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CONTENTS

Message from the Chair

Department History

Department Staff

Faculty List and Contact Information

Research Themes

Faculty Research Programs

Core Facilities

Student and Life in Lexington

Message from the Chair

Modern is advancing at an astounding rate, as new scientific knowledge provides fundamental insight into how biological systems work, leading inevitably to advancements in the cause, diagnosis, and treatment of disease. is fundamental to this progress because, as a discipline, it seeks to understand the molecular mechanisms underlying both normal and pathogenic processes. In the Department of Molecular and Cellular Biochemistry, we see biochemistry as a critical element in devising novel strategies for maintaining human health and combating disease.

As a faculty we have two equally important missions. These are to promote outstanding research with a focus on understanding the molecular basis of fundamentally important life processes and provide the highest quality training for the next generation of biomedical research scientists. We are committed to assisting PhD students in the development of their writing and public speaking skills and as bench scientists. The department provides a supportive working environment, with a friendly atmosphere, a highly innovative and interactive group of 45 faculty (both primary and joint), 30 graduate students, and numerous postdoctoral fellows and staff dedicated to understanding life at the biochemical, genetic and cellular level. With $7.6M in extramural support the department ranks in the top twenty in NIH funding among biochemistry departments in public medical schools. Using cutting-edge technologies and experimental systems, ranging from modern biochemical and biophysical methods to cellular and organismal analyses, our distinguished faculty is focused on elucidating the molecular events involved in diseases such as cancer, neurodegenerative disorders, diabetes, cardiovascular disease, aging, and to investigate the essential properties of disease-causing and protozoan pathogens.

On behalf of the entire Department of Biochemistry and Molecular , I invite you to explore these web pages, which provide information on our research and education missions, and to contact individual faculty for more information about the range of exciting research opportunities in modern biochemistry. Please feel free to come by and visit.

Sincerely,

Douglas A. Andres, Ph.D. Professor & Chair

Department History

Memorial Hall University of Kentucky

The year 1960 marked the beginning of the Medical Center of the University of Kentucky and its basic science departments of the College of Medicine, including the Department of Biochemistry. That year, the department began teaching the first freshman class of medical students. The faculty consisted of George W. Schwert (Ph.D., Minnesota), a World War II Navy veteran who came from Duke University and joined the department as chairman; Richard S. Schwert (Ph.D., Iowa State) a World War II Air Force veteran who was recruited from the City of Hope Hospital in California; and Robert L. Lester (Ph.D., California Institute of Technology), who was recruited from the University of Wisconsin. The faculty reflected the then current research interests of enzymology, synthesis, and . The faculty size increased to six for a number of years and then gradually rose to its current size as the graduate program expanded, additional teaching duties were added, and its research mission became prominent. Michael Wells was the department’s first Ph.D. student and subsequently became a founding member and later head of the Biochemistry Department at the University of Arizona Medical School. Subsequent chairmen were Robert L. Lester, Thomas C. Vanaman (Ph.D., Duke), Louis B. Hersh (Ph.D., Brandeis) and the current chair, Doug A. Andres (Ph.D., Purdue). In 1989, the department changed its name to its current one to reflect the changing spectrum of faculty research interests.

Biomedical and Biological Sciences Research Building (BBSRB)

The department resides principally on the first two floors of this building.

Department Staff

Front row, left to right: Saundra Stinnett, Tonya Simon, James Tribble Second Row, left to right: Brenda , Stephanie Viens, Rachel Putty, Marilou Johnson, Diana Griffieth Back row, left to right: Taha Al-Jumaily, Phil Dickson

BBSRB Name Phone Responsibility Room #

Taha Al-Jumaily (859) 323-9770 167 Information Technology Support Phil Dickson (859) 323-0093 131 Stockroom & Lab Equipment Diana Griffieth (859) 323-5593 178-E Purchasing Marilou Johnson (859) 323-5549 278-E Assistant to the Chair Rachel Putty (859) 323-5492 278-D Faculty Accounts Administration Tonya Simon (859) 323-6048 279 Department Administrator Department & Faculty Accounts Saundra Stinnett (859) 323-5282 277 Administration Assistant to editor, Journal of Biological Donna Sumner (859) 323-8182 148-A Chemistry James Tribble (859) 323-2150 236-B Labwares & Autoclave COBRE Program Administration & Stephanie Viens (859) 323-6050 278-A Assistant to the Director of Graduate Studies Brenda Woods (859) 323-5546 278-B Payroll & Visas

Faculty List and Contact Information *Denotes faculty with secondary appointment in Biochemistry

Andres, Douglas *Kyprianou, Natasha dandres@ uky.edu [email protected] (859) 257-6775 (859) 323-9812 *Bradley, Luke LeVine, Harry [email protected] [email protected] (859) 323-1826 (859) 257-1412 x224 *Chappell, Joe *Li, Guo-Min [email protected] [email protected] (859) 257-5020 x80775 (859) 257-7053 Creamer, Trevor *Li, Zhenyu [email protected] [email protected] (859) 323-6037 (859) 257-0528 Dickson, Bob Liu, Chunming [email protected] [email protected] (859) 323-6052 (859) 323-4588 Dutch, Becky Mendenhall, Mike [email protected] [email protected] (859) 323-1795 (859) 257-5379 *Evers, Mark *Miller, Anne-Frances [email protected] [email protected] (859) 323-6556 (859) 257-9349 Fondufe-Mittendorf, Yvonne Murphy, Paul [email protected] [email protected] (859) 323-0091 (859) 257-1412 x490 Fried, Michael Noonan, Dan [email protected] [email protected] (859) 323-1205 (859) 257-7498 Galperin, Emilia O’Conner, Kathleen [email protected] [email protected] (859) 323-1796 (859) 323-7534 Gao, Tianyan Özcan, Sabire [email protected] [email protected] (859) 323-3454 (859) 257-4821 Gentry, Matthew Rodgers, David [email protected] [email protected] (859) 323-8482 (859) 257-5205 Hersh, Lou Sarge, Kevin [email protected] [email protected] (859) 323-5540 (859) 323-5777 Jia, Jianhang Spielmann, Peter [email protected] [email protected] (859) 323-6236 (859) 257-4790 Korotkov, Konstantin Stamm, Stefan [email protected] [email protected] (859) 323-5493 (859) 323-0896

Turco, Sam Wang, Qingjun [email protected] [email protected] (859) 323-6693 (859) 323-5335 Vanaman, Thomas (post-retirement) Watt, David [email protected] [email protected] (859) 257-1347 (859) 257-7209 Vander Kooi, Craig Whiteheart, Sidney [email protected] [email protected] (859) 323-8418 (859) 257-4882 *van der Westhuyzen, Deneys Zhou, Binhua Peter [email protected] [email protected] (859) 323-4933 x81397 (859) 323-4474 Waechter, Skip Zhu, Haining [email protected] [email protected] (859) 323-6352 (859) 323-3643

Research Themes

The faculty embrace a broad spectrum of research interests, and the following brief list captures some of the categories that best describe their research programs:

Aging and Longevity Molecular Signaling and Cellular Communication Bob Dickson Douglas Andres Qingjun Wang Trevor Creamer Biochemistry of Infectious Disease Bob Dickson Becky Dutch Yvonne Fondufe-Mittendorf Konstantin Korotkov Emilia Galperin Cancer Biology Matthew Gentry Douglas Andres Jianhang Jia Yvonne Fondufe-Mittendorf Guo-min Li Tianyan Gao Chunming Liu Jianhang Jia Dan Noonan Natasha Kyprianou Kathleen O’Connor Guo-min Li Sabire Özcan Chunming Liu David Rodgers Anne-Frances Miller Kevin Sarge Dan Noonan Qingjun Wang Kathleen O’Connor Sidney Whiteheart Binhua Peter Zhou Haining Zhu Michael Fried Neurochemistry and Neurodegenerative Diseases Cardiovascular Biology Douglas Andres Sidney Whiteheart Trevor Creamer Cardiovascular Disease Matthew Gentry Deneys van der Westhuyzen Lou Hersh Harry LeVine Peter Spielmann Guo-min Li David Watt Anne-Frances Miller Joe Chappell David Rodgers Michael Fried Qingjun Wang Drug Design Haining Zhu Luke Bradley Nucleic Acids and Disease Chunming Liu Stefan Stamm Dan Noonan Proteomics and David Rodgers Trevor Creamer Peter Spielmann Yvonne Fondufe-Mittendorf David Watt Qingjun Wang Metabolic Disorders/Diabetes Haining Zhu Sabire Özcan Molecular Basis of Cellular Architecture Luke Bradley Kathleen O’Connor Trevor Creamer Sam Turco Becky Dutch Qingjun Wang Yvonne Fondufe-Mittendorf Sidney Whiteheart Michael Fried Molecular Matthew Gentry Bob Dickson Konstantin Korotkov Guo-min Li Anne-Frances Miller Dan Noonan David Rodgers Stefan Stamm Peter Spielmann Joe Chappell Craig Vander Kooi David Watt Joe Chappell

Faculty Research Programs (listed alphabetically)

The following section of this brochure provides individual faculty research descriptions, a photograph, a list of common techniques used in the laboratory, and recent and representative publications. Some faculty have individual web sites, which are also provided.

The journal covers below feature some of our faculty’s recent research.

Douglas Andres

Our laboratory's research is focused on defining the molecular basis of cellular communication. Our studies center on the identification and biological characterization of several families of Ras-related , to characterize their cellular functions, and to define the cascades which mediate their actions. Our long term goal is to apply this knowledge to understand human diseases that arise from signaling dysfunction and to develop rational for the control of diseases as diverse as heart disease, diabetes, and nerve survival/ . Rit/Rin-mediated Neuronal Survival: Aberrant contributes to various Dr. Andres in back row, second from left. neurodegenerative disorders including stroke, epilepsy, and Alzheimer's disease. Elaborating Contact the signal transduction mechanisms that [email protected], (859) 257-6775 regulate neuronal survival is thus important for understanding both basic biology and for therapeutic intervention. We have discovered that Rit directs neuronal survival and the generation of new adult , while loss of the Rin GTPase is associated the Parkinson’s Disease risk. Regulation of Channels by RGK GTPases: Voltage-dependent calcium channels regulate the flow of Ca2+ across cellular as a of potential. This calcium influx serves to regulate a variety of cellular functions, including muscle contraction, endocrine , and synaptic transmission. We have shown that the Rem and Rad GTPase regulate both cardiac rhythm, muscle contraction, and pressure, and are promising targets for the treatment of cardiac diseases, including heart failure, ischemic dysfunction, and cardiac arrhythmias. The Gem GTPase controls insulin secretion to maintain normal , with alterations in this cascade contributing to diabetes.

Common techniques used in our laboratory

Molecular Biology (e.g., site-directed mutagenesis, expression vector ); immunoblot analysis (Western blotting, ); kinase and GTPase cascade analysis (kinase and GTP/GDP exchange biochemical assays); generation and analysis of transgenic and knockout mouse models; Drosophila genetics; isolation of primary cardiomyocyte and cultures; of calcium channels; (immunohistochemistry).

Recent and representative publications

Gunton, J.E., et. Al.. (2012) Mice deficient in GEM GTPase show abnormal glucose homeostasis due to defects in Beta-cell calcium handling. PLoS ONE 7(6), e39462. Cai, W., Carlson, S. W., Mannon, C., Pleasant, J. M., Saatman, K. E., and Andres D. A. (2012) Rit GTPase Signaling Promotes Immature Hippocampal Neuronal Survival. The Journal of Neuroscience 32(29), 9887-09897. PMID 22815504 Magyar, J., Kiper, C., Shawn, S. M., Cai, W., Li, L., Smith, N., Andres, D. A., and Satin, J. (2012) Rem GTPase Regulates Cardiac Myocyte L-type Calcium Current. Channels 6(3), 166-173. Cai, W., Shi, G-X., and Andres, D.A. (2012) Putting the Rit in Cellular Resistance: Rit, p38 MAPK, and oxidative stress. Comm. & Integrative Biology. In Press. Shi, G,-X and Andres, D.A. (2012) Rit-mediated stress resistance involves a p38-MSK1-dependent CREB activation cascade. J. Biol. Chem. In Press.

Luke Bradley

Our laboratory works at the interface of chemistry, biochemistry, and to advance our understanding of protein and function in the central nervous system. We are using protein design, molecular screening, and techniques to develop with specific biological activities. Detailed biochemical and biophysical characterization of these molecules will contribute towards understanding the basis of the native protein’s function. In addition, these molecules will serve as translational platforms for the development of various medical, biotechnical, Dr. Bradley, second from right. and therapeutic applications. One particular

Contact project of interest is focused on Parkinson’s [email protected], (859) 323-1826 disease (PD), a chronic, disabling neurodegenerative disorder that affects over 1 million Americans and is expected to double in incidence over the next 40 years. This movement disorder, most often diagnosed in people over the age of 50, is characterized by one or more of the following symptoms: bradykinesia, stooped posture, rigidity, resting hand tremors, balance impairments, and gait difficulty. These multisymptoms are linked with the dysregulation of the neurotransmitter dopamine, resulting from the loss of dopamine neurons in the substantia nigra. Glial cell line- derived neurotrophic factor (GDNF), a neurotrophic factor with protective and restorative effects on dopamine neurons and in vivo, was identified as a potential therapeutic agent for PD. However, GDNF did not progress from PD clinical trials, likely due to the pharmacological disadvantages associated with the invasive delivery of large protein molecules directly to the brain. Thus, a viable approach towards the development of potential PD therapeutics is to construct and evaluate novel smaller molecular alternatives with neurotrophic factor activity. We are examining the protective properties of dopamine neuron stimulating - 11 (DNSP-11) and other amidated, small (< 1/25th the size of GDNF) peptides derived from the GDNF proprotein, using molecular, proteomic and cellular biology approaches. This information will pave the way towards utilizing these identified sequences as molecular platforms for the further development of novel parkinsonism/PD therapeutics and age-related neurodegenerative diseases.

