DOCSLIB.ORG

Structural and Biochemical Studies of Rpe65, the Retinoid

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Structural and Biochemical Studies of Rpe65, the Retinoid TITLE PAGE STRUCTURAL AND BIOCHEMICAL STUDIES OF RPE65, THE RETINOID ISOMERASE OF THE VISUAL CYCLE by PHILIP DAVID KISER Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Krzysztof Palczewski Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2010 SIGNATURE PAGE CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. ii DEDICATION I dedicate this work to: My family for their love and support throughout the years and My teachers for inspiration iii TABLE OF CONTENTS TITLE PAGE.......................................................................................................... i SIGNATURE PAGE...............................................................................................ii DEDICATION .......................................................................................................iii TABLE OF CONTENTS .......................................................................................iv LIST OF TABLES ...............................................................................................viii LIST OF FIGURES ...............................................................................................ix ACKNOWLEDGEMENTS....................................................................................xii LIST OF ABBREVIATIONS ................................................................................xiv ABSTRACT .......................................................................................................... 1 CHAPTER 1: BACKGROUND ON RPE65 AND RETINOID ISOMERIZATION ... 3 1.1. Introduction to retinal structure and function .............................................. 4 1.1.1. Structure and function of the photoreceptor and pigment epithelium layers of the retina......................................................................................... 4 1.1.2. The retinoid (visual) cycle.................................................................... 5 1.2. Prior studies on RPE65 function and membrane binding........................... 6 1.2.1. Functional characterization of RPE65 ................................................. 7 1.2.2. Biochemical studies on RPE65 membrane binding........................... 10 1.3. Chemistry of Retinoid Isomerization ........................................................ 14 1.3.1. Substrates and products.................................................................... 14 1.3.2. Thermodynamic and kinetic considerations....................................... 16 1.3.3. Mechanisms of retinoid isomerization................................................ 17 1.3.4. Substrate specificity........................................................................... 21 iv 1.3.5. The requirement of an iron cofactor................................................... 22 1.4. Experimental outline, goals and rationale ................................................ 24 Table............................................................................................................... 27 Figures............................................................................................................ 28 CHAPTER 2: EXPRESSION, PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF RPE65..................................................................... 37 2.1. Expression and purification of Zebrafish RPE65...................................... 38 2.1.1. Rationale and Methods...................................................................... 38 2.1.2. Results and Discussion ..................................................................... 40 2.2. Expression and Purification of Human RPE65......................................... 41 2.2.1. Rationale and Methods...................................................................... 41 2.2.2. Results and Discussion ..................................................................... 43 2.3. Generation of an RPE65 monoclonal antibody and mapping of its epitope ........................................................................................................................ 44 2.3.1. Methods............................................................................................. 44 2.3.2. Results and discussion...................................................................... 47 2.4. Extraction and phase separation studies on native RPE65 ..................... 48 2.4.1. Methods............................................................................................. 48 2.4.2. Results and discussion...................................................................... 50 2.5. Purification of Native RPE65 from Bovine RPE ....................................... 52 2.5.1. Rationale and Methods...................................................................... 52 2.5.2. Results and discussion...................................................................... 55 Table............................................................................................................... 58 v Figures............................................................................................................ 59 CHAPTER 3: CRYSTALLIZATION AND STRUCTURE DETERMINATION OF RPE65 ................................................................................................................ 