE SCRIPPS RESEARCH INSTITUTE 2006

membrane protein transporter involved in multidrug resistance. Also in Science, Bridget Carragher, Clint Potter, Jack Johnson and their colleagues reported the structure of an infectious phage, visualizing the capsid, the tightly spooled, packaged DNA and the tail machinery that senses when packaging is complete. In an article published in Nature, groups headed by Dr. Car- ragher, Dr. Potter, and William Balch described the underlying structure of the coat protein complex-II molecular cage that mediates intracellular transport. Also in Nature, Ron Milligan and colleagues described the mechanism of minus-end directed motion by a microtubule bound kinesin. In Nature Medicine, Mari Manchester’s group reported the use of a fluorescently labeled plant virus as a biosensor for vascular imaging. This novel methodology can effectively image the complete embryonic vasculature and highlight the process of angiogenesis in developing tumors. It was a banner year for Clare Waterman-Storer. Ronald A. Milligan, Ph.D. She received a number of honors and awards including the R.R. Bensley Award in Cell Biology from the American The Center for Integrative Association of Anatomists, the Director’s Pioneer Award from the National Institutes of Health, and an Established Molecular Biosciences Investigatorship from the American Heart Association. During November 2005, 41 students from the he Center for Integrative Molecular Biosciences United States, Canada, and Europe attended a 9-day (CIMBio) was created in 2002 to foster collabo- practical course in molecular microscopy run by the T rative research dedicated to elucidating the high- National Resource for Automated Molecular Microscopy, resolution structures, mechanisms of action, and in vivo our Biomedical Technology Resource Center sponsored dynamic behaviors of the cell’s molecular machines. by the National Center for Research Resources. Leading CIMBio now houses 20 research groups representing scientists in the field participated in lectures, research disciplines including chemistry, cell and molecular biol- seminars, and practical sessions that covered the theory ogy, electron microscopy, x-ray crystallography, advanced and practice of electron microscopy and image analysis. light microscopy, computational biology, and technol- The formal lectures and research seminars attracted ogy development. This year Floyd Romesberg, Philip many attendees from the local scientific community. In Dawson, and Anette Schneemann relocated their research all, 27 instructors and 18 assistants were involved in groups here, occupying new laboratories on the second the course. Financial support for the course was pro- floor of the building and adding strengths in chemistry vided by the National Center for Research Resources, and structural biology. The Agouron Institute, FEI Company, Gatan Inc., Pro- Our faculty members made a number of noteworthy tochips Inc., Tietz Video and Image Processing Systems, scientific advances during the past year, a fact that was and Scripps Research. reflected in the number of papers published in top-rank- The fourth in a series of training workshops on soft- ing scientific journals. The following list highlights some ware for automated molecular microscopy was held dur- of this groundbreaking science. In the journal Cell, ing February 2006. Representatives from 5 institutions Clare Waterman-Storer and her coworkers described (Oxford; Purdue; Brandeis; University of California, fundamental dynamic molecular relationships that under- San Diego; and State University of New York) attended lie rapid cell migration over substrates. Geoffrey Chang and received intensive training on installation and use and members of his laboratory published a paper in of software developed at the National Resource for Science describing the high-resolution structure of a Automated Molecular Microscopy. THE SCRIPPS RESEARCH INSTITUTE 2006 383

In the coming year, Scripps Research will be the Gene to 3D Structure in the Kuhn and Stevens Labo- first research institution worldwide to receive a novel ratories (http://www.atcg3d.org). compact synchrotron light source. This Scripps Cam- These activities and successes during the past year pus Synchrotron, to be housed in CIMBio, will signifi- highlight the collaborative, interdisciplinary nature of cantly accelerate the pace of determining challenging the science being carried out at CIMBio. The enthusiasm protein structures (for example, membrane proteins of our faculty, staff, fellows, and students and their com- and large macromolecular complexes) and structure- mitment to our collaborative mission are also evident at based drug design by enabling real-time experimental the standing-room-only biweekly forums—short seminars evaluation using high intensity, tunable x-rays on cam- designed to promote interdisciplinary interactions. pus. This research is part of the new technology developments of the Accelerated Technologies Center for

land, Sweden, Mexico, and Italy in such disciplines as neurology, immunology, chemistry, molecular biology, and endocrinology to study neurologic disorders. The institute also funds the Helen Dorris Fellow in Schizophrenia, a named fellowship position for a postdoctoral researcher to study aspects of schizophrenia and depression from the neurobiological perspectives. The current fellow is Lisa Sharkey. The visiting professors at the institute in 2005 were the noted electro- physiologist and member of the French Academy of Sciences Henri Korn from the Pasteur Institute in Paris, France; noted pharmacologist and member of the Royal Swedish Academy of Sciences Lars Terenius from the Karolinska Institutet in Stockholm, Sweden; and molecular immunologist Hermann Gram and molecular pharmacologist Daniel Hoyer from Basel, Switzerland. In 2006, the faculty of the institute expanded beyond the founding faculty members with the election of 3 talented young faculty members: Dorian McGavern, Tamas Bartfai, Ph.D. Eric Zorrilla, and Marisa Roberto.

Harold L. Dorris Neurological Research Institute

Tamas Bartfai, Ph.D., Director

ajor depression and schizophrenia affect millions of people. Treatment of these conditions requires M an understanding of their underpinnings, and this need is addressed by The Harold L. Dorris Neurologi- cal Research Institute. Founded in 1999 as the result of a long-term $10 million commitment by Helen L. Dorris through the Harold L. Dorris Foundation, named in her brother’s honor, the institute has attracted an international cadre of scientists from France, Switzer- 384 THE SCRIPPS RESEARCH INSTITUTE 2006

nervous system, in particular, the postgenome era holds the potential to deliver groundbreaking new medicines for previously intractable psychiatric disorders including anxiety, depression, and schizophrenia. However, in order to realize this goal, a new breed of research institute is needed that cultivates cross talk among many experimental disciplines. Indeed, unraveling the complexities of the human brain and behavior can only be achieved by bringing together scientists from diverse backgrounds and expertise, including chemistry, physics, genetics, and behavior. The Helen L. Dorris Child and Adolescent Neuro- Psychiatric Disorder Institute was established with a generous gift from mental health advocate and San Diego State University professor emeritus Helen L. Dorris. Her interest in mental health advocacy led her to provide the funding to establish this institute, which has a strong emphasis on interdisciplinary approaches to studies of Ben Cravatt, Ph.D. neurologic and psychiatric disorders. Specifically, the aim of scientists at the institute is Helen L. Dorris Child and to uncover the pathologic basis of mental disorders and to develop therapies for these disorders. In the past 3 Adolescent Neuro-Psychiatric years, several talented investigators have been recruited Disorder Institute to join the institute. Together, these investigators are addressing many of the most challenging problems fac- he sequencing of the human genome promises ing contemporary molecular and behavioral neuroscience. to propel humans into the age of molecular medi- Their research promises to uncover fundamental mecha- T cine, where complex diseases are diagnosed and nisms for brain function and to reveal novel strategies treated in a patient- and target-specific manner. For the and targets for the treatment of nervous system disorders.

The Institute for Childhood and Neglected Diseases

he Institute for Childhood and Neglected Diseases (ICND) was established to apply cutting-edge T research to understand the basic mechanisms underlying diseases of childhood and orphan diseases that lack efficacious treatments. Diseases in both these categories often affect populations in developing countries, where the health infrastructure may be too poor to support major research efforts on these problems. Examples of such diseases include malaria, epilepsy, mental retardation, cystic fibrosis, chronic pain, and sleep disorders. The human and economic costs of these diseases are staggering. According to the World Health Organization, each year, the microorganism that causes malaria infects 300 million persons, and the Steve Kay, Ph.D. THE SCRIPPS RESEARCH INSTITUTE 2006 385 disease kills approximately 1 million persons. About and to devise treatments for these maladies. ICND sci- 90% of the people who have malaria are in Africa, entists plan to systematically study not only the genes where the annual costs associated with the disease associated with these abnormalities but also the inter- are $12 billion. Malaria is the leading cause of child- actions between the genes in living model systems. hood mortality in African countries. ICND researchers have already been recognized by Epilepsy is another widespread and costly disease. the international scientific community during the first 5 It affects about 2.3 million persons in the United States years of the institute’s existence. In December of 2002, and accounts for $12.5 billion in medical costs and 3 of the highly prized Top Ten Breakthroughs of the Year reduced productivity each year. of Science magazine were results produced by ICND Mental retardation is another condition that affects researchers working on the malarial genome, mecha- children everywhere; about 1% of American children nisms of pain perception, and processes important for 3–10 years old are mentally retarded. During the sleep disorders and seasonal depression. This remark- 1995–1996 school year, about 600,000 6- to 21- able level of achievement speaks to the investment year-olds with mental retardation in the United States made in these research areas and bodes well for fur- received special educational services, at a cost of about ther rapid progress in the near future. $3.3 billion. Housed in a state-of-the-art, 54,000-square-foot Faculty Areas of Research building on the east side of the Scripps Research cam- Steve A. Kay, Ph.D. Molecular mechanisms of circadian pus, the ICND is a focus group within Scripps Research rhythms and sleep disorders, genetics of anxiety, novel targets in neu- for young scientists who are working in areas relevant rodegenerative diseases to the ICND mission. The concept of the ICND grew William E. Balch, Ph.D. Protein trafficking and the molecular out of conversations in 1996 and 1997 among Scripps basis for the hereditary childhood Research president Richard Lerner; John Moores, who disease cystic fibrosis was interested in supporting research on illnesses that Kristin Baldwin, Ph.D. Molecular biology of the sense of affect people in developing countries; and the brothers smell, genetic mechanisms govern- Bernard and Marc Chase, who were interested in sup- ing neural circuit development in porting research on childhood diseases. John and the olfactory system and cortex Rebecca Moores, Bill Bauce, and other automobile Shelley Halpain, Ph.D. Organization and function of the neu- enthusiasts donated a number of vintage automobiles, ronal cytoskeleton, mechanisms underlying potential treatments for which were auctioned to support the ICND. The Moores Alzheimer’s disease and repair of went on to contribute a valuable coin collection, as neuronal damage after trauma well as pledging $5 million to be awarded over 5 years. Mark Mayford, Ph.D. Molecular basis of cognitive function, The Human Genome Project has led to a deeper including learning and memory dis- understanding than ever before of the mechanisms abilities and mental retardation underlying human disease. The ability to study the Ulrich Müller, Ph.D. Molecular cell biology of mechanosensory draft mouse and human genomes in parallel is providing perception and childhood deafness an unprecedented opportunity to create a road map for Ardem Patapoutian, Ph.D. Ion channels and receptors involved in assigning a physiologic function to all of the 35,000- nociception and neuropathic pain 45,000 human genes. This formidable challenge requires Elizabeth Winzeler, Ph.D.Functional genomic approaches to complex multidisciplinary approaches that allow scien- identifying targets in Plasmodium, the parasite that causes malaria tists to create and implement the most powerful research tools available. Investigators at the ICND use genomics, proteomics, and advanced microscopic imaging technologies; develop many novel transgenic animal models; and aggressively apply these technologies in an effort to understand the mechanisms of action of a variety of diseases and conditions—malaria, mental retardation, neurodegenerative diseases, neuropathic pain, deafness, sleep disorders, migraines, and epilepsy, for example— 386 THE SCRIPPS RESEARCH INSTITUTE 2006