Common techniques used in our laboratory

Peptide and protein engineering and design; in vitro and in vivo screens and selections; chemical modification of and ; assays.

Recent and representative publications

Magnani R, Chaffin B, Dick E, Bricken ML, Houtz RL, Bradley LH. (2012) Utilization of a calmodulin lysine methyltransferase co-expression system for the generation of a combinatorial library of post-translationally modified proteins. Protein Expression & Purification, in press. Littrell OM, Fuqua JF, Richardson AD, Turchan-Cholewo J, Hascup ER, Huettl P, Pomerleau F, Bradley LH, Gash DM, Gerhardt GA. (2012) A synthetic five GDNF propeptide increases dopamine neuron differentiation and neurochemical function. Neuropeptides, in press. Sexton T, Boock LJ, Rodgers DR, Bradley LH, Hersh LB. (2012) Active site change the specificity of neprilysin. PLoS ONE, 7(2): e32343. Kelps KA, Turchan-Cholewo J, Hascup ER, Taylor TL, Gash DM, Gerhardt GA, Bradley LH. (2011) Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence. Neuropeptides 45(3): 213-218.

Joe Chappell make a dizzying and dazzling array of chemicals. Well over 100,000 different compounds, compounds that are distinctly different from those needed for general house- keeping functions like amino acids, fatty acids and typical , have been isolated from plants and characterized. In the relevant scientific literature, this richness of chemical diversity is referred to as specialized . Plants have evolved specialized biochemical abilities over geological time that provide them with unique evolutionary advantages. Some of the specialized chemicals serve to attract beneficial insects that might help the plants reproduce more efficiently. Others of these chemicals might serve to ward off pests and pathogens, giving those plants that produce them the ability to occupy habits or niches that otherwise would be uninhabitable. The chemical richness Dr. Chappell in back row, second from right. of plants has not escaped the Contact attention of the biochemical [email protected], (859) 323-1826 and medical communities. About two-thirds of all the drugs currently used in medical therapies are isolated from natural sources, or were inspired by compounds found in nature. Unfortunately, the chemical complexity of natural compounds cannot be easily mimicked by synthetic or organic chemistry, hence understanding the native biochemical machinery provides yet another means for expanding on the chemical warehouses of plants. And of course the chemistry of plants is green chemistry, processes driven by and atmospheric CO2, and yielding the chemicals to make our planet more sustainable in terms of production and industrial manufacturing. These are the aims of the Chappell laboratory, mapping complex biochemical pathways and creating transgenic plants and microbes for the production of new drugs and biofuels.

Common techniques used in our laboratory One of the defense responses of Solanaceous plants to pathogen Transcriptomic and metabolomics analyses, heterologous expression, challenge is production of anti- assays, bio-assays, computational interrogation of biological datasets, genetic microbial compounds. When engineering tobacco cell cultures are treated with fungal elicitors, the cells synthesize and secrete novel Recent and representative publications chemicals into the culture media in a time dependent manner. When Niehaus TD, Okada S, Devarenne TP, Watt DS, Sviripa V, and Chappell J. (2011) aliquots of the media samples are Identification of unique mechanisms for triterpene in separated by thin-layer Botryococcus braunii. Proc. Natl. Acad. Sci.108, 12260-12265. and the TLC Wu S, Jiang Z, Kempinski C, Nybo E, Husodo S, Williams R, and Chappell J. (2012) plates over-laid with fungal spores, Engineering triterpene metabolism in tobacco. Planta 236, 867-877. zones of inhibition (white zones) become apparent wherever the Web Site fungal spores cannot grow. http://www.uky.edu/Ag/Agronomy/Chappell/welcome.htm

Trevor Creamer

Our laboratory is studying the function and regulation of the serine/threonine phosphatase called calcineurin (CaN). CaN is ubiquitously expressed and highly conserved in all . As an integral component of several signaling pathways, this enzyme dephosphorylates, and thereby regulates, a number of important proteins such as the Nuclear Factor of Activated T-cells (NFAT) family of activators, the -associated protein tau, and (NOS). CaN plays central roles in neuronal signaling, cardiac growth, and immune system activation. Dysregulation of CaN contributes to a number of Dr. Creamer in middle. disorders including Alzheimer’s disease, Down syndrome, mental retardation, cardiac hypertrophy, Contact and autoimmune diseases. [email protected], (859) 323-6037 CaN activity is tightly regulated by a number of other proteins, including the ―king‖ of regulatory proteins, calmodulin (CaM). CaM binds to and activates CaN. Other proteins, including Rcan1, CHP1 and cabin/cain, are endogenous inhibitors of CaN. Still others, such as AKAP79, serve to localize CaN to specific regions within a cell. Breakdowns in any of these regulatory systems can lead to associated with the diseases listed above. Following an increase in cellular calcium levels, CaM binds to and activates CaN. CaM binds to a regulatory within CaN, which leads to a conformational change that in turn ejects an autoinhibitory domain from CaN’s active site. The regulatory domain of CaN is devoid of structure prior to CaM binding. In other words, this domain is intrinsically disordered. Upon CaM binding, the CaN regulatory domain gains structure, and we are studying this disorder-to-order transition and how it leads to CaN activation. Rcan1 (regulator of calcineurin 1) is an endogenous inhibitor of CaN that has been associated with Down syndrome and Alzheimer’s disease. This protein is known to bind to the catalytic domain of CaN, blocking access to the active site. Rcan1 possesses two domains: a well-structured domain of unknown function, and the CaN binding domain which appears to be completely unstructured. This interesting situation is best summarized as one in which an enzyme with an intrinsically disordered regulatory domain is inhibited by a protein with an intrinsically disordered inhibitory domain. Rcan1 binding to CaN is a potential drug, but little is known regarding the details of the interactions between these proteins. CHP1 (Calcineurin B Homology Protein 1) is another endogenous inhibitor of CaN. What is remarkable about this protein is that, like the CaN B chain, it is structurally homologous to CaM. CaN is thus regulated by three proteins with the same structure, but with three different roles. CaM activates, the CaN B chain has a structural role, and CHP1 inhibits. Other than the fact that it inhibits CaN, little is known about how CHP1 functions.

Common techniques used in our laboratory

Molecular biology; protein expression and purification; enzyme assays; fluorescence (steady-state and time-resolved); circular dichroism ; analytical ultracentrifugation; NMR; x-ray crystallography.

Recent and representative publications

Smith EC, Gregory SM, Tamm LK, Creamer TP, and Dutch RE (2012) Role of sequence and structure of the hendra fusion peptide in membrane fusion. J Biol Chem 287, 30035-48. Smith EC, Culler MR, Hellman LM, Fried MG, Creamer TP and Dutch RE (2012) Beyond anchoring: the expanding role of the hendra fusion protein transmembrane domain in protein folding and stability. J Virol 86, 3003-13.

Web Site http://www.mc.uky.edu/Biochemistry/labs/creamer/index.html

Robert Dickson Some people grow old yet show few signs of aging, while others show signs of aging long before they grow old. How can this be? It is due to individual variation in the biological networks that regulate cellular processes. For example, as we grow older some of the ability of some networks to maintain normal homeostasis decline leading to the so-called age-related diseases. Our research aims to develop ways to decrease the incidence of age-related diseases including diabetes, cancer, cardiovascular and inflammatory dysfunction and by controlling. Reducing the frequency of these diseases will improve human health and may Dr. Dickson on left. enhance lifespan. We are trying to identify Contact and understand the signal transduction [email protected], (859) 323-6052 pathways and cellular processes that control the networks that regulate the rate of biological aging and lifespan. We use the common baker’s yeast Saccharomyces cerevisiae as a model because it has a short lifespan that can be analyzed by genetic and biochemical techniques. A striking outcome of studies in model is that similar proteins and signaling pathways regulate lifespan. Glucose in yeast and insulin or insulin-like in other organisms regulate signal transduction pathways that limit lifespan (see the figure). Protein kinases are a well-conserved feature of these signaling pathways. The Akt/PKB protein kinases are found in organisms ranging from baker’s yeast, where the protein is called Sch9, to worms and man where these kinases play roles in insulin signaling, apoptosis and cell proliferation, all of which contribute to aging and lifespan. One focus of our research is to understand how a combination of two drugs used at low doses can produce a synergistic increase in lifespan as measured by how long cells survive in stationary phase after division stops. Another focus of our research is to understand how treatment of cells with a drug that down-regulates sphingolipid synthesis is able to enhance lifespan via gobal actions on signaling pathways including the PKA, TORC1, Snf1/AMPK and Sch9 signaling pathways (see figure) Another focus of our research is to understand how signaling pathways control homeostasis and redox balance in order to protect cells from killing during Evolutionary conserved signal transduction pathways that regulate aging iron deprivation, the most common human nutritional and lifespan. disorder in the world.

Common techniques used in our laboratory

Molecular genetic analysis of mutant yeast strains; gene modification by deletion and site-directed techniques; protein isolation and characterization; Western blotting; analysis by high pressure liquid chromatography; chronological lifespan assays; profiling by DNA microarrays; assays for , mitochondrial functions, stress resistance and genomic stability.

Recent and representative publications

Huang, X., Liu, J., Dickson, R. C. (2012) Down-regulating sphingolipid synthesis increases yeast lifespan, PLoS Genet. 8(2): e1002493. Lee, Y-J., Huang, X., Kropat, J., Henras, A., Merchant, S. S., Dickson, R. C., Chanfreau, G. F. (2012) Sphingolipid Signaling Mediates Iron , Cell Metabolism 16:90-96.

Becky Dutch Our research focuses on the molecular details of viral infection, with an emphasis on the entry of paramyxoviruses into cells. Our studies on viral glycoproteins aim to elucidate mechanisms of promotion of membrane fusion and the receptors and pathways utilized for infection, and to provide new targets for antiviral treatments. Part of our work examines the fusion proteins from the Hendra and Nipah viruses, newly emerged diseases in the paramyxovirus family that are highly pathogenic in multiple species, including , and which are classified as Biosafety Level 4 pathogens. Our Dr. Dutch in back row, far right. laboratory has identified cathepsin L, a cellular endosomal/lysosomal protease, as critical protein for Contact activation of the Hendra and Nipah fusion proteins, [email protected], (859) 323-1795 and thus a potential drug target. The Hendra and Nipah fusion proteins undergo a complex intracellular trafficking pathway to allow cleavage by cathepsin L, and analysis of the protein signals involved is providing new insights into the mechanisms of endocytic trafficking and sorting. We have identified key regions within the Hendra F protein that are important for the initiation of membrane fusion, and have been examining the functions of membrane-interacting domains within the protein, including analysis of the transmembrane domain and the fusion peptide domain. Recently, we have been the first to demonstrate that the transmembrane domains of these fusion proteins interact with each other in a monomer-trimer equilibrium in the absence of the rest of the protein, and we are currently studying the role of transmembrane domain interactions. Our laboratory also works on human metapneumovirus (HMPV), a recently identified virus that is a causative agent of severe respiratory disease. We have shown that the HMPV fusion protein can provide binding to target cells in the absence of the viral attachment protein, and identified cell surface heparan sulfate as a critical attachment factor. Our studies of clade A HMPV have shown that of the virus is important for target cell entry. Low pH can trigger membrane fusion promotion by the HMPV fusion protein, and we have identified key residues important for this process.

Common techniques used in our laboratory

Transient expression of proteins in culture cells; metabolic labeling; immunoprecipitation; western blot analysis; expression of recombinant proteins; cell- assays; entry of pseudotype particles (Hendra or Nipah virus); creation and study of recombinant viruses (HMPV), with an emphasis on study of , flow , molecular biology including site-directed mutagenesis, PCR, and rtPCR; immunofluorescence; analytical ultracentrifugation; and circular dichroism.

Recent and representative publications

Chang, A and Dutch, RE (2012). Paramyxovirus fusion and entry: multiple paths to a common end. Viruses 4(4), 613- 36. Chang, A, Hackett, BA, Winter, CC, Buchholz, UJ and Dutch, RE (2012). Potential electrostatic interactions in multiple regions affect HMPV F-mediated membrane fusion. J Virol 86, 9843-53. Chang, A, Masante, C, Buchholz, UJ and Dutch, RE (2012). Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate. J Virol 86(6), 3230-43. Popa, A, Carter, JR, Smith, SE, Hellman, L, Fried, MG and Dutch, RE (2012). Residues in the hendra virus fusion protein transmembrane domain are critical for endocytic recycling. J Virol 86(6), 3014-26.