77 3.1. Construction of an RPE65 homology model ............................................ 78 3.2. Attempted crystallization of recombinant RPE65 ..................................... 79 3.3. Crystallization of native bovine RPE65 .................................................... 79 3.4. Diffraction data collection and phasing .................................................... 80 3.5. Model building, refinement and analysis of the model ............................. 86 Table............................................................................................................... 89 Figures............................................................................................................ 90 CHAPTER 4: DESCRIPTION AND INTERPRETATION OF THE RPE65 CRYSTAL STRUCTURE.................................................................................... 98 4.1. A general description of the structure ...................................................... 99 4.2. The membrane-binding surface ............................................................. 100 4.3. The active site cavity and iron-binding site ............................................ 101 4.4. Dimeric structure found in the asymmetric unit and analysis of crystal packing ......................................................................................................... 106 4.5. RPE65 amino acid substitutions associated with Leber congenital amaurosis ..................................................................................................... 109 4.6. Comparison of the RPE65 and apocarotenoid oxygenase structures.... 110 Figures.......................................................................................................... 114 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS .................................. 142 APPENDIX I: CLASSIFICATION OF MEMBRANE PROTEINS ....................... 152 vi BIBLIOGRAPHY............................................................................................... 155 vii LIST OF TABLES Table 1: RPE65 substrate specificity .................................................................. 27 Table 2: Detergents tested for extraction, purification and crystallization of bovine RPE65 ................................................................................................................ 58 Table 3: Data collection, phasing and refinement statistics. ............................... 89 viii LIST OF FIGURES Figure 1: Role of the RPE in maintaining photoreceptor health and function. .... 28 Figure 2: Schematic representation of the retinoid (visual) cycle........................ 30 Figure 3: Potential mechanisms of RPE65 membrane association. ................... 32 Figure 4: Proposed catalytic mechanisms of retinoid isomerization. .................. 34 Figure 5: Expression of an MBP-His10-zRPE65a fusion protein in E. coli and its purification. ......................................................................................................... 59 Figure 6: Purification of recombinant human RPE65 from insect cells. .............. 61 Figure 7: Immunostaining of a mouse retinal section using the monoclonal RPE65 antibody.................................................................................................. 63 Figure 8: Identification of the epitope-containing RPE65 segment recognized by a RPE65 monoclonal antibody. ............................................................................. 65 Figure 9: SDS-PAGE analysis of bovine RPE microsomal proteins after incubation with reagents
Load more

Recommended publications
  • Dominant Retinas: a Novel Pathway for Visual- Pigment Regeneration in Daylight
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Neuron, Vol. 36, 69–80, September 26, 2002, Copyright 2002 by Cell Press Isomerization and Oxidation of Vitamin A in Cone- Dominant Retinas: A Novel Pathway for Visual- Pigment Regeneration in Daylight Nathan L. Mata,1 Roxana A. Radu,1 cis-retinaldehyde through a multistep pathway called Richard S. Clemmons,3 and Gabriel H. Travis1,2,4 the visual cycle (Figure 1B). Most of what is known about 1Jules Stein Eye Institute the visual cycle has come from the study of rod-domi- 2 Department of Biological Chemistry nant species such as cattle and rodents. UCLA School of Medicine Several lines of evidence suggest that rod and cone Los Angeles, California 90095 photopigments regenerate by different mechanisms. In 3 Center for Basic Neuroscience frog retinas separated from the retinal pigment epithe- UT Southwestern Medical Center lium (RPE), cone opsin, but not rhodopsin, regenerates Dallas, Texas 75235 spontaneously (Goldstein and Wolf, 1973; Hood and Hock, 1973). After bleaching, isolated salamander cones, but not rods, recover sensitivity with addition of Summary 11-cis-retinol (Jones et al., 1989). Cultured Mu¨ ller cells isomerize all-trans-retinol to 11-cis-retinol, which they The first step toward light perception is 11-cis to all- secrete into the medium (Das et al., 1992). Mu¨ ller cells, trans photoisomerization of the retinaldehyde chro- in addition to RPE cells, contain cellular retinaldehyde mophore in a rod or cone opsin-pigment molecule. binding protein (CRALBP), which specifically binds 11- Light sensitivity of the opsin pigment is restored cis-retinoids (Bunt-Milam and Saari, 1983; Saari and through a multistep pathway called the visual cycle, Bredberg, 1987).