15 postdoctoral scholars for professional development coursework. Typical courses included certificate programs like Drug Discovery and Regulatory Affairs held at the University of California, San Diego, Extension. Such formal coursework provided our postdoctoral candidates with an important resume booster and gave them a completive edge in an increasingly competitive job market. This year’s social calendar was again packed with many entertaining events, including trips to the SOCIETY OF FELLOWS EXECUTIVE COMMITTEE: Left to right: back row: Chris Ramsey, George Nicola, Neil Hime, LA County Museum of Art and Universal Studios, a Anne Bellon, Kelly-Anne Purton, Justin Carlson, Ryan Wheeler; whale watching excursion, an historical tour of San front row: Ralph Pantophlet, Adam Mullick, Ron Nepomuceno. Diego, and our annual Big Bear ski trip. Happy hours were held throughout the year to promote interaction among scientists at Scripps Research and those at Society of Fellows nearby science institutions. This year’s Hawaiian-themed summer bash was held at Canes Bar and Grill. In addi- n its 44th year, the Society of Fellows took pride in tion, a sunset yacht cruise around San Diego Bay was maintaining its mission to serve the scientific com- enjoyed by all who attended. Other notable social events munity at Scripps Research, with particular emphasis I this year included a premier showing of the movie The on enhancing the trainee experience of junior scien- DaVinci Code, a day at the Del Mar races, and a World tists. We enjoyed a great year of bringing graduate Cup soccer tipping contest. students, postdoctoral fellows, and faculty members Finally, at the annual spring vendor show, the soci- together in both professional and social arenas. ety played host to approximately 100 scientific vendors, Our Distinguished Lecturer Series brought several who displayed their latest scientific equipment and prominent scientists to lecture and, more importantly, technology. to interact with junior scientists. The society is thankful The Society of Fellows executive committee expresses to the speakers for their excellent presentations: Claire its sincere gratitude to the Office of the President and Fraser, Paul Nurse, and Stanley Plotkin. Postdoctoral Services at Scripps Research for enthusi- The Society also sponsored a wide range of career development workshops and seminars, most notably sev- astic and continued support of the society’s activities. eral postdoctoral career events including Jorge Cham (The Power of Procrastination), Jean-luc Doumont Executive Committee 2005-2006 (Making the Most of Your Presentation), and a grantwrit- Officers ing workshop lead by Luc Teyton. These events were Adam Mullick President sponsored in partnership with Ryan Wheeler, manager Ralph Pantophlet Vice President Florence Brunel Treasurer of the newly formed Office of Postdoctoral Services, which Kelly-Anne Purton Social Chair has greatly expanded our reach by providing help in Anne Bellon Social Co-Chair organizing events. We acknowledge Ryan’s outstanding Neil Hime Distinguished Lecturer Series Chair assistance and appreciate his commitment to fulfilling Reshma Jagasia/ Career Development Committee Chairs the society’s aims. Justin Carlson The Fall Research Symposium was organized to draw Tricia Burdo Vendor Show Chair postdoctoral scholars, graduate students, and faculty Ryan Wheeler Postdoctoral Services Office Liaison Anne Bunner/ Network for Women in Science Liaisons members together in an informal setting. The event, held Jilla Sabeti in the Beckman Galleria, featured 52 poster presenta- Ron Nepomuceno Website Manager tions covering research projects from all departments. Non-Officers With a generous contribution from our Human Ben Croker Chris Ramsey Resources department, we were able to initiate a post- Joerg Hinnerwisch BinQing Wei doctoral tuition reimbursement program in the amount George Nicola Scott Westenberger of $400 to each fellow. Thus far, the program has funded Vandana Ramachandran THE SCRIPPS RESEARCH INSTITUTE 2006 387

Author Index Aschrafi, A. 34 Belvindrah, R. 459 Breton, G. 340 Ashe, M. 224 Benedict, J. 307 Brignole, E. III 24 Abagyan, R. 56, 199 Ashkenasy, G. 75 Benkovic, S.J. 183 Brik, A. 94 Abalos, G. 148 Ashkenasy, N. 75 Bennett, C. 94 Brinson, D.C. 245 Abdulla, B. 278 Ashley, J. 78 Bennett, K. 141 Brogan, A. 78 Abelson, D.M. 135 Asmar-Rovira, G.A. 173 Benning, N. 301 Brooks, C.L. III 183, 193, 218 Abola, E. 37 Astronomo, R.D. 108 Benoit, R.R. 173 Brooks, D. 295 Abola, E.E. 173 Asturias, F.J. 24 Ben-Shir, I. 212 Brooun, A. 37, 173 Abraham, S. 214 Atkins, A. 330 Benson, K. 104 Brown, J. 24 Abu-Jarour, R. 72 Atteberry, B. 141 Ben-Tal, N. 56 Brown, S. 133 Adair, B. 56, 291 Augustyniak, W. 177 Benton, H.P. 201 Browning, S. 356 Adams, H.P. 251 Aujila, H. 319 Berezhna, S.Y. 189 Bruce, R. 37 Adams, M.A. 160 Aur, R. 291 Berger, M. 104 Brudler, R.M. 160 Adusumalli, M. 308 Ayad, N.G. 351, 358 Bernard-Trifilo, J.A. 137 Bruijnzeel, A. 307 Agnelli, F. 202 Baccala, R. 142 Berndt, C. 78 Brunel, F. 30 Agneta, C. 302 Bacconi, A. 29 Berton, F. 314 Bruning, J. 352 Aguilar, E. 33 Bacher, J. 203 Bespalov, A. 307 Brustad, E. 88 Aguilar de Diaz, H.E. 21 Bachovchin, D.A. 86 Beuck, C. 202 Bubeck, A. 34 Aguilar-Sino, R.O. 108 Bader, A. 276 Beuscher, A. Bu, L. 193 Ahamed, J. 134 Badie-Mahdavi, H. 321 Beutler, B. 104 Buchmeier, M.J. 56, 291–293 Ahlquist, M. 89 Bae, S.H. 183 Beutler, E. 24, 270 Buffkins, K. 308 Ahmad, M. 208 Bai, D. 276 Bharati, I.S. 50 Bulger, P. 83 Ahmed, S. 304 Bai, H. 43 Bhattacharjee, G. 111 Bunker, K. 70 Ahn, C. 140 Baik, A. 43 Bieschke, J. 81 Bunner, A. 202 Ahn, E.-Y. 278 Bailey, A.O. 55 Biggs, J.A. 139 Burge, R. 178 Aït-Azzouzene, D. 128, 142 Baillargeon, P. 365 Biggs, J.R. 278 Burns, N.Z. 69 Ajami, D. 15 Bajo, M. 317 Bilbe, G. 322 Burrer,R.J. 291, 292 Albertshofer, K. 208 Bajova, H. 303, 309 Birgbauer, E. 232 Busby, S.A. 341 Alexander, J. 19 Baker, C.A. 356 Birkenfeld, J. 106 Buset, E. 295 Alexandrov, A.I. 173 Baker, K. 56 Biros, S. 15 Burtoloso, A. 83 Aliahmad, P. 119 Balch, W.E. 25 Bisson, W. 199 Burton, D.R. 108, 109, 135, Alirezaei, M. 294 Baldwin, K. 27 Bjoras, M. 167 150 Aller, S. 169 Bandell, M. 46 Blais, V. 227 Burnett, R. 186 Allin, L.K. 233 Banerjee, M. 216 Blixt, O. 233 Bushey, M. 88 Allison, B.Z. 311 Banin, E. 33 Bobardt, M. 113, 114 Butterfield, S. 11,15 Almeida, B. 232 Baptista, M.A. 319 Boddy, M.N. 227 Buxbaum, J.N. 279 Almeida, M. 177 Baran, P.S. 69 Boehr, D. 183 Bychkova, V. 180 Altieri, K. 56 Barbas, C.F. III 208 Boga, S.B. 70 Cabral, K.M.S. 260 Alvarez, D. 186 Barber-Singh, J. 250 Boger, D.L. 70, 300 Cahalan, D. 133 Alvarez, L. 310 Barnes, D. 41 Bohl, B.P. 106 Cai, G. 24 Alves, J. 214 Barnes, M. 104 Bohorov, O.V. 233 Calabrese, B. 36 Ambasudhan, R. 72 Barnett, F. 33 Boitano, A. 88 Calarese, D.A. 108, 160 Ambrus-Aikelin, G. 34 Barr, A. 307 Bokoch, G.M. 106 Calderon, E.M. 205 Amelio, A.L. 351 Barrett, E. 11, 15 Boldt, G. 78 Caldwell-Busby, J.A. 365 Amitai, N. 307 Barnett, K.S. 265 Bongiorni, C. 254 Cameron, M. 343 Ampudia, J. 114 Baronciani, L. 262 Bonham, K. 127 Campbell, D. 264 An, C. 193 Barondeau, D.P. 160, 167 Borelli, I. 193, 218 Canciani, M.T. 262 An, J. 199 Barr, A. 321 Boren, B. 89 Canonigo, V. 314 An, N. 301 Barros, C. 45 Borgstrom, P. 111 Cantin, G. 55 An,Y. 25, 160 Barrowman, P.A. 293 Borrow, P. 295 Cantu, C. 141 Andersson,T. 346 Bartel, R. 264 Borst, P. 357 Capul, A. 295, 298 Andersson-Sand, H. 233 Bartfai, T. 11, 289, 317, 321, Bosco, D.A. 81 Cardenas, J. 36 Anliker, B. 232 322 Bosco, N. 114 Cardoso, R.M.F. 108 ,160 Annalora, A. 170, 221 Baskerville, C. 222 Bostick, D. 193 Carella, A. 11,15 Aoyagi, M. 165 Bates, R. 345 Botten, J.W. 293 Carey, J. 112 Apon, J. 201 Baudry, A. 340 Bouma, B.N. 259, 260 Carlson, J.E. 160 Appadurai, S. 232 Bauer, S. 212 Boutrel, B. 307 Carlton, D. 175 Arandjelovic, S. 111 Baumert, T. 268 Bower, K. 276 Carmel, A. 202 Archer,H.M. 173 Bautchek, R. 33 Bowley, D.R. 108 Carney, P.J. 160 Ardi, V. 47 Beck, A. 201 Boyapati, A. 278 Carragher, B. 28 Arends, M. 304 Bednenko, J. 34 Boyd, J. 294 Cartier, A. 271 Armen, R. 193 Beebe, K. 203 Boyman, O. 140 Case, D.A. 178, 189, 191 Arnaout, M.A. 56 Beierle, J. 75 Bracey, M.H. 173 Cassany, A. 34 Arndt, J.W. 173 Beis, K. 160 Brady, N. 271 Cassidy, M. 89 Arseniyadis, S. 83 Beligni, M. 41 Braga, J. 36 Castellino, F.J. 43 Arvai, A.S. 160, 167 Bell, A. 19 Braun, D. 193 Catz, S.D. 247 Asabe, S. 267 Bellamy, A.R. 56 Breakwell, L. 292 Cauvi, D. 282 Asahara, H. 246 Bellon, A. 148 Brennan, M. 304, 320 Cauvi, G. 282 Asawapornmongkul, L. 208 Belting, M. 111 Brenzovich, W. 83 Cavanagh, J. 254 388 THE SCRIPPS RESEARCH INSTITUTE 2006