Web Site http://www.mc.uky.edu/biochemistry/labs/dutch/default.asp

Yvonne Fondufe-Mittendorf

Central to chromatin's role in regulating a number of basic cellular processes is its ability to adopt different states associated with gene activation and repression. These states may be characterized by differences in nucleosome positions or post- translational modifications (PTMs) of proteins. What is unclear is how these states are passed on from one cell generation to the next, and the molecular mechanisms underlying this epigenetic cellular memory. Although much research in this area has focused on histone modifications, the spatial organization of chromatin into higher-order structure is also a key contributor to regulation. Our laboratory is interested in understanding how specific chromatin components interrelate and integrate to regulate transcriptional activity and maintain cellular memory. The positioning of nucleosomes with respect to the DNA sequence plays an important role in regulating transcription. Our studies in yeast reveal that the overwhelming majority of nucleosome positions are encoded in the genome by specific signals that favor or disfavor nucleosome positions. The extent to which Contact this is true in higher eukaryotes and in with skewed base composition is [email protected] not known. Our research will explore the relationship between nucleosome (859) 323-0091 positions, higher order chromatin fiber structure, and gene regulation, through genome-wide studies of nucleosome positioning in Drosophila melanogaster. We are exploring this relationship over the long term by investigating the role of nucleosome binding proteins histone H1, poly-ADP-polymerase (PARP-1) and high mobility group protein 1 (HMGB-1) on chromatin dynamics and inheritance of chromatin states. Our goal is to characterize and determine the dynamics of these chromatin architectural proteins, their effects on ―higher order‖ chromatin structure, gene regulation and subsequently, cellular memory. Since transcriptional machinery is similar in all eukaryotes, lessons learned from transcription regulation in these model organisms, will apply to humans. This will provide a foundation for understanding how misregulation of can lead to diseases such as cancer.

Common techniques used in our laboratory

Molecular biology, biochemical and biophysical, and genome-wide techniques including PCR, cloning, transformation, restriction enzyme studies, nucleosome mapping, and fluorescence Resonance Energy Transfer measurements (FRET); footprinting; ; assays; chromatin-immunoprecipitation; mass spectroscopy; genome-wide techniques including nucleosome mapping, DNAse footprinting, bisulphate footprinting, and chromatin-capture experiments combined with deep sequencing techniques. We also employ bioinformatics and to analyze results from genome-wide experiments.

Recent and representative publications

Kaplan N, Moore I, Fondufe-Mittendorf Y, Gossett AJ, Tillo D, Field Y, Hughes TR, Lieb JD, Widom J, and Segal E (2010) Nucleosome sequence preferences influence in vivo nucleosome organization. Nat Struct Mol Biol 17, 918- 20. Tillo D, Kaplan N, Moore I, Fondufe-Mittendorf Y, Gossett AJ, Field Y, Lieb JD, Widom J, Segal E, and Hughes TR (2010) Human regulatory sequences have high intrinsic nucleosome occupancy. PLoS One. 2010 Feb 9;5:e9129 Field Y, Fondufe-Mittendorf Y, Moore I, Mieczkowski P, Kaplan N, Lieb J, Widom J, and Segal E (2009) Gene expression divergence in yeast is coupled to evolution of DNA-encoded nucleosome organization. Nat Genet 41, 438-45. Kaplan N, Moore IK, Fondufe-Mittendorf Y, Gossett AJ, Tillo D, Field Y, LeProust EM, Hughes TR, Lieb JD, Widom J, and Segal E (2009) The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458, 362-6. Segal E, Fondufe-Mittendorf Y, Chen L, Thåström AC, Field Y, Moore IK, Wang J-PZ, and Widom J (2006) A genomic code for nucleosome positioning. Nature 442, 772-8.

Michael Fried

Members of the Fried group use biophysical techniques to study protein-DNA and protein-protein interactions. The assembly of macromolecular complexes is an essential step in many cellular processes including transcription, DNA replication, and structure. We are interested in the mechanisms that govern the assembly rates and the equilibrium stabilities of such complexes. We are currently exploring these issues for selected protein-DNA and protein-protein interactions. Our current work is focused on the DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the Dr. Fried in middle. integrity of the genome, but also contributes to the resistance of tumors to DNA- Contact [email protected], (859) 323-1205 alkylating chemotherapeutic agents. This image shows a model of a cooperative AGT- DNA complex based on equilibrium binding data (Adams, C.A. et al. (2009) J Mol Biol 389, 248-263). The repeating unit is one of AGT (colors) plus four base-pairs of DNA (black) derived from the structure of Daniels et al. (2004) Nat Struct Mol Bio. 11, 714-720. Repeating units were juxtaposed with preservation of B-DNA helical parameters between base-pairs of adjacent units. Yet another area of research in our laboratory is focused on the development of new spectroscopic probes for protein-protein and protein-DNA interactions. The hexahistidine (His6)/Nickel (II)-Nitrilotriacetic Acid (Ni2+- NTA) system is widely used for affinity purification of recombinant proteins, and it is also useful for the attachment of chromophores and fluorophores to His6-proteins. We are developing novel applications for bis-NTA-dye derivatives such as (Ni2+-NTA)2-Cy3. These bind His6 motifs with greater stability than mono-NTA compounds. They allow characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions (e.g. high ADP absorbance) that prevent direct detection of protein. In addition, FRET between (Ni2+-NTA)2-Cy3-labeled proteins and suitable donors/acceptors provides a convenient for binding interactions and for measurements of donor-acceptor distances.

Common techniques used in our laboratory

Electrophoresis mobility shift assay (EMSA) (our laboratory is one of the original developers of this method); chemical and nuclease footprinting assays; and ligation assays to measure DNA linking differences and DNA bending; chemical crosslinking/mass spectrometry analyses of protein-protein contacts; fluorescence spectroscopy; circular dichroism spectroscopy; titration calorimetry; analytical ultracentrifugation; thermodynamic linkage analyses; and transient kinetics analyses.

Recent and representative publications

Tubbs JL, Butt A, Kanugula S, Melikishvili M, Marriott A, Watson AJ, Verbeek B, Santibanez-Koref MF, Kraehenbuehl R, Fleck O, Millington C, Arvai AS, Kroeger MD, Peterson LA, Williams DM, Fried MG, Margison GP, Pegg AE, and Tainer JA (2009) Alkylated base flipping bridges distinct DNA base and nucleotide excision repair pathways. Nature 459, 808-813. Hellman LM, Zhao C, Melikishvili M, Tao X., Hopper JE, Whiteheart SW and Fried, MG (2011). Histidine-tag- directed chromophores for tracer analyses in the analytical ultracentrifuge. Methods, 54, 31-38. Adams CA and Fried MG (2011). Mutations That Probe The Cooperative Assembly Of O6-Alkylguanine-DNA Alkyltransferase (AGT) Complexes. Biochemistry 50, 1590-1598 . Tessmer I, Melikishvili M and Fried MG (2012). Cooperative clusters, DNA bending and base-flipping by O6- alkylguanine DNA alkyltransferase bound to double-stranded . Nucl. Acids Res. 40, 8296-8308. Melikishvili M and Fried MG (2012). Lesion-Specific DNA-Binding And Repair Activities Of Human O6- Alkylguanine DNA Alkyltransferase (AGT) Nucl. Acids Res. 40, 9060-9072.

Emilia Galperin

Our research focuses on the functional ―cross-talk‖ between endocytic and signaling cellular machineries and how this applies to cancer development. Endocytosis is a mechanism by which cells remove plasma membrane proteins, ligands, nutrients, and other types of molecules from the cell-surface to the cell interior. Contents of the internalized membranes then are destined to be degraded or recycled, which is facilitated by the complex network of endocytic compartments. Most of the surface signaling receptors internalize rapidly after ligand binding by means of endocytosis. One important aspect of endocytosis that has been appreciated in the past decade is that endocytosis provides spatial and temporal control of signaling. In addition, some endocytic serve as platforms for the assembly of signaling complexes. The beauty of this system is that it controls not only the fate of signaling receptors but also provides an endocytic route for signal to persist on ―signaling .‖ Contact Given the role of endocytosis in various signaling programs, it is expected that [email protected] endocytosis will have an impact on the development of patho-physiological conditions in (859) 323-1796 which signaling is aberrant. In our research, we study the spatiotemporal regulation of Mitogen-Activated Protein kinase (MAPK) signaling in different types of cells. We analyze how endocytic trafficking provides spatial and temporal control of MAPK activated by the EGF , using advanced methods of live-cell fluorescence microscopy. We monitor the dynamic localization of fluorescently-tagged proteins of MAPK signaling cascade upon EGFR stimulation in living cells. Our goal is to test whether the regulatory mechanisms of the spatiotemporal regulation of the components of the MAPK cascade are altered in cancer cells with different levels of EGFR. Endocytosis plays an important role in the regulation of the EGFR signaling network, and understanding the cross-talk between signaling and trafficking machineries is highly important for further understanding the role that the EGFR plays in the development and progression of cancer, as well for further optimization of the strategies of therapeutic inhibition of EGFR overexpressing tumors.

Common techniques used in our laboratory

Live-cell fluorescence microscopy; quantitative Immunofluorescence assays; various cell-based methods (protein expression, , siRNA/shRNA and DNA ); Western blotting; protein and ; and basic molecular biology procedures (DNA purification, cloning, and PCR amplification).

Recent and representative publications

Galperin E and Sorkin A (2008) MEK2 targeting to endosomes requires clathrin-dependent endocytosis and MEK activity. Traffic. 9, 1776-90. Galperin E and Sorkin A (2005) Use of FRET microscopy to visualize Rab5 activity in living cells. Methods Enzymol 403, 119-134. Galperin E, Verkhusha V, and Sorkin A (2004) Three-fluorochrome FRET to analyze protein interactions in living cells. Nature Methods 1, 209-217. Galperin E and Sorkin A (2003) Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma membrane-targeted Rab5 on clathrin-dependent endocytosis. J Cell Sci 116, 4799-4810. Galperin E, Benjumin S, Rappoport D, Rotem-Yehudar R, Tolchinsky S, and Horowitz M (2002) EHD3: A protein that resides in recycling tubular and vesicular membrane and interacts with EHD1. Traffic 3, 575-89.

Tianyan Gao

Our laboratory focuses on elucidating the functional importance of a novel family of protein phosphatase, PHLPP, in regulating tumorigenesis. We use colon cancer as a model system to study how PHLPP functions in suppressing cancer development and progression. The PI3K/Akt signaling pathway has been the focus of numerous studies in recent years as mounting evidence has suggested that signaling components in this pathway are critical regulators of mammalian cell proliferation and survival. Hyperactivation of this pathway is often linked with tumor progression and resistance to cancer drug treatment. PHLPP represents a family of novel Ser/Thr protein Dr. Gao on left. phosphatases that directly dephosphorylates Akt

Contact and terminates Akt-mediated growth and survival [email protected], (859) 323-3454 signals. Recently, we found that loss of PHLPP expression is commonly associated with colon cancers and re-introduction of PHLPP into colon cancer cells inhibits tumorigenesis. The long-term goal of our laboratory is to understand the physiological function of PHLPP and the molecular mechanisms underlying PHLPP- mediated regulation in cancers. In addition, we have developed PHLPP knockout mouse models in our laboratory to investigate the physiological role of PHLPP. The results from our studies will aid in developing novel therapeutic strategies in cancer treatment by using PHLPP as a target.

Common techniques used in our laboratory

Cell culture; SDS-PAGE; Western blotting; PCR; genotyping; cloning; protein purification; transwell migration assay; immunoprecipitation; immunohistochemistry; immunofluorescence ; retroviral production.

Recent or representative publications

Liu J, Weiss HL, Rychahou P, Jackson LN, Evers BM, and Gao T (2009) Loss of PHLPP expression in colon cancer: Role in proliferation and tumorigenesis. Oncogene 28, 994-1004. Li X, Liu J, and Gao T (2009) β-TrCP-mediated ubiquitination and degradation of PHLPP1 is negatively regulated by Akt. Mol Cell Biol 29, 6192-6205. Liu J, Stevens PD, Li X, Schmidt MD, Gao T (2011) PHLPP-mediated dephosphorylation of S6K1 inhibits protein and . Mol Cell Biol. 31(24):4917-27 Li X, Yang H, Liu J, Schmidt MD, Gao T (2011) Scribble-mediated membrane targeting of PHLPP1 is required for its negative regulation of Akt. EMBO Rep. 12:818-24 Li X, Stevens PD, Yang H, Gulhati P, Wang W, Evers BM, Gao T (2012) The deubiquitination enzyme USP46 functions as a tumor suppressor by controlling PHLPP-dependent attenuation of Akt signaling in colon cancer. Oncogene. Mar 5. doi: 10.1038/onc.2012.66. [Epub ahead of print]

Web Site http://ukhealthcare.uky.edu/Markey/research/gao.asp

Matthew Gentry

Our lab studies the role of signal transduction proteins, namely phosphatases and E3 ligases, in both a neurodegenerative epilepsy and in biofuels production. We utilize a multidisciplinary approach that addresses and/or employs methodologies of cell biology, biochemistry, genetics, bioinformatics, and phylogenetic relationships in vertebrate and protozoan model organisms. These applications employ model organisms, cells, and structural biology to study basic cell processes that have direct relevance to neurodegeneration and epilepsy. Lafora disease is caused by Dr. Gentry, fourth from left. perturbations in metabolism that cause human glycogen to resemble . This Contact finding allowed us to extend our work into [email protected], (859) 323-8482 biofuels research because starch is a key component of both first-generation and next- generation biofuels. Epilepsy is a medical condition initiated in the brain and affects both mental and physical functions of the patient. Epilepsy affects nearly 3 million people in the United States at an estimated annual cost of $15.5 billion. Lafora disease (LD) is a rare type of neurodegeneration that results in severe epilepsy and death. Unlike most other forms of epilepsy, LD is only mildly managed by medication for a brief period of time. LD patients develop normally until they present with a single seizure when the patient is 15-20 years old; this single seizure is followed by progressive central nervous system degeneration and the disease ends with the death of the patient within ten years of the first seizure. A hallmark of LD is the accumulation of /glucan granules that form in most cells in the body, called Lafora bodies (LBs). LBs are similar to glycogen, the cell’s normal glucan storage molecule, but unlike glycogen LBs are water insoluble and are more closely related to plant starch than human glycogen. Our goal is to untangle the intercalated events of metabolism, neurodegeneration, and epilepsy utilizing our insights from studying Lafora disease.