    [Show full text]
  • Retinoid Isomerase Inhibitors Impair but Do Not Block Mammalian Cone Photoreceptor Function
    Published Online: 2 March, 2018 | Supp Info: http://doi.org/10.1085/jgp.201711815 Downloaded from jgp.rupress.org on April 2, 2018 RESEARCH ARTICLE Retinoid isomerase inhibitors impair but do not block mammalian cone photoreceptor function Philip D. Kiser1,2, Jianye Zhang2, Aditya Sharma3, Juan M. Angueyra4, Alexander V. Kolesnikov3, Mohsen Badiee5, Gregory P. Tochtrop5, Junzo Kinoshita6, Neal S. Peachey1,6,7, Wei Li4, Vladimir J. Kefalov3, and Krzysztof Palczewski2 Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1−/− mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity.
    [Show full text]
  • Key Enzymes of the Retinoid (Visual) Cycle in Vertebrate Retina☆
    Biochimica et Biophysica Acta 1821 (2012) 137–151 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip Review Key enzymes of the retinoid (visual) cycle in vertebrate retina☆ Philip D. Kiser a,1, Marcin Golczak a,1, Akiko Maeda a,b,⁎, Krzysztof Palczewski a,⁎⁎ a Department of Pharmacology, Case Western Reserve University, Cleveland, OH, 44106-4965, USA b Department of Ophthalmology and Vision Sciences, Case Western Reserve University, Cleveland, OH, 44106-4965, USA article info abstract Article history: A major goal in vision research over the past few decades has been to understand the molecular details of Received 19 January 2011 retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that Received in revised form 8 March 2011 result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a Accepted 22 March 2011 variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is Available online 5 April 2011 essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that Keywords: RPE65 ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal Retinol dehydrogenase back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, Visual cycle which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically Retinoid cycle reactive aldehyde that can form toxic conjugates with proteins and lipids.
    [Show full text]
  • Tsin-CV; Updated 6/94
    Updated June, 2019 Andrew Tsin CURRICULUM VITAE Andrew Tsin, Ph.D. Professor and Chair Deaprtment of Molecular Science And Senior Associate Dean of Research School of Medicine The University of Texas Rio Grande Valley (UT Health – Rio Grande Valley) CONTACT INFO: Department of Molecular Science, School of Medicine The University of Texas Rio Grande Valley 1210 W. Schunior St., Edinburg, TX 78541 Phone: (956) 665-6599 Email: [email protected] CITIZENSHIP: USA and CANADA EDUCATION/TRAINING: 1973 BS in Biology, Dalhousie University, Halifax, Nova Scotia, Canada 1976 MS in Zoology (Physiology), The University of Alberta, Edmonton, Alberta, Canada 1979 Ph.D. in Zoology (Physiology), The University of Alberta, Edmonton, Alberta, Canada 1979-81 Postdoctoral Fellow, Cullen Eye Institute, Department of Ophthalmology, Program in Neuroscience, Baylor College of Medicine, Texas Medical Center, Houston, Texas CONTINUED EDUCTION: 2014 Healing Health Care Disparities through Education: An Interdisciplinary Faculty Development Program, Harvard Medical School, Boston, Massachusetts. 2016 Effective Validation of Commercialization Strategy through tactical collection and analysis of data from direct interaction with customer- a short course by The Southwest Node of the National Science Foundation Innovation Corps (I-Corps) Program, The IC2 Institute of the University of Texas at Austin. 2017 Organizational Leadership in Academic Medicine: An Executive Development Seminar for Associate Deans and Department Chairs, AAMC (Association of American Medical
    [Show full text]