Cavett, V. 365 Chung, J. 178, 180, 183, 267 Dabernat, S. 136 Diaz, A. 254 Censullo, A. 217 Churchill, M. 30 Da Costa, C.P. 205 Dickerson, T. 78 Ceredig, R. 114 Ciccocioppo, R. 319 da Silva Correia, J. 146 Dietz, D. 136 Chaban, Y. 24 Cirulli, V. 256 Dagneau, P. 83 Ding, S. 72 Chahwan, C. 225 Cirz, R.T. 86 Dahlgren, C. 346 Dirksen, A. 30 Chai, Q. 173 Clamme, J.P. 189 Dai, S.Y. 341 Domingo, E. 295 Chalmers, M.J. 341 Clark, P. 37 Dai, X. 160 Don, A. 133 Chamero, P. 50 Clark, R. 70 Dale, T.J. 15 Dong, M.Q. 55 Chang, G. 169 Clayton, T. 175 Dallakyan, S. 195 Donners, H. 108 Chang, J.Y. 92 Cleary, M. 136 Dambacher, J. 92 Dorn, J. 29 Chang, S. 89 Clemente, R. 295, 299 Daneholt, B. 19 Dorrell, M. 33 Chang, S.Y. 141 Cleveland, J.L. 350 Dang, I. 276 Dovey, C. 225 Chang, Y. 274 Cociorva, D. 55 Dang, T. 37 Dowell, A. 306 Chang, Z.-F. 106 Coito, C. 358 Daniels, M. 114 Dresios, J. 331 Chao, J. 202 Colby, D. 70 Daniels, M.J. 56 Drobes, D. 308 Chapados, B.R. 167 Cole, K. 83 Danielson, P.E. 231 Druzina, Z. 203 Chapman, E. 184 Cole, M. 304 Danuser, G. 29 Dryden, K.A. 56 Chapman, J. 133 Coleman, R. 37 Darout, E. 345 Du, L.-L. 225 Chappell, S.A. 331 Collins, B.E. 233 Das, S. 214 Du, X. 104 Chappie, J. 44, 49 Columbus, L. 177 Datta, K. 34 Dubin, A. 46, 232 Chartron, J. 170 Colwell, C.W. 245 Datta, S. 136 Duckett, D. 343 Chase, P. 365 Conkright, M.D. 351 Daudenarde, S. 205 Dunetz, J. 345 Chatterji, A. 216, 217 Conkright-Johnson, J. 25 Davis, C. 322 Duong, B. 128 Chatterji, U. 113, 114 Connelly, S. 160 Davis, S.A. 318 Dupradeau, F. 189 Chavochi, A. 75 Conner, S.D. 49 Dawson, P.E. 30, 145, 150 Dupuy, J. 37 Cheli, Y. 262 Conti, B. 322 Dayas, C.V. 319 Duquette, M. 227 Cheltsov, A. 199 Conti, F. 45 De, S. 214 Durrans, A. 51 Chen, A. 169, 320 Converso, A. 83 de Bruin, R. 224 Dwek, R.A. 108 Chen, B. 34 Cook, R.T. 116 de Graan, P.N.E. 303 Dyson, H.J. 178, 180, 183 Chen, C. 25, 37 Coombs, K. 56 de la Torre, J.C. 295, 298, 299 Earley, T. 46 Chen, E. 55 Coon, S. 37 De Lamo Marin, S. 78 Eam, B. 301 Chen, E.I. 258 Coppinger, J. 55 de Lecea, L. 229, 317 Eberhardy, S. 208 Chen, I. 20 Corey, L. 302 De Noronha, R. 83 Edelman, D.B. 330 Chen, J. 83, 116, 193 Cornell, C. 301 de Parseval, A.P. 113, 220 Edelman, G.M. 328, 330–332 Chen, N. 340 Cornillez-Ty, C.T. 292 De Riccardis, F. 73 Edelmann, K. 295, 296 Chen, S. 88, 330 Corper, A.L. 160 de Rozieres, S. 220, 265 Edgcomb, S. 202 Chen, X.L. 137 Cottell, J. 70 Dean, S. 94 Edgington, T.S. 111, 112 Chen,Y. 169, 171 Cottone, P. 320 DeBaillie, A. 345 Edmonds, D. 83 Chen, Y.H. 94 Cottrell, J.W. 205 Debler, E.W. 160 Ehlers, C.L. 302, 316 Chen, Y.P. 92 Coveney, K. 308 DeCathlineau, A.M. 106 Eichinger, S. 259 Chen, Y.-T. 345 Craig, L. 167 Deguchi, H. 259 Eidenschenk, C. 104 Chen, Z. 214, 216 Crain, K. 270 Dehmelt, L. 36 Eisenbraun, M.D. Chen, Z.Y. 85 Cramer, T. 261 Del Papa, F. 254 Ekholm-Reed, S. 222 Cheng, A. 28, 56 Cravatt, B.F. 19 del Zoppo, G.J. 257 Elias, D.J. 259 Cheng, G. 267, 268 Crawford, E. 304 Delacruz, J.P. 281 Elias, Y. 175 Cheng, Z. 148 Crawford, J. 83 Delahunty, C. 55 Elder, J.H. 113, 220, 265 Cherezov, V.G. 173 Crawford, M. 106 Deller, M. 175 El-Kalay, M. 33 Cherqui, S. 264 Crean, R.D. 308, 318 Delorme, V. 106 Elliott, G. 70 Cherrier, M. 112 Cremeens, M.E. 86 DeMartino, J. 70 Ellis, B.A. 247 Chi, A. 137 Crisa, L. 256, 265 DeMartino, M.P. 69 El-Sheikh, A. 111 Chien, E. 173 Crocker, S. 301 Demczyk, C. 356 Elsliger, M.-A. 160 Chin, J.K. 86 Croker, B. 104 Dendle, M.T.A. 81 Elsner, J. 70 Chintalapati, R.M. 142 Cross, T.H. 160, 165, 167 Deng, Q. 228 Eltepu, L. 92 Chisari, F.V. 56, 262, 267–269 Crossin, K.L. 330 Deniz, A.A. 189 Emre, N. 72 Chitnis, S. 51 Crowley, B. 70 Denley, A. 276 Engstrom, P. 346 Cho, J. 303, 309 Crowley, M. 189 Densley, W.L. 160 Eschenmoser, A. 73 Choe, J.-W. 160 Crowley, M.F. 193 Denton, R. 83 Escher, T. 308 Choi, E. 34 Crozat, K. 104 DerMardirossian, C. 106 Espana, F. 259 Chopp, M. 259 Cruite, J. 148 Dervan, P.B. 186 Espinoza, C.R. 112 Chou, C.-J. 186 Cruz, J. 50 Deryugina, E. 47 Estrada, A. 83 Chow, S. 303 Cryan, J. 307 deSchöpke, A. 340 Estrada, M. 37 Chowdari, N.S. 208 Cubitt, B. 298, 299 Desnues, B. 120 Eubanks, L. 78 Chowdhury, P. 44 Cui, Q. 189 Desplats, P.A. 230 Evans, R. 178 Chrencik, J. 37 Culyba, E. 81 Desponts, C. 72 Ewalt, K. 203 Chu, J. 340 Cunningham, B.A. 330 Destito, G. 37 Ezquerra-Ruiz, L. 274 Chuang, L.-C. 222 Curry, D. 19 Deuel, T.F. 274 Ezzili, C. 70 Chuang, T.-H. 146 Curtiss, L.K. 109, 145 Develioglu, L. 141 Faghihi, M.A. 346 Chun, J. 232 D’Haeze, W. 81 Dhaka, A. 46 Falke, S. 44 Chung, C. 78, 365 D’Lima, D. 245 Diamant, J. 308 Fan, L. 167 THE SCRIPPS RESEARCH INSTITUTE 2006 389

Fang, C. 83 Furihata, K. 262 Grant, A. 89 Harmey, D. 351 Fang, Q. 122 Funk, C. 304 Grant, Y. 304, 320 Harris, D.A. 86 Faraoni, R. 83 Gabriel, R. 280 Grbic, J. 88 Harris, J.L. 214 Farkas, M. 186 Gaillard, P.-H. 225 Greenberg, H.B. 56 Harris, K. 201 Farré, E. 340 Gairin, J.E. 295 Greenberg, W. 94 Harris, R. 195 Fath, T. 32 Gakhal, A.K. 108 Greenman, N. 178 Harrison, S. 83 Fazilleau, N.R. 125 Gale, A.J. 261 Greenwell, T. 304 Hart, M.K. 135 Fearns, C. 146 Galkan, A. 276 Griffin, J.H. 259 Hartley, O. 126 Federici, A.B. 262 Gallay, P.A. 113, 114 Griffin, K.J. 246 Hassenpflug, W. 258 Fedor, M.J. 205 Gallo, G. 279 Griffin, P.R. 341, 357, 364 Haudenschild, D. 245 Fee, J.A. 171,, 191 Gámez, A. 173 Griffith, M.T. 173 Havran, W.L. 117 Feeney, A.J. 112 Gambin, Y. 189 Grittini, C. 173 Havstad, J.W. 302 Feistritzer, C. 132 Ganser, B. 56 Groff, D. 88 Hayashi, M. 121 Fekete, É. 320 Gao, J. 81 Groschel, B. 219 Haynes, M. 117 Felding-Habermann, B.F. 258 Gao, M.-Y. 81 Grover, R.K. 92 Hays A.-M. 221 Feldman, A. 89 Garcin-Hosfield, E.D. 165 Grünewald, J. 88 Hazen, B. 36 Felitsky, D. 180 Gardel, M.L. 53 Gruol, D.L. 303, 309 Hazen, S.P. 340 Fellman, D. 28 Gardel, S. 232 Guaderrama, M. 224 He, X. 169 Feng, A.C. 251 Garfunkel, J. 70 Guan, T. 34 He, Y. 368 Fenton, W.A. 184 Garidou, L. 295, 299 Guerrero, C.A. 69 Head, S. 230, 262, 264 Ferguson, S. 160 Gaskill, P. 294 Guerrero, M. 73 Heaslet, H. 170, 220, 265 Fernández, J.A. 259 Gastaminza, P. 267, 268 Guidotti, L.G. 269 Hedlund, P.B. 231 Ferreon, A.C.M. 189 Gavin, A. 128 Gulati, A. 344 Heeb, M.J. 260 Ferreon, J. 178 Gavin, J. 264 Gunderson, A. 295, 300 Heilig, M. 319 Feuer, R. 301. Gelbart, T. 270 Guo, F. 122, 214 Hein, J. 89 Ficht, S. 94 George, O. 304 Guo, J. 88, 208 Helfer, A. 340 Fields, B. 27 Gerace, L. 34 Gupton, S.L. 53 Hemmers, S. 126 Fine, C. 136 Geralt, M. 177 Gurkan, C. 25 Hennig, M. 202 Finerman, G. 302, 307 Gerber, A. 271 Gustafsson, Å.B. 271 Henriksen, S.J. 231 Finn, M.G. 74, 172 Gertsman, I. 216 Guvench, O. 193 Henry, A.A. 86 Fischer, K.M. 265 Getzoff, E.D. 165 Guy, R. 94 Henry, B. 307 Fischer, R.S. 32 Geyer, M.A. 231 Gymnopoulos, M. 276 Henry, R. 41 Fish, K. 307 Ghadiri, M.R. 75, 268 Haas, C. 11, 15 Henson, K. 54 Fitch, M.J. 51 Ghoneim, O. 85 Hafensteiner, B.D. 69 Henze, M. 222 Flanagan, J. 270 Ghosh, S. 27 Hagihara, K. 314 Herman, D. 186 Flanagan, K. 50 Ghozland, S. 304 Hagiwara, K. 299 Herr, D. 232 Flood, C. 109 Giang, E. 108 Hahm, B. 295 Herradon, G. 274 Flynn, C. 294 Gianneschi, N. 75 Hahm, H.S. 72 Hessell, A.J. 108 Fokin, V.V. 89 Gianni, D. 106 Hall, M.O. 260 Hewel, J. 55, 264 Foos, G.E. 265 Gianniello, F. 262 Hallenbeck, J. 257 Hicks, J. 345 Forsyth, J.S. 258 Gibe, R. 83 Halpain, S. 36 Hilcove, S. 72 Foss, K.L. 359 Giffin, M.J. 220, 265 Halvorsen, G. 345 Hill, D.M. 127 Foss, T.R. 81 Gil-Lamaignere, C. 86 Hamacher-Brady, A. 271 Hill, N. 136 Foster, S. 37 Gilder, D.A. 302 Hamasaki, A. 70 Hills R. 193 Fotsing, J. 89 Gill, J. 187 Hamilton, E. 340 Hime, N.J. 109 Fowler, B. 106 Gillet, A. 195 Hamilton, S.E. 207 Hinnerwisch, J. 184 Fowler, D. 25, 81 Gilmartin, T. 230 Hamilton-Williams, E. 139 Hitomi, C. 165, 167 Fowler, V.M. 32 Gilpin, N. 304 Han, B.-K. 224 Hitomi, K. 165, 167 Fox, H.S. 294 Gingles, N. 86 Han, B.W. 160 Hixon, M. 78 Francesconi, W. 314 Glazer, E.C. 221 Han, G.W. 160 Hoang, L. 201 Franco, S. 45 Gleason, J. 308 Han, J. 116, 281 Hoch, J.A. 252–255 Fraser, R. 89 Go, E. 201 Han, S. 233 Hock, M. 36 Frausto, R. 301 Goergen, C. 114 Han, W. 70 Hodder, P. 365 Frederick, M. 83 Gogol, E. 44 Han, W.-G. 191 Hoebe, K. 104 Freestone, M. 83 Gombosuren, N. 11, 15 Handa, M. 345 Hoffman, J. 233 Freigang, S. 141 Gonias, S.L. 111 Hanekamp, S. 33 Hogg, P.J. 134 Friedlander, M. 32, 134 Gonzalez, B. 208 Hangartner, L. 108 Holmberg, K. 114 Friedlander, S.F. 33 Gonzalez, K.N. 332 Hankel, S. 45 Holmgren, A 183 Friedman, J.S. 280 Gonzalez, M.J. 37 Hanneken, A. 248 Holt, M. 141 Friske, L. 279 Gonzalez-Cabrera, P. 133 Hansen, F. 30 Hom, D. 306 Froestl, W. 307 Gonzalez-Quintial, R. 142 Hanson, D.A. 137 Hong, S. 70 Fu, G. 114 Goodin, D.B. 221 Hanson, M.A. 173 Hong, Z. 94 Fu, Y. 81 Goodsell, D.S. 195 Hanson, S. 94 Hooley, R.J. 11, 15 Fuchs, J. 70 Gordley, R. 208 Haq, N. 70 Hopkins, T. 345 Fusco, M.L. 135 Gosink, M. 368 Haraldsson, M.K. 142 Horne, D. 70 Fusio, M. 94 Gottesfeld, J.M. 178, 186 Harger, J.W. 205 Horne, W.S. 75 Fukushima, T. 252 Gottlieb, R.A. 271 Harismendy, O. 332 Horning, D.P. 207 Fuller, R. 208 Goularte, O. 119 Harkins, S. 301 Horst, R. 177 Fung, M. 119 Grandl, J. 46 Harless, J. 56 Horvath, S. 264 390 THE SCRIPPS RESEARCH INSTITUTE 2006