Common techniques used in our laboratory Protein purification via affinity chromatography, size exclusion, and -exchange; DNA purification; PCR; Western blotting; immunoprecipitation; DNA and protein ; cloning and site-directed mutagenesis; X-ray Crystallography/structural biology; hydrogen deuterium exchange mass spectrometry (DXMS); mammalian cell culture (utilizing cells for microscopy and biochemistry); culture, phosphatase activity assays; phosphorylation (radio-labeling) assays; glucan binding assays; kinase assays; (mice) maintenance, genotyping, and tissue harvesting; glycogen content determination; bioinformatics; microscopy at the cell and tissue level; yeast two-hybrid screens; in vitro transcription and translation (TnT); and in vitro high-throughput drug screening.

Recent and representative publications (out of 27 total) Roma-Mateo C, Sanz P, and Gentry MS (2012) Deciphering the role of malin in the Lafora progressive myoclonus epilepsy. IUBMB Life. In Press. Gentry MS, Roma-Mateo C, and Sanz P (2012) Laforin, a protein with many faces: glucan phosphatase, adapter protein, et alii. The FEBS Journal. In Press. Dukhande VV, Rogers DM, Romá-Mateo C, Donderis J, Marina A, Taylor AO, Sanz P, and Gentry MS. (2011) Laforin, a dual specificity phosphatase involved in Lafora disease, is present mainly as monomeric form with full phosphatase activity. PLoS ONE. 6(8): e24040. Romá-Mateo C, Gimeno-Alcañiz JV, Dukhande VV, Donderis J, Worby CA, Criado O, Koller A, Gentry MS, and Sanz P (2011) Laforin, a dual specificity phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase. Biochemical Journal. 15; 439(2): 265-75.

Web Sites http://www.mc.uky.edu/biochemistry/faculty.asp?fid=10 http://gentrylab.com/wordpress/

Lou Hersh

Research in our laboratory is focused on zinc containing metallopeptidases and their role in normal and pathological . Our focus has been on elucidating the properties of a number of peptidases including neprilysin, a key regulator of many neuropeptides including opioid peptides, substance P, and beta peptides; insulysin (insulin degrading enzyme), which controls cellular insulin levels and amyloid beta peptide levels; nardilysin, which regulates a number of cellular events including amyloid Dr. Hersh, second from right. precursor protein processing, and metalloprotease 1, which appears to be Contact involved in . Our studies [email protected], (859) 323-5549 involve biochemical and crystallographic analyses coupled with cellular and animal studies. We have been exploring the use of neprilysin as a therapeutic to lower brain amyloid beta peptide levels. The enzyme has been expressed in a transgenic mouse model of Alzheimer’s disease and shown to lower brain amyloid levels as well as prevent the cognitive decline characteristic of Alzheimer’s disease. We are producing variants of neprilysin that are more specific for amyloid beta peptide that can be used therapeutically. In another study we are investigating the role of pitrilysin in type 2 diabetes. Pitrilysin is believed to regulate the level of islet amyloid peptide, a peptide that, like amyloid beta peptide, aggregates into toxic species. The aggregation of islet amyloid peptide in pancreatic islet cells is believed to be a causative agent in type 2 diabetes. Insulysin is the only zinc-containing metalloneuropeptidase that is subject to . We are employing a combination of crystallographic and mutagenic studies focused on understanding the regulation of this peptidase. Activators of the enzyme that wok in the brain will be useful for preventing amyloid peptide accumulation in Alzheimer’s disease, while inhibitors of the enzyme that work peripherally would be useful for treating diabetes. The peptidase metalloprotease 1 is loosely related to insulysin and is being used to compare structures to further understand the basis for catalysis and allosteric regulation.

Common techniques used in our laboratory

Site-directed and random mutagenesis; protein purification using affinity chromatography techniques; molecular cloning; enzyme expression using adeno-associated, lenti, and baculo viruses; fluorescent spectroscopy; and gene expression in mice.

Recent and representative publications

Song ES, Rodgers DW, Hersh LB. Mixed dimers of insulin degrading enzyme reveal a cis activation mechanism. Chem. 286: 13852-13858, 2011 Noinaj N, Bhasin SK, Song ES, Scoggin KE, Juliano MA, Juliano L, Hersh LB, Rodgers DW. Identification of the allosteric regulatory site of insulysin. PLoS One.;6:e20864, 2011. Noinaj N, Song ES, Bhasin S, Alper BJ, Schmidt WK, Hersh LB, Rodgers DW. The anion activation site of insulin degrading enzyme. J Biol Chem. 287:48-57 Holler CJ, Webb RL, Laux AL, Beckett TL, Niedowicz DM, Ahmed RR, Liu Y, Simmons CR, Dowling AL, Spinelli A, Khurgel M, Estus S, Head E, Hersh LB, Murphy MP. BACE2 Expression Increases in Human Neurodegenerative Disease. Am J Pathol. 180:337-50201. Sexton T, Hitchcook LJ, Rodgers DW, Bradley LH, Hersh LB. Active site mutations change the cleavage specificity of neprilysin. PLoS One. 2012;7(2):e32343. Guan H, Chow KM, Shah R, Rhodes CJ, Hersh LB. Degradation of islet amyloid polypeptide by neprilysin. Diabetologia. 2012 Aug 17. [Epub ahead of print]

Jianhang Jia

Cellular level signal transduction refers to the movement of signals from outside the cell to inside. Complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include protein interactions and modifications. Eventual outcome of the signal transduction is switching specific genes on or off in a regulated manner. Cancer cells arise when such signal transduction goes awry, and this is often due to incorrect responses to the signals that should normally regulate cell growth and differentiation. The Hedgehog (Hh) pathway is one of the major cell- signaling pathways in animal development, controlling cell growth, proliferation, and differentiation. Aberrant Hh signaling induces numerous human diseases, including cancers in many tissues, so it has been the main Dr. Jia in back row, left. therapeutic target for the treatment of these cancers. The Hh pathway is highly conserved from insect Contact to human, so both vertebrate and invertebrate model [email protected], (859) 323-6236 systems can be used to study its mechanisms. The Hh signal is transduced through a reception system that includes the twelve-span transmembrane protein, Patched (Ptc), and the seven-span transmemberane protein, Smoothened (Smo). In the absence of Hh, Ptc inhibits Smo signaling activity by a mechanism that is still not clear. The presence of Hh relieves the inhibition of Smo by Ptc, activating Smo and allowing Smo to regulate the downstream signaling components. The main outcome of Hh signaling is the modulation of transcriptional responses via the cubitus interruptus (Ci)/Gli family of zinc-finger transcription factors. The goal of our research is to understand the molecular mechanisms of Hh signal transduction. We have shown that the seven-pass transmembrane protein Smo transduces Hh signals by directly recruiting a Costal2-fused (Cos2-Fu) complex, and that Smo activation requires phosphorylation by protein kinase A (PKA) and casein kinase 1 (CK1), leading to increased Smo cell-surface levels and signaling activity. In addition, our laboratory uncovered a feedback mechanism by which Fu promotes Smo hyperphosphorylation and cell-surface accumulation by antagonizing Cos2. We have recently tested the hypothesis that gradient Hh signaling activity is transduced by differentially phosphorylated Smo. Along the other direction, we have identified and characterized PP4 and PP2A as the phosphatases that inhibit the phosphorylation of Smo and Ci, respectively. More recently, we have begun to focus on additional mechanisms, such as the subcellular trafficking of the receptor complex and the regulation of Smo ubiquitination that are likely regulated by Hh. We have found that the of Smo is mediated by ubiquitination, and USP8 prevents Smo ubiquitination and elevates Smo activity by promoting Smo cell surface accumulation. Since abnormal Smo activation results in such cancers as basal cell carcinoma and medulloblastoma, these studies will provide insights into fundamental developmental problems and new avenues for cancer diagnosis and . Current projects in our laboratory are to identify and characterize Smo-interacting genes that play critical roles in Hh signal transduction, and to evaluate the roles of Hh signaling components in cancer formation.

Common techniques used in our laboratory

Cell culture techniques; Western blotting; confocol imaging; molecular cloning; and Drosophila tissue dissection.

Recent and representative publications

Jia H, Liu Y, Yan W, and Jia J (2009) PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development 136, 307-316. Xia R, Jia J, Fan J, Liu Y, and Jia J. (2012) USP8 promotes Smoothened signaling by preventing its ubiquitination and changing its subcellular localization. PLoS Biol 10(1): e1001238. Fan J, Liu Y, and Jia J. (2012) Hh-induced Smoothened conformational switch is mediated by differential hosphorylation at its C-terminal tail in a dose- and position-dependent manner. Dev Biol 366(2): 172-184.

Konstantin Korotkov

Many have developed sophisticated mechanisms of protein transport across membranes. These macromolecular secretory systems play a pivotal role in microbial pathogenesis and are considered as promising targets for a new generation of anti-bacterial therapy. The ESX systems of Mycobacterium tuberculosis, an important human pathogen, secrete multiple proteins that are implicated in host-pathogen interactions. The homologous secretion systems have been also identified in a number of Gram-positive pathogenic bacteria including Staphylococcus aureus and . Although numerous components of the ESX systems have been identified, the structure of individual building blocks, the overall architecture of the system and Dr. Korotkov, second from left. the molecular mechanism of Contact secretion are [email protected], (859) 323-5493 currently unknown. Our research aims to unravel the molecular mechanism of the ESX secretion system using structural biology methods, mainly X-ray crystallography and electron microscopy, in combination with biochemical and biophysical analyses. The ultimate goal of our research is to develop novel tuberculosis therapies.

Common techniques used in our laboratory Bioinformatics and structural modeling, molecular biology, recombinant protein production, protein purification and characterization, protein-protein interaction assays, enzyme activity assays, X-ray crystallography, electron microscopy.

Recent and representative publications Korotkov KV, Sandkvist M, and Hol WGJ (2012) The type II secretion system: biogenesis, molecular architecture and mechanism. Nature Rev Microbiol 10, 336-351. Korotkova N, Hoff JS, Becker DM, Quinn JK, Icenogle LM, and Moseley SL (2012) SpyA is a membrane-bound ADP-ribosyltransferase of pyogenes which modifies a streptococcal peptide, SpyB. Mol Microbiol 83, 936-952. Korotkov KV, Johnson TL, Jobling MG, Pruneda J, Pardon E, Héroux A, Turley S, Steyaert J, Holmes RK, Sandkvist M, and Hol WGJ (2011) Structural and functional studies on the interaction of GspC and GspD in the type II secretion system. PLoS Pathog 7, e1002228. Korotkov KV, Gonen T, and Hol WGJ (2011) Secretins: dynamic channels for protein transport across membranes. Trends Biochem Sci 36, 433-443. Reichow SL, Korotkov KV, Hol WGJ, and Gonen T (2010) Structure of the cholera toxin secretion channel in its closed state. Nature Struct Mol Biol 17, 1226-1232.

Natasha Kyprianou

Our research interests focus on the deregulation of apoptosis and growth factor signaling pathways in benign and malignant prostate, development of molecular therapeutics (via tumor selective apoptosis-targeting) for castration-resistant prostate tumors and development of novel biomarkers of prostate and bladder cancer progression. Specific pursuits include the contribution of anoikis (apoptosis upon cell detachment from extracellular ) resistance and epithelial mesenchymal transition (EMT) to prostate cancer and therapeutic resistance.

Common techniques used in our laboratory

Dr. Kyprianou in front row, middle. Cell migration assay; cell invasion assay; Confocal microscopy for intracellular protein localization; Contact apoptosis analysis using the V staining [email protected], (859) 323-9812 and TUNEL staining; in vivo experimental metastatic assay; real-time PCR; transfections/cloning; transcriptional regulation and characterization of transgenic mice.

Recent and representative publications

Pu, H., Collazo, J., Jones, E., Gayheart, D., Sakamoto, S., Vogt, A., Mitchell, B., and Kyprianou, N (2009). Dysfunctional TGF-β Receptor II Accelerates Prostate Tumorigenesis in the TRAMP Mouse Model. Cancer Res., 69(18):7366-7374. Zhu M and Kyprianou N (2010) Role of androgens and the androgen receptor in epithelial-mesenchymal transition and invasion of prostate cancer cells. FASEB J 24, 769-777. Sakamoto S, McCann RO, Dhir R, and Kyprianou N (2010) Talin1 promotes tumor invasion and metastasis via focal adhesion signaling and anoikis resistance. Cancer Res 70, 1885-1895. Zhu, B., Zhai, J., Zhu, H., and Kyprianou, N (2010). Prohibitin Regulates TGF-β Induced Apoptosis as a Downstream Effector of Smad-dependent and -independent Signaling. The Prostate, 70(1):17-26. Zhu, M., Horbinski, C., Garzotto, M., Qian D., Beer, T. and Kyprianou, N.(2010). -targeting impairs androgen receptor activity in prostate cancer. Cancer Res., 70(20):7992-8002. Horbinski, C., Mojesky, C. and Kyprianou, N. (2010). Live Free or Die: Tales of Homeless Cells in Cancer. Am. J. Pathol., 177(3):1044-52. Wang, Wang-Xia, Kyprianou, N. and Nelson, P. M. (2010). Dysregulation of the mitogen granulin in human cancer through the miR-15/107 microRNA gene family. Cancer Res., 70(22):9137-9142. Sakamoto, S., Schwarze, S., and Kyprianou, N. (2011). Anoikis Disruption of Focal Adhesion-Akt Signaling Impairs Renal Cell Carcinoma. European Urology, 59(5):734-744.