  • ANNUAL REPORT 2014–2015 Stein Eye Institute ANNUAL REPORT
    ANNUAL REPORT 2014–2015 Stein Eye Institute ANNUAL REPORT July 1, 2014–June 30, 2015 Director Bartly J. Mondino, MD Faculty Advisor Debora B. Farber, PhD, DPhhc Managing Editor Tina-Marie Gauthier c/o Stein Eye Institute 100 Stein Plaza, UCLA Los Angeles, California 90095–7000 Email: [email protected] Editors Teresa Closson Susan Ito Rosalie Licht Peter J. López Ellen Pascual Debbie Sato M. Gail Summer Guest Writer Dan Gordon Photography Reed Hutchinson J. Charles Martin Robin Weisz Design Robin Weisz/Graphic Design Printing Colornet Press Send questions, comments, and updates to: [email protected]. To view the Annual Report online, visit: jsei.org/annual_report.htm. For more information about the Institute, see: www.jsei.org. ©2015 by the Regents of the University of California. All rights reserved. On the cover: Signature eyewear worn by philanthropists Edie and Lew Wasserman dominate the lobby of the Edie & Lew Wasserman Building, paying homage to the couple’s infinite vision and long- standing commitment to preventing blindness and restoring eyesight. A Year in Review 1 Stein Eye Institute 2 Transitions 6 Alumni News 6 Honors and Awards 8 Research 11 Education 13 Community Outreach 15 Philanthropy 17 Thank You 19 Jules and Doris Stein 24 Board of Trustees and Executive Committee 26 Faculty 29 Programs 87 Patient Care Services 88 Research and Treatment Centers 90 Clinical Laboratories 95 Training Programs 97 Appendices 101 Volunteer and Consulting Faculty 102 Residents and Fellows 104 Educational Offerings 105 Research Contracts and Grants 107 Clinical Research Studies 116 Publications of the Full-Time Faculty 123 Giving Opportunities 130 Dear Friends, The 2014–2015 academic term stands as a banner year for the Stein Eye Institute.
    [Show full text]
  • Processing and Transport of Retinoids by the Retinal Pigment Epithelium
    Eye (1990) 4, 326-332 Processing and Transport of Retinoids by the Retinal Pigment Epithelium DE AN BOK Los Angeles Summary Recent developments regarding our understanding of retinoid processing and trans­ port during the visual cycle and related events are reviewed. Retinoids are bound and protected by a cohort of retinoid binding proteins, each of whiCh is unique. The production of retinol (Vitamin A) derivatives is accomplished by a group of mem­ brane-bound enzymes, some of which appear to be coupled in their actions. Historical Perspectives on a series of biochemical reactions involving The photopigments of all organisms studied the coordinated activity of the retinal pigment to date consist of a chromophore derived from epithelium (RPE) and the photoreceptors.3 Vitamin A (Retinol)! covalently bound to a These events begin with the photobleaching protein by a protonated aldimine bond of rhodopsin to form opsin and all-trans·ret· (Schiff's base).2 The chromophore or pros­ inal, the production of various retinol deriva­ thetic group is, in all cases, retinaldehyde (ret­ tives, the regeneration of ll-cis-retinal and inal) the oxidised form of retinol. Retinol and ultimately, the regeneration of the photopig­ its derivatives are collectively referred to as ment itself. This complex series of interac­ retinoids and the proteins that covalently bind tions is known as the visual cycle. In retinal are called opsins. Both the retinoids Dowling's original observations, light was and the opsins are hydrophobic molecules, found to cause a cis to trans isomerisation of retinol being a fat-soluble vitamin, and the opsin-bound retinal following which retinal opsins belonging to a class of membrane com­ dissociated from the protein and was reduced ponents collectively known as intrinsic pro­ to form all-trans-retinol.