Horwich, A.L. 184 Jin, W. 70 Kimball, F.S. 70 Lam, P. 199 Hou, S. 137 Jo, E. 133 Kimber, T. 304 Lambert, W. 345 Hoyer, J.D. 322 Johns, G.C. 207 Kimberlin, C.R. 135 Lambolez, B. 315 Hsu, J. 72 Johnson, A. 33, 83 Kingsbury, M. 232 Lamoureux, J. 112 Hsu, M.H. 246 Johnson, E.F. 202, 246 Kinney, J. 321 Landais, E. 141 Hsu, T.-L. 94 Johnson H. 175 Kirkland, T. 145 Lander, G. 28, 216 Hu, H. 46 Johnson, J. 54 Kislukhin, A. 83 Landes, M. 178 Hu, K. 53 Johnson, J.E., Jr. 56, 216–218 Kita, K. 53 Langley, E. 227 Hu, Y. 195 Johnson, J.L. 247, 248 Knaus, U.G. 120 Lanigan, C. 264, 294 Hua, H. 136 Johnson, J.R. 55 Kocerha, J. 346 Lanman, J. 216 Hua, Y. 56 Johnson, M. 177 Koculi, E. 184 Lanver, A. 83 Huang, C. 271 Johnson, S.M. 81 Koehler, J. 36 LaPointe, P. 25 Huang, I. 70 Jonkman, S. 307 Koehntop, B.B. 178 Lasmézas, C.I. 357 Huang, R. 216 Joseph, J. 37, 173 Koenig, M. 368 Lau, P. 302 Huang, T. 106 Joyce, G.F. 207 Koh, D.C.Y. 331 Lauterbach, H. 295, 299 Huang, T.-H. 178 Kalashnikova, T. 224 Kolatkar, A. 37 Law, B.J. 207 Huang, Z.-Z. 75 Kalisiak, J. 89 Kolkowski, E. 117 Law, M. 108 Huber, M. 34, 128 Kamenecka, T. 343 Komives, E.A. 183 Lawhorn, B. 70 Huey, R. 195 Kanelakis, K. 34 Komori, H.K. 117 Lay, C.C. 318 Huffman, J.L. 167 Kang, S. 276 Kompfner, E. 202 Layton, B. 104 Huh, C.-W. 345 Kang, Y. 116 Kondreddi, R. 73 Lazarus, N. 37 Huitrón-Reséndiz, S. 231, 294 Kannemeier, C. 228 Kono, D.H. 142 Leach, M. 37 Huminiecki, L. 346 Kao, M.-C. 250 Koob, G.F. 304 Lebus, D. 233 Hung, H.-C. 114 Kao, Y.-Y. 106. Korn, H. 322 Leconte, A.M. 86 Hunsicker-Wang, L. 191 Kapadia, S.B. 268 Korzus, E. 42 Lederman, M. 126 Huntoon, J. 195 Kaplan, C.D. 131, 149 Kossoy, E. 212 Lee, A. 276, 295 Hunziker, I. 301 Kapoor, M. 203 Kostic, M. 178 Lee, B. 178 Hur, W.Y. 88 Karin, M. 112 Kota, S. 358 Lee, C.W. 178, 232 Hutt, D. 25 Karnati, S. 195 Koudelka, K. 39 Lee, H.-K. 145 Hwa, T. 253 Karst, U. 276 Koulov, A. 25 Lee, J. 88, 193, 219 Hwang, D.-R. 94 Karyakin, A. 169 Kovacs, J. 199 Lee, J.-C. 94 Hwang, E. 36 Kass, K.E. 230 Koziol, J.A. 251 Lee, J.-D. 121 Hwang, G.T. 86 Kassmann, C.J. 160, 167 Kralli, N. 36 Lee, J.E. 135 Hyun, K. 199 Kastrinsky, D. 70 Krasnova, L. 89 Lee, J.-S. 88 Iannacone, M. 269 Katner, S.N. 318 Kraus, M. 37 Lee, J.Y. 140 Im, W. 193 Kato, D. 70 Kravchenko,V.V. 146 Lee, K. 83 Imaizumi, T. 340 Kato, N. 54 Krawczuk, P.J. 69 Lee, K.-B. 88 Imming, P. 75 Kaufmann, G. 78, 146 Krieg, C. 119 Lee, K.K. 216 Ino, A. 78 Kay, S.A. 340 Krishnamurthy, R. 73 Lee, M.B. 44 Inoue, K. 304, 320 Kaye, J. 119 Kritzik, M. 136 Lee, P. 270 Inoue, O. 262 Kazmierczak, P. 45 Kroener, J.F. 258 Lee, S. 70, 116 Ishido, M. 49 Keck, J. 222 Kroon, G. 183 Lee, S.H. 131, 149 Ishikawa, H. 70 Keinan, E. 212 Krueger, J.A. 131 Lee, Y.M. 259 Isogawa, M. 266, 267 Kelly, J.W. 81 Krueger, J.S. 258 Lefebvre-Roque, M. 357 Issafras, H. 141 Kelso, M. 70 Ku, S. 186 Lehmann, M. 120 Ito, T. 246 Kennedy, G. 232 Kubli, D. 271 Leissring, M.A. 343 Iwasaki, S. 320 Kennedy, J. 78 Kufareva, I. 199 Lekic, D. 314 Iwasawa, T. 11, 15 Kenny, P.J. 307, 346 Kuhn, P. 37 Leman, L. 75 Iwata, K. 146 Kerkow, D. 202 Kumar, G. 73, 218 Lemire, A. 83 Jaakola, V.-P. 173 Kerr, T.M. 319 Kunicki, T.J. 262 Lemke, E. 88, 189 Jackson, T.A. 207 Kerver, M. 134 Kunken, J. 29 Le Moal, M. 304 Jacobson, D. 279 Khandogin, J. 193 Kunz, S. 295, 300 Lempens, E. 30 Jacobson, R. 33 Khavrutzi, I. 193 Kuo, P. 111 Lenhard, B. 346 Jahnz, M. 88 Khayat, R. 216 Kuo, T. 88 Lenta, R. 132 Jahrling, P.B. 109 Kickhoefer,V. 130 Kupriyanova, T. 47 Lenzen, A. 83 Jameson, J.M. 117, 118 Kidd, L. 123 Kurian, S.M. 264 Leonard, M. 49 Janda, K.D. 78, 111, 146, 258 Kidgell, C. 54 Kurokawa, T. 134 Lerner, R.A 141, 258 Janjic, C. 352 Kiessling, R. 295 Kurokawa, Y. 134 Lerner, R.L. 208 Jaramillo, F. 36 Kim, C.H. 117 Kutilek, V.D. 265 Lesley, S.A. 175 Jaqaman, K. 29 Kim, D. 83, 140 Kuzelka, J. 74 LeVine, W. 51 Jenkins, B. 340 Kim, D.-H. 208 Kuzmin, Y.I. 205 Levy, C.L. 313 Jensen, R. 108, 150 Kim, G. 70 Kwan, S. 202 Lewicki, H. 295, 296 Jenssen, K. 186 Kim, H.-O. 114 Kwok, P.Y. 264 Lewis, J.D. 51 Jeso,V. 83 Kim, J.-H. 278 Kwok, S.-W. 89 Lewis, J.R. 319 Ji, L. 29 Kim, J.-S. 106 Kyle, M. 308 Lewis, W. 340 Jiang, H. 276 Kim, L. 37 Kyrle, P. 259 Li, A. 83 Jiang, R. 343 Kim, M. 72 Lad, S.P. 121 Li, E. 121 Jiang, Z. 104 Kim, S.-W. 121 Lai, C. 306 Li, H. 83 Jiminez-Dalmaroni, M.J. 160 Kim, Y. 78 Lai, C.Y. 130 Li, I. 167 THE SCRIPPS RESEARCH INSTITUTE 2006 391