Web Site http://www.mc.uky.edu/surgery/Uro/research.asp

Harry LeVine

Our laboratory seeks to elucidate the contribution of misfolded proteins to chronic neurodegenerative diseases and to develop therapeutic agents capable of modifying the course of these diseases. Our focus is on the Alzheimer’s β-peptide (Aβ) and its role in the cognitive decline in Alzheimer’s disease. As the average age of the of the developed nations continues to rise, the societal impact of chronic diseases with onset later in life increases. Multiple chronic neurodegenerative diseases, led in prevalence by Alzheimer’s disease (AD) and Parkinson’s disease, exact an escalating economic and personal toll on families and on society. Currently, 5.3 million Americans suffer from AD, and by 2050, 11-16 million people Dr. Levine on right. will have AD and an additional 2 million will be stricken with Parkinson’s disease. Current FDA-approved therapies attempt Contact to address the cognitive deficits which are the symptoms of [email protected], (859) 257-1412 x224 AD, with limited success. Multiple approaches for disease- modifying therapies are being tested in preliminary clinical trials but none as yet are reproducibly effective. A potential reason for the ineffectiveness of the disease-modifying interventions is that by the time cognitive deficits can be detected the disease is far advanced. Biomarkers for very early detection, perhaps 15-20 years before clinical symptoms, will be essential for successful prevention and early treatment of AD. One of the difficulties with developing therapies for AD is the lack of animal models that faithfully recapitulate both the neuropathology and the clinical dementia of the disease. While both natural (non-human primates) and genetically-modified animal models show similar neuropathology and perhaps early cognitive deficits, only humans 1) develop a significant content of the amyloid imaging ligand (PIB) binding of their amyloid plaques, and 2) progress to the final phase with neuronal death leading to profound dementia. Our laboratory is involved at multiple levels in elucidating the basis of Aβ oligomer-induced neuronal deficits, biomarkers for very early stage disease, therapeutic approaches to alleviating Aβ and tau toxicity, and improving animal models of AD pathogenesis.

Common techniques used in our laboratory

Bacterial protein expression; protein separation and purification from native and recombinant sources; ELISA immunoassays; radioligand binding; enzymology; HPLC and FPLC chromatography; electrophoresis; subcellular tissue fractionation; and ultracentrifugation, chemical compound library screening.

Recent and representative publications

Rosen, R. F., Walker, L. C., and LeVine, III, H. ―PIB Binding in Aged Primate Brain: Unique Enrichment of High Affinity Sites in Humans with AD", Neurobiology of Aging 32: 223-234 (2011). LeVine H III, Ding Q, Walker JA, Voss RS, and Augelli-Szafran CE Clioquinol and other hydroxyquinoline derivatives inhibit Aβ(1–42) oligomer assembly. Neurosci Lett 465, 99-103 (2009). Rosen RF, Ciliax BJ, Gearing M, Dooyema J, Wingo TS, Lah JJ, Ghiso J, LeVine H III, and Walker LC Deficient high-affinity binding of Pittsburgh Compound B in a case of Alzheimer's Disease. Acta Neuropathol 119, 221-233 (2010). Niedowicz, D. M., Beckett, T. L., Matveev, S., Weidner, A. M., Baig, I., Abner, E. L., Kryscio, R. J., Mendiondo, M. S., LeVine, III, H., Keller, J. N., and Murphy, M. P. ―Pittsburgh Compound B and the Postmortem Diagnosis of Alzheimer’s Disease‖, Annals of Neurology, in press. Weidner, A. M., Housley, M., Murphy, M. P, and LeVine, III, H. ―Purified High Molecular Weight Synthetic Aβ(1-42) and Biological Aβ Oligomers are of Comparable Potency in Rapidly inducing MTT Formazan Exocytosis‖, Neurosci. Lett., 497(1): 1-5 (2011). LeVine, III, H., Lampe, L., Abdelmoti, L., and Augelli-Szafran, C. E. ―Dihydroxybenzoic Acid Isomers Differentially Dissociate Soluble Biotinyl-Aβ(1-42) Oligomers‖, Biochemistry, 51(1): 307-312 (2012).

Web Site http://www.mc.uky.edu/coa/faculty/levine.html

Guo-Min Li

Our laboratory studies DNA repair and genome instability, with a focus on two DNA repair pathways and their roles in human diseases. First, we are studying DNA mismatch repair (MMR). MMR maintains genomic stability primarily by correcting mispairs generated during DNA replication. Defects in MMR predispose to cancer. Using a functional in vitro assay, our group has identified and characterized individual components required for MMR in human cells, and we have successfully reconstituted the MMR reaction in a defined system. However, the reconstituted MMR system cannot correct DNA heteroduplexes assembled Dr. Li on far right. into nucleosomes, suggesting that additional

Contact factor(s) are needed for MMR in vivo at the [email protected], (859) 257-7053 chromatin level. Our recent studies reveal that histone modifications controls the MMR function in vivo. We are in the process of identifying and characterizing these important epigenetic marks that regulate MMR in human cells. Second, we are studying the mechanism of nucleotide repeat instability and large DNA loop/hairpin repair. Expansion of simple nucleotide repeats in DNA, e.g., trinucleotide repeats (TNRs), is the genetic basis for more than 40 human familial neurological, neurodegenerative and neuromuscular disorders, including Huntington's disease, myotonic dystrophy, Friedreich ataxia, and fragile X syndrome. The repeat expansion can occur in any part of a gene and leads to a defective gene product. However, how the repeat units expand and what cellular mechanism(s) prevent such an expansion in normal remain unknown. Our laboratory has recently identified a DNA repair pathway (referred to as DNA hairpin repair) that specifically removes trinucleotide repeat-formed hairpin structures to prevent TNR expansions. We are identifying protein components involved in this DNA hairpin repair pathway. This work will provide significant insight into the pathogenesis underlying diseases associated with repeat expansions.

Common techniques used in our laboratory

Protein expression in bacteria and insect cells; protein purification and characterization; protein-DNA interaction; cell and tissue culture; gene and cloning; gene knockdown by siRNA; in vitro microRNA processing; and in vitro DNA repair assays.

Recent and representative publications

Zhang Y, Yuan F, Presnell S, Tian K, Gao Y, Tomkinson A, Gu L, and Li G-M (2005) Reconstitution of 5’ directed human mismatch repair in a purified system. Cell 122, 693-705. Hou C, Chan N, Gu L, and Li G-M (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nature Struct Mol Biol 16, 869-875. Li F, Tian L, Gu L, and Li G-M (2009) Evidence that nucleosomes inhibit mismatch repair in eukaryotic cells. J Biol Chem 84, 33056-33061. Mao, G., Lee, S., Ortega, J., Gu, L., and Li, G.-M. (2012) Modulation of microRNA processing by mismatch repair protein MutL. Cell Res. 22, 973-85 Chan, N., Hou, C., Zhang, T., Yuan, F., Machwe, A., Huang, J., Orren, D.K., Gu, L., and Li, G.-M. (2012) The Werner syndrome protein promotes CAG/CTG repeat stability by resolving large (CAG)n/(CTG)n hairpins. J. Biol. Chem. 287: 30151-30156.

Chunming Liu

The Wnt/β- signaling pathway plays important roles in early development, renewal, and tumorigenesis. In addition, Wnt signaling is crucial in the organization and maintenance of the human intestinal . In this pathway, many different components work together to transduce an external signal into changes in gene expression within the target cell. Upon binding its receptor, the Wnt ligand ultimately results in the stabilization of cytoplasmic β-catenin, which is then free to enter the nucleus and activate transcription through its interaction with the TCF/LEF family of transcription factors. Somatic mutations that result in deregulated Wnt signaling are an early event in the development of colorectal Dr. Liu on far left. cancer, which is the second leading cause of cancer death in the United States. About 80% of colorectal Contact cancers have APC (adenomatous polyposis coli) [email protected], (859) 323-4588 mutation, and about 15% have β-catenin mutation. We have identified novel molecules in the Wnt pathways, and have provided insights into how mutations cause β-catenin accumulation that leads to cancer. We are also interested in the crosstalk between Wnt signaling and other signaling pathways. We have identified KLF4 as a novel inhibitor of β-catenin. KLF4 is a tumor suppressor and a key factor for stem cell re-programming. We found that KLF4 controls transcription by regulating chromatin-remodeling. We are currently investigating the molecular mechanisms of KLF4 in cancers and in iPS cells (induced pluripotent stem cells). We are collaborating with Dr. Mark Evers to study Wnt/β-catenin signaling in human cancers, especially GI cancer. We are collaborating with Dr. David Watt to develop small molecular β-catenin inhibitors. Our long-term goal is to investigate the molecular mechanisms of human cancers and cancer stem cells, and use our knowledge of to develop therapeutics and diagnostics for human cancers.

Common techniques used in our laboratory

DNA cloning; protein purification; protein-protein interaction; Chromatin Immunoprecipitation (ChIP assay); adenovirus and lentivirus; in vitro assay (phosphorylation, ubiquitination, , transcription and translation); cell and tissue ; reporter assay; Western blot; RT-PCR; insect and mammalian cell culture; generation; tumor xenograft; high throughput screening.

Recent or representative publications

Liu C, Li Y, Semenov M, Han C, Baeg G, Tan Y, Zhang Z, Lin X, He X. Control of -catenin phosphorylaton/degradation by a dual-kinase mechanism. Cell 108:837-847, 2002. Zhang W, Chen X, Kato Y, Evans PM, Yuan S, Yang J, Rychahou PG, Yang VW, He X, Evers BM and Liu C. Novel crosstalk of Krüppel-like factor 4 and -catenin regulates normal intestinal homeostasis and tumor repression. Mol Cell Biol 26:2055-2064, 2006. Evans PM, Chen X, Zhang W and Liu C. KLF4 interacts with -catenin/TCF4 and blocks recruitment of p300/CBP by -catenin. Mol Cell Biol. 30: 372-381, 2010. Zhang W, Sviripa V, Kril LM, Chen X, Yu T, Shi J, Rychahou P, Evers BM, Watt DS, and Liu C. Fluorinated N,N- Dialkylaminostilbenes for Wnt Pathway Inhibition and Colon Cancer Repression. J Med Chem, 54: 1288– 1297, 2011. Yu T, Chen X, Zhang W, Colon D, Shi J, Napier D, Rychahou P, Lu W, Lee EY, Evers BM and Liu C. Regulation of the Potential Marker for Intestinal Cells, Bmi1, by beta-Catenin and the Zinc Finger Protein KLF4: Implications for Colon Cancer. J Biol Chem. 287: 3760-8, 2012.

Mike Mendenhall

In the past, our laboratory has studied the roles of cyclin-dependent protein kinases, their inhibitors and ubiquitin ligases on the decision to enter the DNA replicative phase of the eukaryotic . More recently, we have shifted our focus to the function of a novel class of ubiquitin ligases on the initiation of differentiation of neural and cell precursors. In addition, we operate the Viral Synthesis Core which packages constructs provided by researchers throughout the department and beyond into infective, recombinant lentivirus, adenovirus, adeno-associated virus, and baculovirus derivatives.

Common techniques used in our laboratory Dr. Mendenhall on left. Yeast genetics; general molecular biology; tissue culture; Contact viral packaging and analysis; and recombineering. [email protected], (859) 257-5379

Recent and representative publications

Mendenhall MD (1996) ―From Start to S phase: Early regulatory events in the yeast cell cycle.‖ In Cellular and Molecular Regulation of Testicular Cells. Claude Desjardins, ed. p.11-25. Springer-Verlag Inc.: New York, NY. Mendenhall MD (1998) ―The cyclin dependent kinase inhibitors of Saccharomyces cerevisiae and Schizosaccharomyces pombe” In Current Topics in and . p.1-24. S.I. Reed and P. Vogt, eds. Springer-Verlag, Inc.: New York, NY. Schneider BL, Patton EE, Lanker S, Mendenhall MD, Wittenberg C, Futcher B, and Tyers M (1998) Yeast G1 cyclins are unstable in G1 phase. Nature 395:86-89. Mendenhall MD and Hodge AE (1998) Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1191-1243. Hodge A and Mendenhall M (1999) The cyclin-dependent kinase inhibitory domain of the yeast Sic1 protein is contained within the C-terminal 70 amino acids. Mol Gen Genet 262:55-64.