    [Show full text]
  • O O2 Enzymes Available from Sigma Enzymes Available from Sigma
    COO 2.7.1.15 Ribokinase OXIDOREDUCTASES CONH2 COO 2.7.1.16 Ribulokinase 1.1.1.1 Alcohol dehydrogenase BLOOD GROUP + O O + O O 1.1.1.3 Homoserine dehydrogenase HYALURONIC ACID DERMATAN ALGINATES O-ANTIGENS STARCH GLYCOGEN CH COO N COO 2.7.1.17 Xylulokinase P GLYCOPROTEINS SUBSTANCES 2 OH N + COO 1.1.1.8 Glycerol-3-phosphate dehydrogenase Ribose -O - P - O - P - O- Adenosine(P) Ribose - O - P - O - P - O -Adenosine NICOTINATE 2.7.1.19 Phosphoribulokinase GANGLIOSIDES PEPTIDO- CH OH CH OH N 1 + COO 1.1.1.9 D-Xylulose reductase 2 2 NH .2.1 2.7.1.24 Dephospho-CoA kinase O CHITIN CHONDROITIN PECTIN INULIN CELLULOSE O O NH O O O O Ribose- P 2.4 N N RP 1.1.1.10 l-Xylulose reductase MUCINS GLYCAN 6.3.5.1 2.7.7.18 2.7.1.25 Adenylylsulfate kinase CH2OH HO Indoleacetate Indoxyl + 1.1.1.14 l-Iditol dehydrogenase L O O O Desamino-NAD Nicotinate- Quinolinate- A 2.7.1.28 Triokinase O O 1.1.1.132 HO (Auxin) NAD(P) 6.3.1.5 2.4.2.19 1.1.1.19 Glucuronate reductase CHOH - 2.4.1.68 CH3 OH OH OH nucleotide 2.7.1.30 Glycerol kinase Y - COO nucleotide 2.7.1.31 Glycerate kinase 1.1.1.21 Aldehyde reductase AcNH CHOH COO 6.3.2.7-10 2.4.1.69 O 1.2.3.7 2.4.2.19 R OPPT OH OH + 1.1.1.22 UDPglucose dehydrogenase 2.4.99.7 HO O OPPU HO 2.7.1.32 Choline kinase S CH2OH 6.3.2.13 OH OPPU CH HO CH2CH(NH3)COO HO CH CH NH HO CH2CH2NHCOCH3 CH O CH CH NHCOCH COO 1.1.1.23 Histidinol dehydrogenase OPC 2.4.1.17 3 2.4.1.29 CH CHO 2 2 2 3 2 2 3 O 2.7.1.33 Pantothenate kinase CH3CH NHAC OH OH OH LACTOSE 2 COO 1.1.1.25 Shikimate dehydrogenase A HO HO OPPG CH OH 2.7.1.34 Pantetheine kinase UDP- TDP-Rhamnose 2 NH NH NH NH N M 2.7.1.36 Mevalonate kinase 1.1.1.27 Lactate dehydrogenase HO COO- GDP- 2.4.1.21 O NH NH 4.1.1.28 2.3.1.5 2.1.1.4 1.1.1.29 Glycerate dehydrogenase C UDP-N-Ac-Muramate Iduronate OH 2.4.1.1 2.4.1.11 HO 5-Hydroxy- 5-Hydroxytryptamine N-Acetyl-serotonin N-Acetyl-5-O-methyl-serotonin Quinolinate 2.7.1.39 Homoserine kinase Mannuronate CH3 etc.
    [Show full text]
  • 12) United States Patent (10
    US007635572B2 (12) UnitedO States Patent (10) Patent No.: US 7,635,572 B2 Zhou et al. (45) Date of Patent: Dec. 22, 2009 (54) METHODS FOR CONDUCTING ASSAYS FOR 5,506,121 A 4/1996 Skerra et al. ENZYME ACTIVITY ON PROTEIN 5,510,270 A 4/1996 Fodor et al. MICROARRAYS 5,512,492 A 4/1996 Herron et al. 5,516,635 A 5/1996 Ekins et al. (75) Inventors: Fang X. Zhou, New Haven, CT (US); 5,532,128 A 7/1996 Eggers Barry Schweitzer, Cheshire, CT (US) 5,538,897 A 7/1996 Yates, III et al. s s 5,541,070 A 7/1996 Kauvar (73) Assignee: Life Technologies Corporation, .. S.E. al Carlsbad, CA (US) 5,585,069 A 12/1996 Zanzucchi et al. 5,585,639 A 12/1996 Dorsel et al. (*) Notice: Subject to any disclaimer, the term of this 5,593,838 A 1/1997 Zanzucchi et al. patent is extended or adjusted under 35 5,605,662 A 2f1997 Heller et al. U.S.C. 154(b) by 0 days. 5,620,850 A 4/1997 Bamdad et al. 5,624,711 A 4/1997 Sundberg et al. (21) Appl. No.: 10/865,431 5,627,369 A 5/1997 Vestal et al. 5,629,213 A 5/1997 Kornguth et al. (22) Filed: Jun. 9, 2004 (Continued) (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2005/O118665 A1 Jun. 2, 2005 EP 596421 10, 1993 EP 0619321 12/1994 (51) Int. Cl. EP O664452 7, 1995 CI2O 1/50 (2006.01) EP O818467 1, 1998 (52) U.S.