Li, J. 121 Luxen, S. 120 Matsuno-Yagi, A. 250 Morris, K.V. 281 Li, K. 69 Luyendyk, J.P. 123 Matsuo, N. 42 Morris, M.A. 351 Li, L. 343 Luz, J.G. 160 Matteson, J. 25 Morita, H. 201 Li, S. 259 Lyssiotis, C. 88 Mattock, M. 320 Moser, B.A. 225 Liang, C. 368 Ma, B. 233 Maue, M.K.-D. 69 Mosier, D.E. 126 Liang, P.-H. 94 Ma, H. 78 Mauro, V.P. 331 Mosnier, L. 259 Liang, Z. 346 Ma, X. 172 Mayfield, S.P. 41 Motiei, L. 75 Liao, L 55, 233 Machacek, M. 29 Mayford, M. 42 Motta, C. 203 Liao, R. 228 Machetti, V. 33 McAllister, L. 78 Mowen, K.A. 127 Liberal, V. 222 Mackman, N. 111, 123 McCarty, O.J. 262 Moy, K. 173 Lichti, M. 307 MacLaren, A. 227 McClatchy, D. 55 Moyer, J. 32 Liebler, D. 145 MacMillan, K. 70 McClintock, P.A. 265 Mu, T. 81 Light, J. 308 Macpherson, L. 46 McCreight, M. 232 Mueller, B.M. 134 Lim, J. 53 Madamba, S. 317 McDonald, P. 346 Mukherji, M. 88 Lim, M. 128 Madoux, F. 365 McElhaney, G. 78 Mukhopadhyay, S. 189 Lim, S.T. 137 Madsen, M. 47 McGavern, D.B. 295, 299 Müller, U. 45 Lim, Y. 83 Maeda, S. 112 McGowan, C.H. 227 Mullick, A.E. 109 Lim, Y.M. 137 Magee, J. 193 McHeyzer-Williams, L.J. 125 Mulligan, S. 44 Lin, D.W. 69 Mahajan, S. 78 McHeyzer-Williams, M.G. 125 Munafo, D.B. 247 Lin, J. 50 Mahal, S.P. 356 McKay, D.B. 125 Munshi, A. 114 Lin, L. 343 Mahen, E.M. 205 McKeatin, M. 268 Muto, M. 41 Lin, R. 112 Maier, H. 266 McKenzie, K. 78 Myles, A. 11, 15 Lin, T. 217 Maimone, T.J. 69 McKeown, C. 32 Nahmias, C. 358 Lin, Y.-C. 265 Mainolfi, N. 83 McLeod, I. 55 Nakamura, T.M. 225 Lin, Y.-H. 74 Malakhova, O.A. 278 Mee, J. 78 Nakamaru-Ogiso, E. 250 Lindstrom, W. 195 Malherbe, L.P. 125 Meech, R. 332 Nalbant, P. 106 Lintz, R. 304 Mallick, S. 28 Meehan,T. 117 Nam, J. 70 Liou, L. 295 Manayani, D.J. 219 Meijler, M. 78, 146 Nagai, K. 89 Lira, R. 345 Manchester, M. 39, 218 Mejuch, T. 212 Nangle, L. 203 Lis, E.T. 86 Mandell, J. 208 Mendez-Dias, M. 314 Narayan, S. 89, 230 Lister, T. 83 Mandyam, C. 304 Mendoza-Fernandez, V. 314 Nash, S. 308 Littlefield, R. 53 Manetsch, R. 89 Mercer, B.A. 351 Natarajan, P. 216 Liu, C. 70, 88, 111, 112, 122 Manige, R. 193 Mercurio, P. 28 Nauli, A. 25 Liu, C.Y. 340 Manlapaz, E. 178 Merriman, E. 203 Navarro, S. 259 Liu, E. 276 Mann, E. 11, 15 Metanis, N. 30, 212 Nedellec, R. 126 Liu, F. 301 Mannige, R. 218 Meyers, A. 201 Nelson, J.D. 108, 150 Liu, J. 78 Manuell, A. 37 Michino, M. 193 Nelson, M. 173 Liu, L. 94 Manukyan, M. 120 Mico-Alvarez, X. 83 Nelson, N. 104 Liu, T. 191 Marchese, P. 269 Mikolosko, J.R. 160 Nelson, T.E. 303, 309 Liu, W. 88 Marchetti, V. 33 Mikulecky, P. 202 Nemazee, D. 128 Liu, X. 233 Marcondes, C. 294 Milburn, R. 83 Nemerow, G.R. 129, 130, 218 Liu, Y. 78, 89, 111, 122 Marella, M. 250 Mileni, M. 173 Nepomuceno, R. 129 Lizos, D. 83 Marin-Navarro, J. 41 Miles, L.A. 43 Nettles, K.W. 352 Lo, C.H. 212 Markou, A. 231, 307 Millar, D.P. 187, 357 Neuman, B.W. 291–293 Lo, E. 257 Marleau, A. 136 Miller, B.H. 344 Nguyen, C. 345 Lo, J.-F. 121 Marrinucci, D. 37 Miller, S. 42 Nguyen, D. 189 Lo, M.-C. 278 Marsh, C. 264 Milligan, R.A. 44, 291 Nguyen, H.D. 193 Loerke, D. 29 Marshall, D. 219 Mills, J. 88 Nguyen, N. 298 Logan, D. 50 Marsolais, D. 133 Mills, R. 117, 118 Nguyen, S. 69 LoGrasso, P. 34 Marquardt, K.L. 139 Milner, R. 257 Nicola, G. 199 Loizidou, E. 83 Martin, V. 225 Minond, D. 365 Nicolaou, K.C. 67, 83 Loo, J. 218 Martin-Fardon, R. 319 Mitchell, J.W. 43 Nicoletti, D. 75 Lopez, S.L. 302 Martina, Y. 264 Mitra, S.K. 137 Nie, Z. 317 Loren, J. 89 Martinez, C. 85 Mitsumori, S. 208 Niessen, F. 134 Lotz, C. 114 Martinez, X. 137 Mitsutake, A. 193 Nieva, J. 3, 927 Lotz, M. 230 Martinez-Iacobelli, M. 129 Miyamoto, T. 46 Nishikawa, T. 178 Louis-Dit-Sully, C.A. 142 Martinez-Yamout, M. 178, 183 Mizutani, M. 149 Nishimura, C. 180 Loutchnikov, A. 75 Marton, T. 50 Mizutani, N. 149 Nold, A. 83 Lovell, T. 191 Marucci, K. 264 Moisan, L. 11, 15 Nom, T. 88 Lowe, R. 201 Maruszak, B.M. 258 Molina, J.E. 137 Nomura, W. 208 Lowery, C. 78 Mason, B.J. 308 Mols, J. 116 Noncovich, A. 83 Lu, B.W. 55 Masuda, K. 173 Moncalian, G. 167 Noodleman, L. 191 Lu, M. 232 Mathison, C. 83 Mondala, T.S. 262 Nordstrom, A. 201 Lu, Q. 343 Mathison, J.C. 146 Montero, A. 75, 268 Norikane, Y. 75 Luna, V.M.M. 170 Matho, M. 56 Montminy, M. 178 Norledge, B. 195 Lund, C. 208 Matov, A. 29 Moon, S. 189 Northen, T. 201 Luo, J.-K. 278 Matsuda, S. 86 Moore, S. 317 Nowak, R. 32 Luo, K. 222 Matsui, S. 56 Moroi, M. 262 Nussbaum, A. 301 Luo,Y. 111, 149 Matsui, T. 216 Morris, G.M. 195 O’Maille, G. 201 392 THE SCRIPPS RESEARCH INSTITUTE 2006

O’Malley, D.P. 69 Pelletier, N. 125 Quello, S. 308 Rosario, D. 298, 299 O’Neill, B.A. 86 Pelmentschikov, V. 189, 191 Quigley, J. 47 Rosen, H. 133, 271 O’Reilly, M. 233 Pendyala, G. 294 Quiroz, A.L. 340 Rosenstein, R. 195 O’Sullivan, D. 258 Peng, H. 270 Quispe, J. 28, 291 Roth, C. 173 Oakman, E.L. 86 Peram, M.M.R. 92 Radakovits, R. 45 Roth, J. 302, 316 Ober, M. 345 Perego, M. 252, 254 Radu, D. 75 Roush, W.R. 345 Odegard, A. 216 Perera, R. 88 Rae, C. 39 Roy, R.S. 172 Odermatt, S. 11 Perez, M. 299 Rahe, N. 75 Roychowdhury-Saha, M. 205 Offord, R. 126 Perez-Pinera, P. 274 Rahimipour, S. 75 Rozenshteyn, D. 261 Ojakian, R. 294 Pestonjamasp, K. 106 Ramachandran, R. 49 Rudyak, S. 222 Okamura, A.J. 278 Peters, F. 88 Ramachandran, V. 54 Ruf, W. 33, 111, 134 Okamura, F. 278 Petersen, H. 134 Ramachary, D.B. 208 Ruggeri, A.M. 269 Okram, B. 88 Peterson, L.F. 278 Ramasatry, S.S.V. 208 Ruse, C. 55 Oldstone, M.B.A. 295, 296 Peterson, S. 232 Ramos, A. 126 Ruse, M. 120 Olson, A.J. 195 Petrillo, J.E. 219 Ramos, C. 45, 50 Russell, P. 225 Olson, B. 222 Petrovan, R.J. 109 Ramsey, C. 140 Rutschmann, S. 104 Olson, E. 276 Petrovic, G. 83 Rao, S. 308 Ryan, K. 365 Omelchenko, A. 195 Philipson, L.H. 56 Rasmussen, L.K. 89 Ryba, T. 345 Orahovats, P. 345 Phillips, E. 302 Ratner, L. 126 Ryu, Y. 88 Ortiz, A. 83 Picuri, J. 75 Ratner, T. 212 Saá Prieto, P. 357 Ortiz, B. 104 Pilotte, J. 330, 332 Raushel, J. 89 Saban, S. 130 Osornio, T. 73 Piomelli, D. 315 Rayon, E. 37 Sabatini, R. 357 Ospina, H. 264 Piper, J. 83 Razvi, A. 25 Sabeti, J. 303 Oswald, W.B. 109 Pique, M.E. 160, 167, 195 Razi, N. 233 Sabino, V. 320 Ota, M. 141 Piran, R. 212 Reader, J. 203 Sabouri, M. 29 Ota, T. 128 Pitram, S. 89 Reany, O. 212 Salazar, R. 306 Otero, F. 203 Placzek, W.J. 177 Rebek, J., Jr. 13, 15 Salès, N. 357 Otsuka, M. 116 Pletcher, M.T. 344 Reddy, R. 203 Salomon, D.R. 262, 264 Overbaugh, J. 126 Pljevalj˘ci´c, G. 187 Reddy, V. 130, 193 Sagle, L.B. 86 Owen, R. 345 Plutner, H. 25 Reddy, V.S. 218 Saikatendu, K. 37, 173 Owens, G.C. 330 Polat, T. 94 Reed, S.I. 222 Sainz, B. 268 Ozaki, Y. 262 Polet, D. 83 Reedy, M. 44 Saldana, A. 199 Pache, L. 129 Police, S. 130 Reedy, M.K. 44 Salerno, S. 112 Packowski, C. 232 Polich, J. 311 Rehen, S.K. 232 Salkowitz-Bokal, J. 126 Pacquelet, S. 120, 247 Polis, I. 310 Reijmers, G.J. 42 Salvio, R. 11, 15 Paegel, B.M. 207 Pollard, K.M. 282 Rein, A. 56 Sanathara, N. 119 Pagarigan, R. 301 Pollard, T.D. 56 Reisfeld, R.A. 111, 131 Sánchez, A.B. 295, 298 Page, L. 25 Pollock, S. 108 Reiter, J. 308 Sánchez-Alavez, M. 322 Palida, F.A. 56 Pond, S. 187 Reixach, N. 279 Sanna, G. 133 Palmer, T. 104 Pontow, S. 126 Reyes, C.R. 169 Sanna, P.P. 314 Palomique, J. 32 Pontremoli, G. 83 Reynald, R.L. 246 Sanner, M.F. 195 Pañeda, C. 313 Popkov, M. 208 Reynolds, A. 45 Sansen, S. 246 Panopoulos, P. 331 Pottekat, A. 25 Rice, K. 320 Saphire, E.O. 109, 135 Pantophlet, R.A. 108 Potter, C. 28 Riceberg, J. 261 Saraiva, M.J. 279 Papageorgiou, C. 83 Powell, E. 39 Richards, M.R. 109 Sarlah, D. 83 Papp, J. 264 Powers, E.T. 81 Richardson, H. 304 Sarvetnick, N. 136 Pappo. D. 83 Pragani, R. 345 Richter, J.M. 69 Saunders, A.A. 293 Para, A. 340 Prasad, S. 341 Ridgeway, W. 202 Savage, J.H. 265 Parapera, A. 53 Prasuhn, D. 74 Riewald, M. 132 Savas, Ü. 246 Park, J. 78 Pratt, B. 83 Riley, E. 304 Sawa, M. 94 Park, M. 247 Preece, N.E. 183 Ritter, M. 33 Sawkar, A. 81 Park, R. 55 Presolski, S. 74 Rivera, R. 232 Sayen, M.R. 271 Parmer, R.J. 43 Price, D.J. 193 Roberto, M. 312, 315, 317 Scaramozzino, F. 252 Parren, P.W.H.I. 109 Prieto, J. 55 Roberts, A.J. 313, 317 Schaeffer, M.-T. 133 Parsons, L.H. 310, 312, 317 Prinsen,R.C. 256, 265 Roberts, E. 85 Schaffer, L. 230 Partridge, J.P. 47 Prudden, J. 228 Roberts, T.C. 86 Scampavia, L. 365 Pastore, C. 126 Pruneda, J. 340 Robertson, M.W. 283 Scheele, C. 346 Pasunoori, L. 83 Przydzial, M. 346 Rodionov, V. 74 Scheerer, M. 276 Patapoutian, A. 46 Puckett, J. 186 Rodriguez, O. 53 Scheppke, L. 33 Patel, P. 70 Puga, M. 92 Rodriguez-Carreno, M.P. 301 Scheraga, H.A. 193 Patel, S. 193 Pulokas, J. 28 Rodriguez-Gabriel, M.A. 225 Scherer, E.M. 108 Paterson, N.E. 307 Pulvirenti, L. 304 Roeper, S. 89 Schettini, J. 142 Paulson, J.C. 233 Purcell, R. 267 Rogel, J. 92 Schiefner, A. 160 Pawlinski, R. 123 Purdy, R. 304 Rojek, J.M. 300 Schiller, S. 88 Payton, S. 308 Purse, B. 15 Rome, L. 130 Schimmel, P.R. 11, 203 Pebernard, S. 227 Purton, J. 140 Romeo, E. 123 Schlaepfer, D.D. 137 Pecheniuk, N. 259 Purton, K. 267 Romero, A. 70 Schmid, S.L. 22, 49 Pedrini, B 177 Qi, J. 345 Romesberg, F.E. 86 Schneemann, A 56, 218, 219 Pellequer, J.-L. 261 Qin, C. 201 Romijn, E. 55 Schneider, I.C. 53 THE SCRIPPS RESEARCH INSTITUTE 2006 393