Anne-Frances Miller Redox reactions are an important foundation of life in that they are the basis for much of the energy used to support life: reduction of O2 to 2H2O in respiration, oxidation of to CO2, and countless chemi- autotrophic modes of metabolism. Some of the most demanding reactions in biology are catayzed by redox- active , for example, reductive cleavage of the triple bond of nitrogen gas, N2, which is the second strongest bond known. In many redox-active enzymes, it is not the protein but an inorganic or organic cofactor that actually executes the chemistry. However, having incorporated reactive cofactors, the protein must be able to control the activated intermediates that occur during turnover, ensure that only a specific substrate has access Dr. Miller, second from left. to the cofactor, and contrive that a single specific reaction follows. Thus, instead of mediating wholesale oxidation Contact of all the many susceptible molecules that make up a cell, [email protected], (859) 257-9349 redox enzymes permit utilization of specific reactions as sources of energy, in parallel with separate utilization of other similar molecules to build and run the cell. We seek to understand how enzymes can both accelerate difficult reactions by many orders of magnitude (factors of up to x1019), yet on the other hand retain very tight control over what compounds react and what course the reaction takes. These tasks are especially challenging for redox enzymes, since they require control over the transfer of electrons. Electrons are miniscule and virtually intangible in contrast to the enzymes' comparatively large size and their inherently flexible long-range structures. Our research addresses fundamental elements of enzymatic redox catalysis. We are concentrating on two enzymes, superoxide dismutase (SOD) and nitroreductase (NR) using NMR (nuclear magnetic resonance) spectroscopy. In summary, we are learning in atomic detail how mild-mannered proteins can handle the hottest intermediates and super-cofactors, bend them to their will, and turn around ready to do it all again in as little as 20 ns. We are working to implement the lessons from our research in engineered enzymes with applications in waste detoxification and clinical applications.

Common techniques used in our laboratory

High-resolution NMR; solid-state NMR; stopped-flow kinetics; mechanistic enzymology; characterization of binding equilibria; redox equilibria and pH equilibria; cloning; site-directed mutagenesis; and protein overexpression.

Recent and representative publications

Miller A-F, Yikilmaz E, and Vathyam S (2010) 15N-NMR characterization of His residues in and around the active site of FeSOD. Biochim. Biophys. Acta 1804, 275-284. Koder, Jr, RL, Walsh JD, Pometun MS, Dutton PL, Wittebort RJ, and Miller A-F (2006) 15N Solid-State NMR provides a sensitive probe of oxidized flavin reactive sites. J Am Chem Soc 128, 15200-15208. Miller A-F (2008) Redox tuning over almost 1 V in a structurally-conserved active site: lessons from superoxide dismutase. Acc Chem Res 41, 501-510. Yikilmaz E, Porta J, Grove L, Vahedi-Faridi A, Bronshteyn Y, Brunold TC, Borgstahl GEO, and Miller A-F (2007) How can a single second sphere amino acid change cause a reduction midpoint potential change of hundreds of mV? J Am Chem Soc 129, 9927-9940. Yikilmaz E, Rodgers DW, and Miller A-F (2006) The crucial importance of chemistry in the structure-function link: Manipulating H-bonding in Fe-superoxide dismutase. Biochemistry 45, 1151-1161.

Web Site http://www.chem.uky.edu/research/miller/

Paul Murphy

Age-related disease is a major public health problem. As global population demographics shift towards a relatively older population, these diseases will become a catastrophic burden on both health care resources and on human well-being. Our laboratory is interested in Alzheimer’s disease and in the molecular pathways that it shares with other disorders. For example, the amyloid precursor protein (APP) is potentially involved in a variety of cellular processes, but it is best known for its role as the source of a small peptide fragment known as the amyloid β peptide (Aβ). This peptide plays a major role in the development of Alzheimer’s disease in the brain. Recently, we have also begun to explore the connections between Alzheimer’s disease and the metabolic factors involved in Dr. Murphy in front row, middle. type II diabetes, a condition known to confer Contact significant risk for a variety of age-related [email protected], (859) 257-1412 x490 conditions. We believe that these seemingly disparate diseases are connected not only at the level of shared molecular pathways, but also at the level of transcriptional and translational regulation. Our work seeks to understand these factors. Recent studies include the use of NSAIDS (such as ibuprofen) and dietary modifications (such as low and high fat diets) to modify these processes. Our ultimate aim is to use the knowledge gained from these studies to refine model systems in which to develop novel therapeutic approaches that will one day lead to effective clinical treatments.

Common techniques used in our laboratory

ELISA; RT-PCR; Western blotting; Mammalian tissue culture; and immunohistochemistry.

Recent and representative publications

Niedowicz DM, Beckett TL, Matveev S, Weidner AM, Baig I, Abner EL, Kryscio RJ, Mendiondo MS, LeVine H, Keller JN, Murphy MP (2012). Pittsburgh Compound B and the Postmortem Diagnosis of Alzheimer’s Disease. Ann Neurol, in press. Holler CJ, Webb RL, Laux AL, Beckett TL, Niedowicz DM, Ahmed RR, Liu Y, Dowling AL, Martin SB, Spinelli A, Khurgel M, Head E, Hersh LB, Murphy MP (2012). BACE2 Increases in Human Neurodegenerative Disease. Am J Pathol, 180(1): 337-50. Weidner AM, Bradley MA, Beckett TL, Niedowicz DM, Dowling ALS, Matveeva SV, LeVine H, Lovell MA, Murphy MP (2011). RNA oxidation adducts 8-OHG and 8-OHA change with Aβ42 levels in late-stage Alzheimer's Disease. PLoS ONE, 6(9): 1-6; e24930. Beckett TL, Niedowicz DM, Studzinski CM, Weidner AM, Webb RL, Holler CJ, Ahmed RR, Levine H, Murphy MP (2010). Effects of Nonsteroidal Anti-Inflammatory Drugs on Amyloid-β in Mouse . Neurobiol Dis, 39: 449-456. Ahmed RR, Holler CJ, Webb RL, Li F, Beckett TL, Murphy MP (2010). BACE1 and BACE2 enzymatic activities in Alzheimer’s Disease. J Neurochem, 112: 1045-1053.

Dan Noonan

We study the molecular mechanism by which steroid mediate their pleiotropic effects. The intracellular receptors for a variety of steroid and complex lipid based hormones comprise the largest known family of human transcription factors. These include receptors for , , , cortisone, vitamin A, vitamin D, oxysterols, prostaglandins, bile acids, and long chain fatty acids. Cognate ligand binding of an stimulates receptor dimerization, binding to DNA, recruitment of a variety of regulatory proteins and targeting of RNA polymerase (the engine of gene transcription) to its proper place on the gene (Fig. 1). Due to the various roles these transcription factors play in differentiation, development, inflammation and homeostasis, they have been implicated in the pathology of many diseases and cancers. Our most recent studies have centered on two aspects of estrogen signaling and its intracellular receptor (ERα). The first is the recruitment of the regulatory Contact proteins following DNA binding by the receptor. These studies identified a [email protected], (859) 257-7498 unique regulator of this process, the tuberous sclerosis complex gene 2 (TSC2), which plays a key role in regulation of nutrient signaling and protein translation. Our ongoing studies in this area (York et al., Noonan et al.) attempt to elucidate how the mutations in the TSC2 gene might alter estrogen signaling in the female-specific lung cancer Lymphangioleiomyomatosis (LAM). In a second and fairly new direction for our studies, we have initiated a drug discovery program for identifying novel steroid hormones that would be predicted to modulate receptor signaling and activities. These are exciting collaborative studies with David Watt in our department. Dr. Watt is an established researcher in the organic synthesis of steroids. We have initiated an investigation into the development and analysis of bivalent steroid hormones (SH). It is being hypothesized that these molecules will either facilitate or inhibit hormone receptor (SR) dimerization, dependent upon the size and composition of the linker moiety connecting the 2 hormones (Fig. 2). We recently published a manuscript demonstrating the feasibility of this concept using as our working model (Wendlandt et al.). These initial studies open the door to a variety of future studies in which we will insert specific targeting sequences into the linker moiety allowing the selective targeting of hormone or anti-hormone activities to specific tissues, cells, tumors, protein complexes, or perhaps even genes. Fig. 1. Steroid Receptor Signaling Fig. 2. Bivalent Steroid Mechanisms

Common techniques used in our laboratory

Nucleic acid cloning, purification, and analysis technologies; protein purification and analysis technologies; gene expression technologies; transcription regulation analyses; cell and tissue culture technologies; mammalian cell transfection technologies; high throughput screening technologies; and lipid analysis technologies.

Recent and representative publications

York B, Lou D, Panettieri RA, Krymskaya VP, Vanaman TC, and Noonan DJ (2005) Crosstalk between tuberin, calmodulin and estrogen signaling pathways. FASEB J 19:1202-1204. PMID: 15851513 Noonan DJ and Lou D (2010) Evidence for epigenetic changes in the alpha promoter in the female-specific lung disease lymphangioleiomymatosis. TOLCJ 3:1-19. Wendlandt AE, Yelton SM, Lou D, Watt DS, and Noonan DJ (2010) Synthesis and functional analysis of novel bivalent estrogens. Steroids 75:825-853 PMID: 20685325.

Kathleen O’Connor

Our laboratory is interested in understanding how integrins and integrin- mediated signaling contribute to the late stages of carcinoma progression where cells acquire the ability to invade into the surrounding tissues. Integrin receptors, which link the to the and various signaling pathways, are essential for cells to sense and integrate cues from the extracellular matrix. Signaling from integrin receptors is critical for carcinoma cell invasion. One integrin species, the integrin α6β4, can promote this process. Integrin α6β4 confers an invasive and metastatic phenotype in many types of , including breast, colon and pancreas. Dissecting the pathways altered by integrin α6β4 has given and will continue to contribute to great insight into the processes that perpetuate an invasive and metastatic phenotype. Our laboratory is dedicated to furthering an understanding of integrin signaling in cancer since my pre-doctoral studies. During my post-doctoral studies, I Contact discovered links between integrin signaling and the cAMP pathway, and also [email protected], (859) 323-7534 determined that integrin α6β4 could stimulate the small GTPase RhoA leading to lamellae formation. Our laboratory continues to expound upon these findings in the context of chemotactic migration and invasion toward tumor-relevant attractants such as LPA, EGF and HGF, with a concentration on how RhoA signaling is regulated and contributes to invasion. We have further expanded our work to include investigations on how integrin α6β4 modifies the toward an invasion signature. Through these studies, we have identified the first transcriptional targets for NFAT1 and NFAT5 in cancer and provided evidence that integrin α6β4 can stimulate select demethylation of the promoters of pro-metastatic genes, including S100A4. The overarching goal of our work is to better understand the contributions of integrin α6β4 to tumor invasion and understand how this ―oncogenic‖ function differs from its normal functions in order to better target integrin α6β4 for therapeutic intervention.

Common techniques used in our laboratory

Immunoblotting; Rho, Rac and Ras GTPase activity assays; PKA assays (radioactive and non-radioactive); and chemoinvasion assays; cell culture (including 2D and 3D); immunoprecipitations; (cells staining in 2D and 3D); immunohistochemistry; microscopy: live cell videomicroscopy, TIRF, FRET, morphological assessments; fusion protein purification; siRNA and shRNA transfection; gene transfer/cloning; molecular cloning; and gene profiling.

Recent and representative publications

Chen M, Bresnick AR, O’Connor KL. Coupling S100A4 to Rhotekin alters Rho signaling output in breast cancer cells. Oncogene. 2012. In Press. O'Connor KL, Chen M, and Towers LN. Integrin α6β4 cooperates with LPA signaling to stimulate Rac1 through AKAP-Lbc-mediated RhoA activation. Am J Physiol Cell Physiol; 302(3):C605-14. 2012. Chen M, Sastry SK, and O’Connor KL. Src kinase pathway is involved in NFAT5-mediated S100A4 induction by hyperosmotic stress in colon cancer cells. Am J Physiol Cell Physiol; 300(5):C1155-63; 2011. Chen M, Sinha M, Luxon BA, Bresnick AR, O’Connor KL. Integrin α6β4 controls the expression of genes associated with cell , invasion and metastasis including S100A4/metastasin, J Biol Chem; 284: 1484-1494; 2009. Paulucci-Holthauzen AA, Vergara LA, Bellot LJ, Canton D, Scott JD, O’Connor KL. Spatial distribution of PKA activity during cell migration is mediated by A Kinase Anchoring Protein AKAP-Lbc. J Biol Chem; 284(9): 5956-67; 2009. (Reviewed by ―Faculty of 1000‖, Feb 2009) Cruz-Monserrate Z, O’Connor KL. Integrin α6β4 promotes migration, invasion through Tiam1 upregulation, and subsequent Rac activation. Neoplasia; 10:408-417; 2008. PMCID: PMC2373869.

Web Site

http://ukhealthcare.uky.edu/Markey/research/oconnor.asp

Sabire Özcan

The production and secretion of insulin from pancreatic beta cells is essential for maintaining normal blood glucose levels. Defects in insulin production and secretion lead to the development of diabetes. Diabetes has reached epidemic proportions, affecting over 150 million people worldwide (over 20 million in the United States), increasing the risk for a number of serious life-threatening complications—heart diseases (account for about 65% of death in people with diabetes), high blood pressure, kidney failure, blindness, and amputations. The long-term goal of our research is to gain insight into the molecular mechanisms by which increases in blood glucose Dr. Özcan in front row, middle. levels stimulate insulin production and secretion by the pancreatic beta cells. Our laboratory is currently focusing on Contact two major projects. First, we are interested in glucose-regulation [email protected], (859) 257-4821 of insulin gene transcription in pancreatic beta cells. Glucose- induction of insulin gene expression is regulated by the cooperative interaction of three beta-cell specific transcription factors, Pdx-1, MafA, and NeuroD1. The long-term goal of this project is to understand how glucose modulates the function of Pdx-1, MafA, and NeuroD1 to stimulate insulin gene expression. The second project focuses on microRNA function in pancreatic beta cells. MicroRNAs (miRNAs) are small non- coding ribonucleotides that bind mRNAs and function mainly as translational inhibitors. Dysregulation of miRNA levels are associated with many diseases including cancer, neurodegeneration and diabetes. We have identified several glucose-regulated miRNAs from pancreatic beta cells and are currently studying their function in insulin production and secretion. The identified miRNAs may have an important role in the establishment of diabetes and may serve as potential biomarkers for diabetes. In summary, the information obtained in pursuit of these projects should significantly advance our understanding of how glucose metabolism regulates insulin production and insulin secretion in pancreatic beta cells, and should enable the development of novel strategies for the treatment and prevention of diabetes and its associated complications.