    [Show full text]
  • UNIVERSITY of CALIFORNIA Los Angeles New Mechanisms for Regeneration of Visual Pigments in the Vertebrate Retina a Dissertation
    UNIVERSITY OF CALIFORNIA Los Angeles New Mechanisms for Regeneration of Visual Pigments in the Vertebrate Retina A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular, Cellular, and Integrative Physiology by Tongzhou Xu 2017 © Copyright by Tongzhou Xu 2017 ABSTRACT OF THE DISSERTATION New Mechanisms for Regeneration of Visual Pigments in the Vertebrate Retina by Tongzhou Xu Doctor of Philosophy in Molecular, Cellular, and Integrative Physiology University of California, Los Angeles, 2017 Professor Gabriel H. Travis, Chair Vision begins when a photon is captured by a visual pigment in a photoreceptor cell. For most vertebrates, the light-sensitive chromophore in the visual pigment is 11-cis- retinaldehyde (11-cis-retinal), which is covalently bound to the protein moiety, opsin, through a Schiff base linkage. The absorption of a photon isomerizes 11-cis-retinal to its all-trans-isomer and activates the visual pigment. In vertebrate ciliary photoreceptors, after a brief activation, the pigment decays to yield apo-opsin and free all-trans-retinal. Light sensitivity restoration in photoreceptor cells occurs following re-isomerization of all-trans-retinal to 11-cis-retinal by enzymatic pathways termed “visual cycles”. At present, two visual cycles have been established: (1) A well understood canonical visual cycle between RPE cells and photoreceptors, with RPE65 as its retinoid isomerase (Isomerase I); (2) A relatively under characterized alternate visual cycle between Müller cells and cones, with an unknown retinoid isomerase (Isomerase II). Recently, we have identified dihydroceramide desaturase-1 (DES1) as a novel retinoid isomerase and a strong candidate for Isomerase II.
    [Show full text]
  • Transforming Eye Care
    Stein Eye Institute Annual Report 2013–2014 Annual Report 2013–2014 TRANSFORMING EYE CARE LOS ANGELES AND BEYOND The Stein Eye Institute is a proud affiliate of the Doheny Eye Institute. David Geffen School of Medicine at UCLA University of California, Los Angeles Stein Eye Institute Annual Report July 1, 2013–June 30, 2014 Director Bartly J. Mondino, MD Faculty Advisor Debora B. Farber, PhD, DPhhc Managing Editor Tina-Marie Gauthier c/o Stein Eye Institute 100 Stein Plaza, UCLA Los Angeles, California 90095–7000 Email: [email protected] Editors Teresa Closson Ellen Haupt Rosalie Licht Peter J. López Debbie Sato M. Gail Summers Guest Writer Dan Gordon Photography Reed Hutchinson J. Charles Martin ASUCLA Photography Design Robin Weisz/Graphic Design Printing Colornet Press Send questions, comments, and updates to: [email protected]. To view the Annual Report online, visit: jsei.org/annual_report.htm. For more information about the Institute, see: www.jsei.org. ©2014 by the Regents of the University of California. All rights reserved. A Year in Review 1 Stein Eye Institute 2 Transitions 4 Honors and Awards 5 Research 8 Education 10 Community Outreach 13 Philanthropy 15 Thank You 18 Jules and Doris Stein 25 Board of Trustees and Executive Committee 26 Faculty 29 Programs 81 Patient Care Services 82 Research and Treatment Centers 84 Clinical Laboratories 89 Training Programs 91 Appendices 95 Volunteer and Consulting Faculty 96 Residents and Fellows 98 Educational Offerings 99 Research Contracts and Grants 101 Clinical Research Studies 106 Publications of the Full-Time Faculty 112 Giving Opportunities 118 Dear Friends, In November 1966 the doors of the Jules Stein Eye Institute opened on the grounds of UCLA, and with dedication of purpose, the Institute has continued to grow in its commitment to further vision-science re- search, ophthalmic education, and patient care.