Schnermann, M. 70 Sitia, G. 269 Supekova, L 88 Tran, M. 37 Schork, N. 279 Siuzdak, G. 201 Surh, C.D. 140 Trauger, S. 201 Schramm, M. 11, 15 Skog, P. 128 Suri, J. 208 Treadaway, J.C. 359 Schrantz, N. 141 Slattery, D. 307 Surka, M. 49 Trenney, R.L. 139 Schroeder, R. 304 Slavin, D. 227 Sutcliffe, J.G. 229–231 Treweek, J. 78 Schuepbach, R.A. 132 Slawecki, C.J. 316 Suzuki, T. 83 Tria, G. 83 Schultheisz, H. 202 Slown, C. 70 Suzuki-Inoue, K. 262 Trifilo, M. 295, 296 Schultz, P.G. 88 Smith, A. 351 Svensson, T. 307 Tripp, J. 89 Schultz, T.F. 340 Smith, C. 201 Swairjo, M. 203 Tripp, M.C. 193, 218 Schwander, M. 45 Smith, J.G. 130 Swan, C.H. 265 Tripurenani, S. 92 Schweitzer, P. 312, 315, 317 Smith, P.A. 86 Szainer, P. 25 Trombley, J. 33 Schwimmer, L.J. 209 Smith, R. 304 Szewczyk, P. 169 Troseth, R. 92 Scorah, J 227 Snyder, E.Y. 258 Szurmant, H. 25, 253, 255 Truksa, J. 270 Scott, L.G. 202 Sobel, D.F. 251 Szymczyna, B. 202 Truong, P. 299 Sczaniecka, A. 45 Sobieszczuk, P. 233 Tabarean, I. 322 Trzupek, J. 70 Secrest, P. 136 Solel, E. 212 Tabarin, A 320 Tsai, S. 186. Segatori, L. 81 Solforosi, L. 148 Taffe, M.A. 318 Tsao, M.-L. 88 Seit-Nebi, A. 228 Solorio-Alvarado, C. 83 Tagoe, C. 279 Tsatmali, M. 120, 330 Selvarajah, S. 113, 114 Sonderegger, M. 201 Tainer, J.A. 56, 167 Tschan, M.P. 265 Semenova, S.G. 231, 307 Song, B.D. 49, 92 Takahashi, S. 180 Tschulena, U. 208 Seo, B.B. 250 Soragni, E. 186 Takanashi, S. 72 Tsinoremas, N.F. 368 Seo, J.-Y. 106 Soreni, M. 212 Takao, K. 345 Tsuda, M. 246 Serrano, P. 177 Sorg, A. 345 Takaoka, L. 70 Tsvetanova, B. 252 Sette, A. 292, 293 Soulet, F. 49 Tam, K. 220 Tubbs, J.L. 160 Sever, M. 88 Sovath, S. 104 Tama, F. 193 Turner, C. 83 Sevilla, N. 295 Speir, J. 216 Tamura, K. 203 Tuttle, L.M. 183 Sferrazza, G. 357 Spencer, K. 36 Tan, J. 306 Tzima, E. 203 Shabat, D. 208 Sperling, E. 202 Tanaka, F. 208 Ulevitch, R.J. 102, 146 Shadan, F. 308 Spicer, T. 365 Tang, A. 37 Umezawa, T. 83 Shafton, A. 92 Spiropoulou, C. 295 Taniguchi, N. 246 Underwood, L. 313 Shaginian, A. 70 Springsteen, G.G. 207 Tao, H. 70 Unger, V.M. 56 Sharkey, L. 317, 321 Stagg, S. 25, 28 Tassew, N. 187 Unwin, N. 52 Sharpless, K.B. 89 Stanfield, R.L. 108, 160 Tate, S. 27 Uritboonthai, W. 201 Sharpless, W. 89 Stathakis, C. 83 Tateno, H. 233 Uryu, S. 137 Shaw, D. 83 Steardo, L. 320 Taylor, E. 227 Utsintong, M. 195 Shekhter,T. 30, 212 Stefanko,R.S. 141, 160 Taylor, J.A. 56 Utsumi, N. 208 Shen, Z. 201 Steiniger, S. 78 Taylor, K. 118 Uusitalo, T. 21 Shenoy, S. 11 Stengel, G. 187 Tedesco, D. 222 Uusitalo-Jarvinen, H. 33 Shenvi, R.A. 69 Sternik, G. 114 Tellinghuisen, T.L. 359 Uy, H. 104 Shepard, C. 193 Stevens, J. 160 Tennant, L.L. 178, 180, 183 Uzawa, T. 180 Sherman, A. 356 Stevens, R.C. 173 Teyton, L. 141 Uzzell, V. 46 Sherman, L.A. 139 Stewart, L. 233 Thalji, R. 345 Valbracht, J. 245 Shi, J. 276 Stewart, P. 130 Thayer, D. 94 Va, P. 345 Shi, Y. 72 Stinus, L. 304 Theofilopoulos, A.N. 142 Valenta, D.T. 109 Shigeoka, A. 125 Stotland, A. 136 Thielges, M.C. 86 Valente, D. 264 Shikhman, A.R. 245 Stouffer, D. 310 Thomas, D. 39 Vallee, S. 114 Shimada, S. 116 Stout, C.D. 170, 220, 221, Thomas, E.A. 230 Valo, M. 120 Shin, D.S. 167 265 Thompson, K. 29 Van Anda, H. 15 Shin, J. 75 Stowers, L. 50 Thonberg, H. 346 Van der Schans, E.J.C. 187 Shin, W. 53 Strosberg, A.D. 358 Thorpe, I.F. 193 van der Stap, L. 314 Shivakumar, D. 189 Stroupe, M.E. 160 Thurbon, D. 314 van Drogen, F. 222 Shore, D.A. 160 Stuempfig, N.D. 319 Tian, H. 233 Van Leeuwen, E.M.M. 140 Shumilak, K. 122 Stuhlmann, H. 51 Tian, X. 283 Vanderklish, P.W. 330, 332 Shur, O. 279 Sturny, A. 357 Tichenor, M. 70 Vareille, G. 195 Sidhpura, N. 319 Subauste, C. 47 Tiefenbrunn,T. 30 Vanhnasy, J. 160 Sidney, J. 292 Subramaniam, P. 357 Tilley, R.E. 123 Varga, J. 111 Siefker, D. 41 Subramanian, V. 37, 173 Timmons, J.A. 346 Varughese, K.I. 255 Siegel, S. 81 Sue, S.C. 183 Ting, J.P.C. 291–293 Vasilu, D. 233 Siggins, G.R. 312, 315, 317, Sugase, K. 178 Tippmann, E. 88 Vela, J. 128 321 Sugawara, A. 89 Tipton, J.D. 365 Velasquez, J. 173 Siladi, M.E. 219 Sugiyama, M. 94 Tiraby-Nguyen, C. 36 Velcicky, J. 70 Silva, F. 208 Suk, J.Y. 81 Tishon, A. 295 Venable, J. 55 Sim, J. 141 Sullivan, N.L. 109 Tobias, P.S. 109, 145 Venkataiah, B. 74 Simanski, S. 351 Summerer, D. 88 Tonnu, L. 260 Venter,P.A. 219 Singh, P. 39, 218 Sun, C. 111 Torbett, B.E. 220, 256, 265 Verdino, P. 160 Sinha, M. 212 Sun, P. 228 Torres-Bacete, J. 250 Vereyken, E. 303 Sinha, S. 85 Sundaresan, V. 170 Tortosa, M. 345 Versteeg, H. 134 Sinha, S.C. 214 Sundheim, O. 167 Toulon, A. 117 Vetter, S. 221 Sipe, J.C. 271 Sundstrom, M. 220 Tran,H.G. 340 Villena, J. 36 394 THE SCRIPPS RESEARCH INSTITUTE 2006

Vlkolinsky, R. 317 Wills, D. 302, 316 Yin, X. 278 Vogt, P.K. 276 Wilson, A. 252 Yin, Y. 169 Vojkovsky, T. 368 Wilson, C.A. 264 Ying, G. 295, 296 Volonterio, A. 151 Wilson, I.A. 108, 150, 160 Yonemoto, I. 25, 81 Von Allmen-Zurcher, N. 141 Wilson, R.F. 221 Yoo, Y.S. 75 Von Huben, S.N. 318 Wilson-Kubalek, E.M. 44 Yoon, S.-H. 106 von Loehneysen, K. 120 Winbush, S. 345 York-DeFalco, C. 294 Voytek, S.B. 207 Winzeler, EA. 54 Yoshida, K. 141, 246 Waalen, J. 270 Wise, E. 160 Yoshimoto, K. 193 Waas, W. 203 Wiseman, R.L. 81 Yoshioka, C. 28, 44, 291 Wada, S. 169 Witherden, D. 117 Yoshizuka, N. 228 Wagner, S. 251 Witkowski, J.A. 265 Young, B.M. 344 Wahlestedt, C. 346 Wittenberg, C. 224 Young, J. 54 Walker, B. 304 Wohlschlegel, J. 55, 222 Young, T. 88 Walker, R.C. 189 Wojciak, J. 178 Yu, J. 169 Wang, A. 137 Wong, C. 55 Yu, W. 86 Wang, C. 141 Wong, C.-H. 94 Yu, Z. 81 Wang, H. 92 Wong, D. 201 Yuan, Y. 208 Wang, J. 83, 88, 278 Wong, J. 139 Yuem, D. 72 Wang, L. 173, 270 Wood, T.I. 165, 167 Yung, Y. 232 Wang, M. 108 Wright, P.E. 158, 178, 180 Zajonc. D.M. 160 Wang, S.-K. 94 Wu, C.C. 116 Zak, M. 83 Wang, X. 89 Wu, C.-Y. 94 Zal, M.A. 114 Wang, Z. 283 Wu, D. 94 Zal, T. 114 Want, E. 201 Wu, P. 89 Zandonatti, M. 294 Ward, A.B. 44, 169 Wu, W. 111, 122 Zastrow, G.M. 344 Warrington, J. 264 Wu, X. 276 Zeeb, M. 178 Wassenaar, J. 89 Wuchrer, M. 94 Zelder, F. 11, 15 Watanabe, M. 117 Wüthrich, K. 177 Zeng, Y. 233 Waterman-Storer, C. 53 Xia, Y. 104 Zhang, D.-E. 278 Watry, D. 294 Xiang, R. 111, 131, 149 Zhang, E. 340 Watson, L. 112 Xie, C. 116 Zhang, H. 106, 208 Watson, S.P. 262 Xiong, W. 72 Zhang, H.-Y. 346 Webb, S. 45 Xu, C.R. 112 Zhang, Q. 88, 172, 195 Webb, W. 201 Xu, H. 83 Zhang, W. 189, 274 Weber, J.L. 306 Xu, L. 160 Zhang, Y. 70 Weber, K. 32 Xu, T. 55 Zhang, Y.Q. 136 Wee, S. 304 Xu, X. 160, 259 Zhao, J. 343 Wei, C.H. 139 Xu, Y. 72, 78, 116 Zhao, L. 276 Weide, T. 89 Yachi, P. 114 Zhao, T. 106 Weinkam, P. 86 Yadav, D. 136 Zhao, Y. 72, 195, 320 Weiss, F. 319 Yadav, M. 37, 294 Zhong, J. 267 Weissman, C. 355, 356 Yadav, M.K. 173 Zhou, B. 78 Welsh, D.K. 340 Yagi, T. 250 Zhou, H. 78, 131, 149 Wennmalm, K. 346 Yamada, Y. 225 Zhou, M. 357 Wentworth, A.D. 92 Yamagata, A. 167 Zhu, L. 232 Wentworth, P. Jr. 92, 271, 357 Yamaguchi M. 170 Zhu, P. 189 West, C. 270 Yamasaki, R. 75 Zhu, S. 88 Whalen, L. 94 Yamashita, T. 250 Zhu, W.H. 160 Wheeler, A. 53 Yan, M. 278 Zhu, X. 72 Wheeler, R. 193 Yang, A.H. 232 Ziegler, J. 189 Whiby, L. 70 Yang, G. 29 Zijlstra, A. 47 White, R.A. 253 Yang, X. 259 Zimmerman, J. 86 Whitefield, B. 69 Yang, X.-L. 203 Zlokovic, B.V. 259 Whitelock, J. 117 Yang, Y.-Y. 94 Zoni, C. 85 Whiting, M. 89 Yano, J.K. 246 Zorrilla, E.P. 319, 320 Whitley, K. 104 Yao, S. 72 Zou, Z. 51 Whitmire, J.K. 269, 301 Yao, Y. 180, 183 Zuniga, E. 295 Whitton, J.L. 293, 301 Yarar, D. 49 Zwick, M.B. 108, 150 Wieland, S.F. 267 Yasuda, M. 42 Wiethoff, C. 130 Yasuda, R. 42 Wikoff, W. 201 Yates, J.R. III 55, 258, 264 Wildman, C. 193 Ye, M. 120 Williams, A. 345 Ye, X.Q. 232 Williams, J. 225 Ye, Y. 208 Williams, R.C. 167 Yeager, M. 56, 271, 291 Williamson, J.R. 202 Yegneswaran, S. 259 Williamson, R.A. 148 Yeung, B. 278 Willis, A. 78 Yi, J. 280 THE SCRIPPS RESEARCH INSTITUTE 2006 395