Common techniques used in our laboratory

Western blotting; co-immunoprecipitation; staining of paraffin-embedded tissues; immunohistochemistry; cell culture; isolation of pancreatic islets; realtime qPCR; miRNA assays; RT-PCR; silencing using siRNA and shRNA; luciferase assays; site-directed mutagenesis; insulin secretion assay; in-situ hybridization; adenoviral and lentiviral gene transfer and cloning; chromatin immunoprecipitation assay (ChIP).

Recent and representative publications

Sampley, M.L, and S. Özcan. 2012. Regulation of insulin gene transcription by multiple histone acetyltransferases. DNA Cell Biol. 31:8-14. PMID: 21774670 Cantrell Stanford, J., A.J. Morris, M. Sunkara, G.J. Popa, K.L. Larson, and S. Özcan. 2012. Sphingosine 1- (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells. J. Biol. Chem. 287: 13457-13464. PMID: 22389505. Zhao, X., R. Mohan, S. Özcan, and X. Tang. 2012. MicroRNA-30d induces insulin MafA and insulin production by targeting mitogen-activated protein 4 kinase4 (MAP4K4) in pancreatic beta cells. J. Biol. Chem. 287: 31155-31164. PMID: 22733810.

Web Site http://aggregate.org/ozcan/

David Rodgers

We focus on understanding the basis for enzyme function and developing the ability to manipulate for the treatment of diseases and other practical applications. One project is to explore the unusual level of substrate recognition in neuropeptidases, enzymes that inactivate or modify the activity of peptide signaling molecules and peptide hormones. Neuropeptidases cleave only small peptide substrates, not larger peptides or proteins, and they recognize a variety of seemingly unrelated cleavage sites. Our work suggests possible molecular mechanisms for these unusual properties, and efforts are underway to test our hypotheses. Ultimately, we hope to engineer peptidases that are specific for particular peptides and may be used as therapeutics to treat disease. For example, we and our collaborators are attempting to alter the specificity of one neuropeptidase to better target the A peptide that causes Alzheimer’s disease. In Dr. Rodgers in back row, left. collaboration, with groups in Engineering, we are also involved in developing delivery vehicles for therapeutic enzymes. Contact In addition, we are attempting to design inhibitors of two [email protected], (859) 257-5205 neuropeptidases that control the levels of a particular peptide associated with psychotic disorders, pain perception, and addiction. Our goals are to understand the action of these inhibitors and to produce highly specific and bioavailable molecules that can be tested in clinical trials. We are also studying the basis for allosteric modulation of the neuropeptidase that metabolizes insulin, hoping to develop new therapeutics for diabetes and other disorders. In other work, we are exploring the molecular bases for sever neuromuscular disorders caused by inherited mutations in choline acetyltransferase, the enzyme responsible for the synthesis of the neurotransmitter acetylcholine. Our work indicates that structural defects in the enzyme make it particularly susceptible to disruption by mutation and suggests a possible approach for stabilizing the enzyme to treat the motor disorders.

Common techniques used in our laboratory

X-ray crystallography; enzyme kinetics; molecular biology/mutagenesis; fluorescence; light scattering; calorimetry, molecular visualization and computational chemistry; and single molecule fluorescent microscopy.

Recent and representative publications

Brown CK, Madauss K, Lian W, Beck MR, Tolbert WD, and Rodgers DW (2001) Structure of neurolysin reveals a deep channel that limits substrate access. Proc Natl Acad Sci USA 98, 3127-3132. Cai Y, Cronin CN, Engel AG, Ohno K, Hersh LB, and Rodgers DW (2004) Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. EMBO J 23, 2047-2058. Lim E-J, Sampath S, Coll-Rodriguez J, Schmidt J, Ray K, and Rodgers DW (2007) Swapping the specificity of the neuropeptidases neurolysin and thimet oligopeptidase. J Biol Chem 282, 9722-9732. Song ES, Rodgers DW, and Hersh LB (2010) A monomeric form of insulysin (IDE) loses its regulatory properties. PLoS One 16, e9719. Song, E.S., Rodgers, D.W. and Hersh, L.B. (2011) Mixed dimers of insulin-degrading enzyme reveal a cis activation mechanism. J. Biol. Chem. 286, 13852-13858. Noinaj, N., Bhasin, S.K., Song, E.S., Scoggin, K., Juliano, M.A., Juliano, L., Hersh, L.B. and Rodgers, D.W. (2011) Identification of the allosteric regulatory site of insulysin. PLoS ONE. 6,e20864. Sexton, T, Brock, L.J., Rodgers, D.W., Bradley, L.H. and Hersh, L.B. (2012) Active site mutations change the cleavage specificity of neprilysin. PLoS One 7, e32343. Noinaj, N., Song, E.S. Bhasin, S. Alper, B.J., Schmidt, W.K., Hersh, L.B. and Rodgers, D.W. (2012) The anion activation site of insulin degrading enzyme. J. Biol. Chem. 287, 48-57

Web Site http://www.mc.uky.edu/biochemistry/labs/rodgers

Kevin Sarge

Our laboratory is focused on two research interests: protein sumoylation and its role in human diseases; and ―gene bookmarking,‖ an epigenetic process that preserves the ―memory‖ of active gene expression. Sumoylation, the covalent attachment of Small Ubiquitin-like Modifier (SUMO) proteins to specific lysine residues in target proteins, regulates many aspects of normal protein function. Cells express three major SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3, with SUMO-2 and SUMO-3 being much more similar to each other than to SUMO-1. The SUMO E2 enzyme, called ubc9, attaches the SUMO proteins to a lysine or lysines in the target protein that are typically, but not always, found within the consensus sequence ΨKXE Ψrepresents hydrophobic amino acids). SUMO E3 proteins stimulate protein sumoylation by associating with both ubc9 (the E2 enzyme) and substrates to increase the efficiency of the modification reaction. Our laboratory recently discovered that a protein that plays an important role in nuclear architecture, A, is sumoylated and that this modification is required for the proper localization of this protein to the nuclear periphery. We also found that two mutations in lamin A that cause a heart disease Contact called cardiomyopathy, mutations that change the conserved glutamic acid residue [email protected] immediately adjacent to the sumoylation site of lamin A, result in this protein being (859) 323-5777 unable to be sumoylated. This is the first known example of a human disease that is caused by a mutation that prevents sumoylation of a protein. Several years ago, we found that a protein called HSF2 functions to prevent the promoter of the gene encoding the critical stress-protective protein hsp70i from being compacted during , in contrast to most of the genomic DNA which is tightly compacted. This lack of compaction is important because it provides cells with the ability to turn on transcription of the hsp70i gene even in early G1 phase if a stress occurs. Otherwise, the cell would be unable to protect itself until it could de-compact the promoter region. HSF2 prevents compaction of the hsp70i promoter by binding to its HSEs at the beginning of mitosis, recruiting protein phosphatase 2A, and interacting with a subunit of the enzyme called condensin that is important for mitotic DNA compaction, so that the HSF2-associated PP2A can dephosphorylate and inactivate the condensin to prevent compaction of this specific region of the chromosome. Our studies on HSF2-mediated bookmarking of the hsp70 promoter led us into a second major area of investigation for our laboratory. This area is the study of the epigenetic mechanism called gene bookmarking, which functions to precisely transmit the ―memory‖ of what genes were active prior to entry into mitosis to their daughter cells, despite the fact that transcription ceases and are highly compacted during this stage of the cell cycle. Knowledge of the gene bookmarking mechanism could lead to advances in generating stem cells from adult cells and cloning via nuclear transfer, because failure to properly reprogram gene bookmarking is believed to be a key barrier limiting the success of both of these processes.

Common techniques used in our laboratory

Western blot; immunoprecipitation; chromatin immunoprecipitation; PCR; fluorescence microscopy; and recombinant protein expression.

Recent and representative publications

Xing H, Wilkerson DC, Mayhew CN, Lubert EJ, Skaggs HS, Goodson ML, Hong Y, Park-Sarge OK, Sarge K (2005) Mechanism of hsp70i gene bookmarking. Science 307, 421-423. Zhang Y and Sarge KD (2008) Sumoylation regulates lamin A function and is lost in lamin A mutants associated with familial cardiomyopathies. J Cell Biol 182, 35-39. PMCID: PMC2447889 Zhang Y and Sarge KD (2008) Sumoylation of amyloid precursor protein negatively regulates Aβ aggregate levels. Biochem Biophys Res Commun 374, 673-678. PMCID: PMC2596940. Xing H, Vanderford NL, and Sarge KD (2008) The TBP/PP2A mitotic complex bookmarks genes by preventing condensin action. Nat Cell Biol 10, 1318-1323. PMCID: PMC2577711 Xing H, Hong Y, and Sarge KD (2010) PEST sequences mediate Heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING . Cell Stress Chaperones 15, 301-308. PMCID: PMC2866995

Pete Spielmann

Our laboratory studies isoprene metabolism that contributes to human health and disease with specific projects in the areas of cancer and cardiovascular disease. We use the techniques of organic synthesis, mass spectrometry and biochemistry as tools to study and manipulate complex cellular processes. Much of our research involves protein isoprenylation important to intra-cellular signal transduction and cancer development and progression. In addition to our work in the chemical biology of isoprenylation, we have a collaborative research program in protein and nucleic acid structure and dynamics in the University of Kentucky Center for Structural Dr. Spielmann in front row, left. Biology. The research projects in our laboratory use concepts from Contact synthetic chemistry to develop new experimental approaches to [email protected], (859) 257-4790 study biological systems (chemical biology). Protein isoprenylation is a major focus area, with recent projects oriented toward the identification of the prenylated for use as cancer biomarkers and understanding the mechanism of the enzyme protein farnesyltransferase (Ftase). of protein isoprenylation is the process of altering prenylated protein function by biosynthetic incorporation of unnatural isoprenoids containing unique chemical functional groups. We introduced alternative isoprenoid substrates for Ftase in 2000 and have extended and applied the technology to several biological problems, including proteomic analysis of protein isoprenylation and characterizing the cellular function of prenylated proteins. We discovered that aromatic groups are an ideal structural substitute for the C-5 isoprene subunit of isoprenoids due to similar surface areas, ease of functionalization, and similar conformational properties. Our expanding toolbox of modified isoprenoids includes structural analogues of C10 geranyl, C15 farnesyl and C20 geranylgeranyl diphosphates with a wide range of physical properties. Using these tools, we are working to determine the prenylated proteome in order to identify new targets for drug development and as biomarkers for early detection of cancer to improve the accuracy of diagnosis and long-term patient survival. In parallel, we are investigating isoprenoid metabolism to open new avenues for treating cardiovascular disease. Our projects in this area focus on the identification of enzymes and metabolites critical for interconversion of isoprenols with their diphosphates. One critical element of this research is synthesis of isoprenoid analogues that are substrates for cellular isoprenoid metabolizing enzymes and mass spectrometric identification of the resulting metabolites. Finally, we are developing new technologies to characterize protein and nucleic acid structure and dynamics.

Common techniques used in our laboratory

NMR; mass spectrometry; organic synthesis; and enzymology.

Recent and representative publications

Troutman J, Subramanian T, Andres DA, and Spielmann HP (2007) Selective Modification of CaaX Peptides with ortho-Substituted Anilinogeranyl Lipids by Protein Farnesyl Transferase: Competitive Substrates and Potent Inhibitors from a Library of Farnesyl Diphosphate Analogues. Biochemistry 46, 11310-11321. Subramanian T, Liu S, Troutman J, Andres, DA, and Spielmann HP (2008) Protein Farnesyltransferase Catalyzed Isoprenoid Transfer To Peptide Depends On Lipid Size and Shape, not Hydrophobicity. Chembiochem 9, 2872- 2882. Onono FO, Morgan MA, Subramanian T, Spielmann HP, Andres DA, Ganser A, and Reuter CWM (2010) A tagging- via-substrate approach to elucidate the farnesylated proteome using two-dimensional electrophoresis (2DE) coupled with Western blotting. Mol Cell Proteomics 9, 742-751. Miriyala S, Subramanian T, Panchatcharam M, Ren H, Sunkara M, Drennan T, Smyth SS, Spielmann HP, and Morris AJ (2010) Functional characterization of the atypical integral membrane lipid phosphatase PDP1/PAPDC2 identifies a pathway for interconversion of isoprenols and isoprenoid in mammalian cells J Biol Chem 285, 13918-13929. Subramanian, T., Pais, J., Suxia Liu, Troutman, J.M., Suzuki, Y., Subramanian, K. L., Fierke, C. A., Andres, D. A. and Spielmann, H. P. (2012) Farnesyl Diphosphate Analogues with Aryl Moieties are Efficient Alternative Substrates for Protein Farnesyltransferase Biochemistry. In press,

Stefan Stamm

Our laboratory works on RNA. We aim to understand the rules that govern alternative splice site selection and to understand how cellular signals change alternative splice site selection in human cells. Almost every human protein- coding gene undergoes alternative splicing. The laboratory applies this knowledge to understand human diseases caused by missplicing events to pave the development of rationale therapies. We focus on Prader-Willi Syndrome. Prader-Willi Syndrome (PWS) is the most frequent genetic cause for type II diabetes. It is now clear that the loss of small nucleolar located in the PWS critical region on is the cause of the disease. These C/D box snoRNAs fall into two clusters: SNORD115 and Dr. Stamm in third row, left. SNORD116. We showed that these RNAs function in pre- mRNA processing and discovered a new class of non- Contact coding RNAs, called psnoRNAs for processed snoRNAs. [email protected], (859) 323-0896 Processed snoRNAs are generated from canonical C/D box snoRNA expressing units. psnoRNAs influence both alternative pre-mRNA splicing and gene expression by binding to RNA and DNA, respectively. SNORD115 regulates the alternative splicing of the serotonin receptor 2C and promotes its most active form, which could explain the hyperphagia observed in patients with PWS. Using chemical screens and oligonucleotide walks, we identified substances that inhibit mice from eating by influencing the alternative splicing of the serotonin receptor 2C. SNORD116 regulated gene expression by binding to chromatin and influencing promoter activity through an unknown mechanism. Genes that are regulated by SNORD116 act mainly in energy and fat metabolism, suggesting that SNORD116 regulated a functionally related set of genes. We are now studying the detailed molecular mechanism for psnoRNA action, with the aim of substituting these short RNAs with oligonucleotides.