    [Show full text]
  • Identification of the 11-Cis-Specific Retinyl-Ester Synthase in Retinal Müller Cells As Multifunctional O-Acyltransferase (MFAT)
    Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT) Joanna J. Kaylora, Jeremy D. Cooka, Jacob Makshanoffa, Nicholas Bischoffa, Jennifer Yonga, and Gabriel H. Travisa,b,1 aJules Stein Eye Institute and bDepartment of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, CA 90095 Edited by Robert S. Molday, University of British Columbia, Vancouver, Canada, and accepted by the Editorial Board April 11, 2014 (received for review October 11, 2013) Absorption of a photon by a rhodopsin or cone-opsin pigment pathway is dihydroceramide desaturase (DES1) (11). DES1 cata- isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to lyzes rapid equilibrium isomerization of retinol (11). At equilibrium, all-trans-retinaldehyde (all-trans-RAL), which dissociates after a 11-cis-ROL is much less abundant than all-trans-ROL, due to the brief period of activation. Light sensitivity is restored to the 4.1 kcal/mole difference in free energy between these isomers (14). resulting apo-opsin when it recombines with another 11-cis-RAL. Accordingly, a secondary source of energy is required to drive Conversion of all-trans-RAL to 11-cis-RAL is carried out by an en- the conversion of all-trans-ROL to 11-cis-ROL by DES1. zyme pathway called the visual cycle in cells of the retinal pigment Retinas from cone-dominant species contain 11-cis-retinyl esters epithelium. A second visual cycle is present in Müller cells of the (11-cis-REs), whereas retinyl esters are much less abundant in retina.
    [Show full text]
  • Substrate Specificity and Reaction Mechanism of Vertebrate Carotenoid Cleavage
    Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Carlo C. dela Seña The Ohio State Biochemistry Program The Ohio State University 2014 Dissertation Committee: Earl H. Harrison, Ph.D., Adviser Robert W. Curley, Jr., Ph.D. Ross E. Dalbey, Ph.D. Steven J. Schwartz, Ph.D Copyright by Carlo C. dela Seña 2014 i Abstract Carotenoids are yellow, orange and red pigments found in fruits and vegetables. Some carotenoids can act as dietary precursors of vitamin A. Humans and other animals generate retinal (vitamin A aldehyde) from provitamin A carotenoids by oxidative cleavage of the central 15-15′ double bond by the enzyme β-carotene 15-15′-oxygenase (BCO1). Another carotenoid oxygenase, β-carotene 9′-10′-oxygenase (BCO2), catalyzes the oxidative cleavage of the 9′-10′ double bond of various carotenoids to yield apo-10′- carotenals and ionones. In this dissertation, we elucidate the substrate specificity of these two enzymes. Recombinant His-tagged human BCO1 was expressed in Escherichia coli strain BL21- Gold (DE3) and purified by cobalt ion affinity chromatography. The enzyme was incubated with various dietary carotenoids and β-apocarotenals, and the reaction products were analyzed by reverse-phase high-performance liquid chromatography (HPLC). We found that BCO1 catalyzes the oxidative cleavage of only provitamin A dietary carotenoids and β-apocarotenals specifically at the 15-15′ double bond to yield retinal. A notable exception is lycopene, which is cleaved by BCO1 to yield two molecules of acycloretinal.
    [Show full text]