Subject Index Autoimmunity 112, 136, 139, Cheminformatics 199 Docking 196, 200 141, 282, 301 Chemobodies 210 Doublecortin 45 Acetogenins 215 antitumor 144 Chemokine receptors 265 Drug abuse 273, 302, 312, Actin 29, 32, 53 genetics of 142 Chemokines 313, 318, 319 Actin filaments 36 inhibition of cell cycle in 143 in the CNS 309 mechanisms of 317 Activated protein C 132, 259 role of p21 143 Chlamydial infection 122 Drug addiction 273 Acute respiratory distress Autophagy 272 Chloroplasts 41 Drug dependence 314 syndrome 133 B cells Cholecystokinin 85 Drug discovery 346, 357, 365 Adaptive immunity 125, 131, antigen recognition by 128 Chondrocytes 245 Dynamin 49 134, 141 immune learning in 128 Chondrogenesis 246 Dyslipoproteinemia 260 Adenovirus gene transfer 129 memory 125 Chromosome segregation 30 Dystroglycan 257 Addiction 304, 310, 313, 319 repertoire 112 Chronic wasting disease 296 Ecstasy 318 Adhesion 262 Bacteria Circadian clocks 340 Electron cryomicroscopy 56 Aging 142, 168, 343 pilus 168 Clathrin 49 Electrostatics 191 AIDS. See HIV infection. sporulation of 252, 254 Click chemistry 74, 89 Emerging viruses 109 Alcohol 305, 313, 316, 318, Base J DNA 94 CNS development 45 Emotion 307 320 Biocatalysis 205 Coagulation 111, 123, 132, Encephalitis 294 animal models of exposure Biofilms 255 134, 259, 260, 261 Endocannabinoids 310 during adolescence and Bioinformatics 199, 218 Cocaine 161, 304, 310, 313, Endocytosis adulthood 316 Biological chemistry 88 319 regulation of 49 neurobiological mechanisms Biomarkers 368 Cocaine addiction 79 Endoglin 149 of consumption 316 Biomolecular computing 212 Cocaine antibodies 198 Endothelial cells 51, 132 Alcohol. See also Ethanol. Biomolecular sensors 40 Cognition 42 Endotoxemia 124 Alcohol dependence 308 Bioorganic chemistry 70, 94 Combinatorial chemistry 72, 88 Endotoxin 146 Alcoholism 302, 309 Biosensors 166 Combinatorial libraries 214 Energy transduction 171 Algae 41 Botulinum neurotoxin 78 Complex I defects 250 Enzyme inhibitors 95 Allergy 283 Bovine spongiform Computer modeling Enzymes Alloantigens 125 encephalopathy 148 of proteins and nucleic acids DNA 207 Allostasis 305 Brain-computer interface 311 189 evolution of 87 Alzheimer’s disease 81, 93, 223, Brain peptides 315 Conformational diseases 25 RNA 207 343 Breast cancer 121, 147, 149, Consortium for Functional structure and dynamics of 183 Amygdala 317 258, 275 Glycomics 235 synthetic 212 Amyloid diseases 81, 279 Calcium channels 303 Copper-catalyzed cycloadditions Epilepsy 85 Amyloidogenesis 81 Cancer 38, 47, 122, 131, 134, 90 Ethanol 310, 312 Analytical chemistry 74 142, 187, 223, 227, 228, Coreceptor switching 126 CNS action of 317 Anemia 280 266, 274, 276, 278, 368 Creutzfeldt-Jakob disease 148 Evolution 86 Angiogenesis 33, 47, 51, 123, design of therapeutic Cyclins 223 Familial amyloidotic 134, 275 antibodies 209 Cystic fibrosis 25 polyneuropathy 81 Anthrax 219 Cannabinoids 273, 315 Cytochrome ba3 171 Familial amyotrophic lateral Anthrax toxins 106 Carbohydrate chemistry 95 Cytochrome oxidases 170 sclerosis 167 Antibiotics 84 Carcinogenesis 121 Cytochrome P450s 170, 246 Fatty acid amide hydrolase 174, Antibodies 161 Cardiac gap junctions 57 Cytokines 127, 274 273 expression in chloroplasts 41 Cardiac remodeling 124 gene expression 116 Fatty acid synthase 25 phage display 209 Cartilage 245 in T-cell homeostasis 140 Feeding 320 to HIV 108, 209 Catalysis 74, 90 Cytolysins 45 Feeding behavior 229 to tumors 209 Catalytic antibodies 88, 161, Cytoskeleton 29, 32, 53, 106 Feline immunodeficiency virus to viruses 109 208, 215 Cytotoxic T lymphocytes as AIDS model 220 Antibody genes 112 in transgenic plants 212 in HBV virus infection 266, proteases of 221 Antibody repertoire 112 Cavitands 15 267 receptors for 220 Antibody-catalyzed water Cell cycle Deafness 46 Fiber-optic array scanning oxidation pathway 92 checkpoints 225, 227, 276 Dendrimers 91 technology 38 Anticancer agents 84 control in mammalian cells Demyelination 292 Flavonoids 248 (+)-CC-1065 70 223 Dendrites 333 Flexibility modeling 196 chemistry of 70 control in yeast cells 222 Dendritic cells 125, 131 Fluorescence spectroscopy duocarmycins 70 regulation of 224 Dendritic spines 36 of nucleic acids 187 Antidepressants 322 ubiquitin-mediated proteolysis Dengue fever 136 Fluorescent speckle microscopy Antisense therapy 291 in 351 Depression 231, 321 53 Antiviral responses 116 Cell migration 29, 53, 137 genetics of 344 Focal adhesion kinase 137 Apomyoglobin 180 Cell morphogenesis 31 Diabetes 37, 118, 136, 139, Forward genetics 104 Apoptosis 43, 123, 259, 271 Cell motility 31, 53 141, 256, 341, 343 Fragile X syndrome 332 Arousal 229 Cell recognition molecules 46 Differentiation 351 Friedrich’s ataxia 187 Arthritis 280 Cellular differentiation Directed evolution 207, 209 Functional genomics 264 Asthma 212 sporulation 252 DNA alkylating agents 70 Galanin 85, 318, 321 Asymmetric synthesis 208 Chaperones 82, 182 DNA damage 87, 225, 227 Gap junctions 57 Ataxia telangiectasia–like Chaperonins 177, 184 DNA enzymes 188, 207 Gaucher disease 82, 197, 270 disorder 276 Charcot-Marie-Tooth disease DNA repair 167, 225, 227 Gelsolin 82 Atheronals 93 205 DNA replication 276 Gene delivery 122, 265 Atherosclerosis 93, 109, 146 Chemical biology 83 DNA sensors 76 Gene expression profiles 369 Autism 85 Chemical synthesis 83 DNA vaccines 131, 149, 301 Gene silencing 281 396 THE SCRIPPS RESEARCH INSTITUTE 2006

Genetic alphabet 87 Innate immunity 106, 114, Molecular microscopy 28 Nucleic acids 205 Genetic code 203 116, 120, 125, 129, 131, Molecular neurobiology 230 computer modeling of 189 Genetic diseases 270, 279 134, 141, 145, 146, 160 Molecular recognition 15 function and dynamics of 87 Genomic stability 227 Insulin-degrading enzyme 343 Molecular wires 222 structure of 73, 187 Genomics 54, 72, 175, 346 Insulin resistance 341 Monocytes 43 Nucleocytoplasmic transport 34 Ghrelin 321 Insulin signaling 55 Motivation 229, 307 Nucleus accumbens 317 Glaucoma 248 Integrins 57, 138, 215, 257, Mouse models of behavior 313 Obesity 273 Glioblastoma multiforme 34 262 Multidrug resistance 45, 169 Possessive-compulsive disorder Glitazones 341 in breast cancer 258 Multiple sclerosis 251 231 Glucose transporters 245 in the CNS 46 Muscle repair 332 Olfaction 27, 50 Glutaredoxin 31 Interferons 143, 301 Muscular dystrophies 35 Oncogenesis 228, 276 Glycobiology 95, 233 Intravasation 48 Myelin 230 Opioids 85, 317 Glycoproteins 135, 263 Ion channels 47, 52, 303 Myeloid development 265 Organic chemistry 74 Griscelli syndrome 248 Iron metabolism 270 Myocardial infarction 271 Organic synthesis 212, 214 GTPases 25, 120 Iron overload 279 Myocarditis 301 Organocatalysis 209 Hair cells 46 Ischemia 123, 257, 271 Myosin 44 Organometallic chemistry 212 Heart disease 279, 296 Joint injury 245 NADH dehydrogenases 250 Osteoarthritis 245 α-Helix mimetics 15 Kinesins 44 NADPH oxidase 248 Osteoarthritis. See also arthritis. Hemangiomas 34 Lassa fever 300 Nanodiscs 170 Oxidative metabolites 82 Hematopoiesis 265, 278 Learning 43 Nanomedicine Oxidative stress 225, 248, 280 Hemochromatosis 270 Legumain 111, 122 Nanoparticles 130 Ozone scavengers 212 Hemophilia 262 Leishmaniasis 357 Nanotechnology 39, 217, 220 p21-Activated kinases Hemorrhagic fever 135 Leukemia 278 Nanotubes 76 in cellular regulation 106 Hepcidin 270 Leukocytes 106, 120 Natural killer cells 131 P450s 222 Hepatitis B virus 58, 266, 267 Ligand discovery 199 Natural products 69, 70, 83, Pain 47, 85 Hepatitis C virus 267, 268, Ligand recognition and 215, 345 Parkinson’s disease 81, 223, 358, 359 specificity 162 Necrosis 271 343 Hepatocellular carcinoma 359 Lipid chemistry 172 Neural aneuploidy 232 Phage display 79 Heroin 310 Lipoproteins 260 Neural circuits 27 Pharmacogenomics 346 High-throughput screening 365 Lung epithelium 120 Neuregulins 306 Phenylalanine hydroxylase 173 Hippocampus 303 Lymphocyte trafficking 133 NeuroAIDS 294 Phenylketonuria 173 drug effects on 317 Lymphocytes Neuroactive steroids 303 Pheromones 50 Histone deacetylase inhibitors regulation of function 119 Neuroadaptation 304 Photoactive proteins 165 187 Lysophospholipids 232 Neurodegeneration 343 Photoreceptors 340 HIV infection 113, 114, 126, α2-Macroglobulin 111 Neuronal differentiation 330 Plasminogen 43 170, 220, 265, 281 Macular degeneration 248 Neuronal plasticity 314 Platelets 262, 269 design of therapeutic Malaria 54 Neurons 27, 50, 257, 309 Pleiotrophin 274 antibodies 210 MAP kinases 116, 120 cytoskeletal organization and Polyamides 186 human antibodies to 108 Mass spectrometry 55, 365 function of 36 Prebiotic chemistry 77 neutralizing antibodies to 162 of human metabolites 201 Neuropeptides 305, 312, 316, Prion diseases 148, 357 SIV model of 294 of peroxisome proliferator- 317, 321 Prions 148, 183, 296, 356, 357 HIV proteases 197 activated receptors 342 Neurotoxins 174 Prodrug therapy 111, 215 HIV vaccines 31, 150 of viruses 201 Neurotransmitters 173 Programmed cell death. HLA-G 256 on silicon 201 Neutrophils 247 See Apoptosis. Homeostasis 140 Materials chemistry 74 Neurovascular unit 257 Prohormone processing 43 in cartilage 245 MDMA 318 Nicotine 305, 307 Proteases 214 of T cells 142 Mechanosensory perception 45 animal models of exposure Proteasomes 222 of T-cell memory cells Mechanotransduction 245 during adolescence and Protein C 132, 259 Homoserine lactones 147 Medicinal chemistry 345 adulthood 316 Protein design 89, 218 Host resistance 104 Membrane channels dependence 308 Protein engineering 30, 221 Human-computer interfaces 195 cardiac gap junctions 57 Nijmegen breakage syndrome Protein folding 81, 180, 184, Huntington’s disease 230 Membrane proteins 169, 170, 276 189, 193 Hydrogen-deuterium exchange 172 Nitric oxide synthases 165, Protein kinase inhibitors 368 342 topogenesis of 33 167, 221 Protein kinases 222 Hypocretins 229 Memory 42, 303 Nitrogenase 191 Protein misfolding 93 Image processing 56 Metalloenzymes 191, 221 Nociceptin/orphanin FQ 319 Protein misfolding diseases 81, Immune memory 125 Metalloproteins 166 Nuclear envelope 34, 115 279 Immune privilege 256 Metastasis 38, 47, 123, 258 Nuclear lamina 34 Protein modification 278 Immunocytotherapy 299 MHC molecules 162 Nuclear magnetic resonance Protein phosphatase 2C Immunodeficiency 247 MicroRNA 116 spectroscopy 177 inhibitors 198 Immunodominance 301 Microtubules 29, 36 , 44, 53 Nuclear magnetic resonance Protein-protein interactions 38, Immunoglobin E 283 Microvessels 257 189 358 Immunoglobulins 162 Mitochondria 36, 250 of enzyme catalysis 183 Protein S 260 Immunologic synapse 115 Molecular assemblies 194 of IκBα 184 Protein structure 38, 163, 177 Immunosuppression 133 Molecular biophysics 193 of prions 183 Protein trafficking 163 Inflammation 92, 109, 120, Molecular dynamics 189 of protein folding 180 and conformational diseases 123, 132, 145, 146, 259 Molecular graphics 195 of proteins in solution 178 25 Infections 104, 120 Molecular imaging 28 Nuclear pore complexes 34 Protein Z-dependent protease Influenza virus 160, 217, 235 Molecular machines 24, 29, 44 Nucleic acid enzymes 188 inhibitor 261 THE SCRIPPS RESEARCH INSTITUTE 2006 397

Proteins Ribozymes. See also RNA activation of 114, 117 Tumor eradication 111, 112 carbohydrate binding 233 enzymes. antiviral function of 301 Tumor immunity 139 computer modeling of 189 RNA enzymes 207 development of 114 Tumor markers 112 design of 166 RNA interference 116, 189, helper 127 Tumor targeting 39 function and dynamics of 87 281, 346 homeostasis of 140, 142 Tumorigenesis 121, 138, 228 glycan binding 233 RNA in autoimmunity 142 Tyrosine protein kinases 137 predicting interactions of 197 mechanisms of assembly and in regulation of B-cell Urocortins 321 structure and function 175 catalysis 205 immunity 125 Vaccine design 135 structure of 165, 178, 197, noncoding 112 in SIV infection 294 Vaccines 292 199 RNA-binding proteins 330 in transplantation 256 antiviral 301 Proteolysis 222 Rubinstein-Taybi syndrome 42 in tumor immunity 131 to HIV 108 Proteomics 37, 55, 365 San Diego Alcohol and in viral infections 296 Vascular imaging 39 Purkinje neurons 303 Minorities Project 302 in viral persistence 295, 299 Vasculogenesis 51 Quantum chemistry 191 Schizophrenia 230, 231 in wound healing 118 Vesicular transport Quorum sensing 182 Schwann cells 306 induction of tolerance 125 GTPase regulation of 26 Reactive oxygen species 120, Scrapie 296 intraepithelial γδ T cells 117, Viral clearance 269 330 Seco-sterols 93 118 Viral nanoparticles 40, 51 Receptors Selective serotonin reuptake memory 140 Viral pathogenesis 301 design of inhibitors 95 inhibitors 344 regulation of development 119 Viral persistence 267, 295, 299 endothelial cell protein C 132 Senescence 229 regulatory 369 Virtual ligand screening 199 ErbBs in the nervous system Sensor kinases 252 selection of 125 Viruses 306 Sensory neurons 46 thymic selection 119 adenoviruses 122, 129, 130 estrogen-related receptor α 37 Sepsis 123, 132 Tangible interfaces 195 antibodies to 109 for acetylcholine 52 Severe acute respiratory Tetratricopeptide repeats 254 arenaviruses 293, 298 for adenoviruses 129 syndrome 177, 291 Thermoregulation 322 assembly of 219 for γ-aminobutyric acid 317 Shock 105 Thermosensation 46 Borna disease virus 299 for B cells 233 Sialosides 234 ThermoTRPs 47 canine parvovirus 40 for cannabinoids 310, 315 Siglecs 233 Thrombosis 111, 123, 260 coronaviruses 177, 291, 292 for corticotropin-releasing Signaling 42, 51, 105, 106, Thymus 119 cowpea mosaic virus 40, 217 factor 305, 316, 318, 321 116, 120, 121, 129, 133, Tissue factor 111, 123, 134, coxsackievirus 301 for FIV 220 134, 138, 146, 162, 174, 197 Dengue virus 136, 214 for galanin 321 205, 232, 253–255, 274, Tissue plasminogen activator DNA virus 216 for HIV 113, 126 303, 306, 310, 351 260 Ebola virus 109, 135 for IgE 283 by proteases 132 Tobacco. See Nicotine. feline immunodeficiency virus for IL-2 160 in coagulation 132 Tolerance 125, 131, 139, 144 220 for Lassa virus 300 in immune tolerance 125 Toll-like receptors Flock House virus 217, 219 for NMDA 317 in 2-component systems 253 in coronavirus infection 292 for delivery of anthrax for nociceptin/orphanin FQ 319 response regulators in 254 Total synthesis 69 antitoxins 219 for opioids 317 Single-molecule analysis 76 Toxins 252 for treating cocaine addiction for sphingosine 1-phosphate Single-molecule biophysics 189 Transcription 277, 346, 351 79 133 SIV infection and adaptation to environmental hepatitis B virus 58, 266, 267 G protein–coupled 232, 347 as AIDS model 294 stimuli 224 hepatitis C virus 267, 268, in innate immunity 141 Sleep 231 in chondrogenesis 246 358, 359 in nicotine withdrawal 307 Sodium-calcium exchangers 34 in vertebrate development 332 HK97 bacteriophage 216 metabolic glutamate 319 Stem cells 33, 51, 72, 330 regulation of 24, 178, 187, icosahedral 219 NKG2D 131 in treatment of brain 224 influenza virus 217, 296 Nod proteins 146 metastases 258 Transcription factors 178, 277, Lassa virus 293, 300, 301 on T cells 162 Stress 36, 304, 320 332 lentiviruses 221 peroxisome proliferator-activated Stroke 257, 260 in myeloid development 265 lymphocytic choriomeningitis 341 Structural biology 24, 167, Transcriptional coactivators 37 virus 269, 293, 296, 298, protease activated 124, 132, 173, 177, 195 Transformation 229 299 134 Structural genomics 177 Transgenic mice measles virus 296 T cell 114, 141 Superoxide dismutases 167, 280 as models for chronic wasting mouse cytomegalovirus 105 Toll-like 105, 109, 116, 120, Synapses 36, 42, 332 disease 296 mouse hepatitis virus 291, 292 129, 145, 146, 160, 292 Synaptic plasticity 314, 332 Transhydrogenase 170 nodaviruses 219 Regenerative medicine 72 Synaptic transmission 52 Translation 330–332 P22 bacteriophage 216 Reperfusion injury 123, 271 Syndecans 113 in algae 41 retroviruses 220 Response regulators 254 Synthetic chemistry 70, 94 in chloroplasts 41 RNA viruses 217 Restriction factors 114 Synthetic methods 345 role of tRNA 203 severe acute respiratory Retinoid homeostasis 78 Synthetic systems 75 Translational neurophysiology syndrome–associated corona- Retinopathies 33 α-Synucleinopathies 82 302 virus 38, 177, 291, 292 Reverse genetics 298, 299 Systemic lupus erythematosus Transmissible spongiform simian immunodeficiency Reward 307 interferons in 143 encephalopathies 296, 356, virus 294 Rho GTPases 106 mouse models of 142 357 structure of 177, 218, 291, Ribonucleoproteins 188 role of cell-cycle genes 144 Transplantation 125, 256, 264 293 Ribonucleotide reductases 192 role of T cells 144 Transporters 169 structure and function of 216 Ribosomes 41, 189, 331 Systems biology 54 Transthyretin 81, 279 sulfolobus turreted icosahedral assembly of 202 Sumoylation 55 tRNA synthetases 203 virus 216 Ribozymes 188, 205 T cells 139 Tropomodulins 32 tetraviruses 217 398 THE SCRIPPS RESEARCH INSTITUTE 2006

tomato bushy stunt virus 219 Zinc fingers 178, 210 Visualization 195 von Willebrand disease 263 Werner syndrome 168 Wound healing 118 Xenobiotics 282 Xenotransplantation 264 Xeroderma pigmentosum 227 X-ray crystallography 160 of cryptochrome 166 of cytochrome oxidases 170 of cytochrome P450s 170 of fluorescent proteins 166 of nitric oxide synthases 165 of photoactive yellow protein 165 of transhydrogenase 170

ACKNOWLEDGMENTS Editor DEPARTMENTAL The Scientific Report is The scientists who have Barbara L. Halliburton, Ph.D. COORDINATION published annually by The contributed sections to this Scripps Research Institute and report wish to acknowledge Associate Editor is available on request from Ruby Blair the dedication and hard work Linda Wood, M.A. Department of Molecular Biology Office of Communications of the laboratory technicians TPC-20 who helped bring the Project Manager Dian Caudebec The Scripps Research Institute research to fruition, the Jann Coury Department of Immunology 10550 North Torrey Pines Road administrative assistants who Office of Communications La Jolla, CA 92037 have made it presentable for Janette Lundgren (858) 784-2171 publication, and the support The Skaggs Institute for Photography e-mail: [email protected] personnel who provided Chemical Biology Michael Balderas critical specialized services Marcia McRae and equipment. Biomedical Graphics Molecular and Integrative Department, Scripps Research Sciences Department

Bruce Hibbs Photography Cheryl Negus Mark Dastrup Department of Cell Biology

Lucien Capehart Photography Vicky Nielsen Armstrong Department of Chemistry Printing and duplication Lynn Oleski Maryland Composition Department of Molecular and Experimental Medicine

Michelle Platero Department of Neurobiology

Candy Walker Program Administrator Scientific Operations, Florida