Common techniques used in our laboratory

In vivo splicing assays; in vitro splicing assays; splicearray analysis; RNAse protection, non-coding RNAs, uptake, RNA cloning.

Recent and representative publications

Zhang, Z., Kelemen, O., van Santen, M.A., Yelton, S.M., Wendlandt, A.E., Sviripa, V.M., Bollen, M., Beullens, M., Urlaub, H., Lührmann, R., Watt, D.S. and Stamm, S. (2011). Synthesis and Characterization of Pseudocantharidins, Novel Compounds That Promote of the SMN2 Exon 7. J Biol Chem 286, 10126-10136. Shen, M., Eyras, E., Wu, J., Khanna, A., Josiah, S., Rederstorff, M., Zhang, M.Q., and Stamm, S. (2011). Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression. Nucleic acids research 39, 9720-9730. Stamm, S., Smith, C.W.J., and Lührmann, L. (2012). Alternative Splicing: Theory and Protocols (Wiley). Kelemen, O., Convertini, P., Zhang, Z., Wen, Y., Shen, M., Falaleeva, M., and Stamm, S. (2012). Function of alternative splicing. Gene, in press Falaleeva, M., and Stamm, S. (2012). Processing of snoRNAs as a new source of regulatory non-coding RNAs. Bioessays, in press.

Web Site http://www.stamms-lab.net

Sam Turco

Protozoan parasites of the genus Leishmania are responsible for a spectrum of human diseases, termed leishmaniasis. Depending on the species involved, leishmaniasis appears clinically in three forms: cutaneous, mucocutaneous, and visceral, the last being fatal if untreated. The parasites have a remarkable capacity to avoid destruction in the hostile environments encountered in their life cycle, alternating between intracellular macrophage and extracellular life in the gut of the sand fly vector. As in other microbial pathogens, the development of genetic tools for the study of these parasites promises to help unravel the molecular details of how these persevere under such harsh circumstances. Advances in functional genetic analysis provide a Dr. Turco in second row. new avenue for identifying genes implicated in the parasite’s infectious cycle, such as those necessary for the synthesis and expression of the key surface virulence Contact factor, lipophosphoglycan (LPG) (see figure for structure and electron microscopy [email protected], (859) 323-6693 showing density of LPG on the parasite’s surface). LPG has been implicated in binding and release of the parasite in the midgut of the sand fly (see figure), resistance to complement, binding and uptake by macrophages, modulating macrophage signal transduction, resistance to oxidative attack, and, ultimately, allowing the parasite to establish successful infections. The overall objective of our research is to elucidate the genetic and biochemical details of the biosynthesis of the Leishmania lipophosphoglycan and related , primarily through the exploitation of LPG-defective mutants. The functions of the proteins encoded by LPG genes recovered by genomic and bioinformatic approaches is a primary focus of our laboratory. These functions and eventual characterization of the encoded proteins will be determined through the development and use of in vitro enzymatic assays. From our studies, we hope to contribute to the understanding of the role glycoconjugates play in the pathogenesis of leishmaniasis and to provide a biochemical rationale for the design of chemotherapeutic regimens that exploit complex carbohydrate differences between those of the mammalian host and of the parasite.

Common techniques used in our laboratory

Capillary electrophoresis; fluorophore-assisted carbohydrate electrophoresis (FACE); organic solvent extractions for carbohydrates and lipids; gene transfections and expressions; protein purification; enzyme assays; carbohydrate structure elucidation; cell culture techniques; various chromatographic techniques.

Recent and representative publications

Coelho-Finamore, J. M., Freitas, V. C., Assis, R. R., Melo, M., N., Novozhilova, N. M., Secundino, N., Pimenta, P., Turco, S. J., and Soares, R., Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts. (2011) Intl. J. Parasitol. 41, 333-342. NIHMSID: 370480. Vinet, A. F., Janaji, S., Turco, S. J., Fukuda, M., and Descoteaux, A., Exclusion of Synatotagmin V Lipophosphoglycan results in decreased Promastigote Internalization. (2011) Microbiology, 157, 2619-2928 NIHMSID: 370461. Assis RR, Ibraim IC, Noronha FS, Turco SJ, and Soares RP , Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: modulation of innate immune system and variations in carbohydrate structure. (2012) PLoS Negl Trop Dis. 6: e1543. PMC3289616.

Deneys van der Westhuyzen

Our laboratory is focused on and ―scavenger‖ receptors and how such receptors contribute to or protect against the disease of atherosclerosis. Oxidized and modified -containing play a key role in the development of atherosclerosis, and our laboratory is studying various scavenger receptors that mediate the metabolism of such lipoprotein particles. One such receptor, the scavenger receptor-BI (SR-BI), is also a known receptor for native HDL. SR-BI mediates the selective delivery of HDL cholesterol esters from circulating HDL to cells and therefore mediates the important process of "reverse cholesterol transport" from extrahepatic tissues to the liver. We have shown that SR-BI exists as alternatively-spliced variants, and we are currently studying the significance and distinct biological functions of these receptor isoforms which are differentially targeted in cells. This includes an Dr. van der Westhuyzen in front row, left. investigation of the regulation of expression of SR-BI isoforms in

different tissues and their regulation by cholesterol and oxidized Contact [email protected], (859) 323-4933 x81397 lipids. The effect of dietary cholesterol and high fat ―Western Diet‖ on the expression of scavenger receptors is being studied, together with their role in the development of atherosclerosis. Studies are also addressing the role of SR-BI and CD36, another Class B scavenger receptor, in genetic mouse models of atherosclerosis formation, and focus on defining how these scavenger receptors contribute to or protect against cholesterol and lipid accumulation in macrophages in the arterial wall. A central hypothesis being examined in our laboratory is that modifications of HDL during inflammation leads to alterations in HDL metabolism that impact cholesterol trafficking in the circulation and cholesterol accumulation in cells. Inflammation, as occurs in the atherosclerotic lesion, results in a number of metabolic changes that can lead to significant modulations of HDL structure and function. Such changes appear to have important implications for HDL cholesterol metabolism in affecting HDL-mediated cholesterol delivery to the liver, as well as cholesterol from cells to HDL.

Common techniques used in our laboratory

Ligand binding assays to cultured cells; labeled lipoprotein clearance studies in cells and mice; confocal microscopy studies of receptor and ligand trafficking in cells; cellular lipid efflux measurement; ligand induced cellular signaling assays; atherosclerosis measurements in mouse models.

Recent and representative publications

Shetty S, Eckhardt ER, Post SR, and van der Westhuyzen DR (2006) Phosphatidylinositol-3-kinase regulates scavenger receptor class B type I subcellular localization and selective lipid uptake in . Arterioscler Thromb Vasc Biol 26, 2125-2131. Cai L, Ji A, de Beer FC, Tannock LR, and van der Westhuyzen DR (2008) SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance. J Clin Invest 118, 364-375. PMID: 18064300 Meyer JM, Ji A, Cai L, van der Westhuyzen DR. High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation. J Lipid Res. 2012 Oct;53(10):2081-91. PMID: 22833685 Cai., L., Wang, Z., Ji., A., Meyer, J.M., van der Westhuyzen, D.R. Macrophage SR-BI regulates pro-inflammatory signaling in mice and isolated macrophages. J. Lipid Res. 2012. 53; 1472-81. PMID: 22589557. Ji, A, Wroblewski, JM, Cai, L, de Beer, MC, Webb, NR, van der Westhuyzen DR, Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1 and SR-BI. J. Lipid Res. 2012. 53:446-55 PMCID: PMC3276468

Craig Vander Kooi

Our laboratory focuses on understanding the basic mechanisms by which secreted cytokines and cell surface receptors function in the formation of new blood vessels (angiogenesis) and the guidance of . We also study how these natural signaling pathways become perturbed in human diseases. Our primary interest centers on the essential human called neuropilin. Neuropilin is a conserved bi-functional mammalian cell surface receptor and has fundamental biomedical importance since it is essential to two distinct biological processes, angiogenesis and guidance. During angiogenesis, neuropilin binds VEGF and functions as a co-receptor for the VEGF-R Dr. Vander Kooi in back row, left. receptor kinases. During neural development, neuropilin binds semaphorin and functions as a co-receptor for members of Contact [email protected], (859) 323-8418 the plexin and L1CAM family of receptors. We seek to understand neuropilin ligand binding, specificity, and receptor activation. Fundamentally, our goal will be to answer the following questions: How does neuropilin activate and regulate VEGF dependent angiogenesis, and how does it function in semaphorin dependent axon guidance? In addition to its normal roles, neuropilin also functions in tumor angiogenesis. Neuropilin expression is observed in tumor vasculature, and overexpression promotes tumorigenesis in vivo in a variety of solid tumors including those of the pituitary, prostate, breast, and colon. Recent evidence has also demonstrated a role for neuropilin in hematological malignancies where neuropilin overexpression is observed in both multiple myeloma and acute myeloid leukemia. In contrast, a soluble splice form containing only the ligand binding region of the extracellular domain of neuropilin inhibits tumorigenesis. Additionally, a number of peptides that block VEGF binding to neuropilin exhibit anti-tumorigenic activity. Strategies to inhibit neuropilin activity are being developed in the laboratory as potential antitumor therapies. Finally, a third area of interest is neuropilin and spinal cord injury. The semaphorins represent one of the largest families of cytokines that directly guide axon growth. Neuropilin functions as the high-affinity cell surface receptor for class III semaphorin family members. While repulsive cues are essential during development, they pose a significant barrier to axonal regrowth following injury. This is particularly the case in repair following spinal cord injury. Following spinal cord injury a glial scar forms. Class III semaphorins are produced by meningeal cells located in the glial scar and are a major component of the repulsive cues which prevent axon regeneration. Thus, there is strong interest in furthering our knowledge of the semaphorin signaling system. We are working to both enhance our understanding of the mechanisms underlying semaphorin mediated axon guidance, as well as to develop new targeted inhibitors for use in the treatment of spinal cord injury.

Common techniques used in our laboratory

X-ray crystallography; mutagenesis; eukaryotic/prokaryotic protein production; protein purification; angiogenesis assays; and axon guidance assays.

Recent and representative publications

Parker M.W., Xu P., Li X., Vander Kooi C.W.‡. (2012) Structural Basis for Selective Vascular Endothelial Growth Factor-A (VEGF-A) Binding to Neuropilin-1. J. Biol. Chem. 287, 11082-11089. Parker MW, Hellman LM, Xu P, Fried MG, and Vander Kooi CW (2010) Furin processing of semaphorin 3F determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin. Biochemistry 49, 4068-4075. Vander Kooi C.W.‡, Taylor A.O., Pace R.M., Meekins D.A., Guo H-F., Kim Y., Gentry M.S.‡ (2010) Structural basis for the glucan phosphatase activity of Starch Excess4. Proc Natl Acad Sci U S A. 107, 15379-15384. Merte J, Wang Q, Vander Kooi CW, Sarsfield S, Leahy DJ, Kolodkin AL, and Ginty DD (2010) A forward genetic screen in mice identifies Sema3A(K108N), which binds to neuropilin-1 but cannot signal. J Neurosci 30, 5767- 5775. Vander Kooi CW, Jusino MA, Perman B, Neau DB, Bellamy HD, and Leahy DJ (2007) Structural basis for ligand and heparin binding to neuropilin B domains. Proc Natl Acad Sci USA 104, 6152-6157.

Skip Waechter

The major interest of this laboratory is to understand the regulation of enzymes involved in the biosynthesis of dolichyl phosphate (Dol-P) and protein N- glycosylation in the (ER). We are particularly interested in genetic defects in lipid-mediated protein N-glycosylation leading to diseases known as Congenital Disorders in Glycosylation. The co-translational addition of N-linked oligosaccharide chains is critical for the proper folding, intracellular translocation, and function of many membrane glycoproteins mediating essential neurobiological functions in the central nervous system (CNS). Because of the importance of this modification, the long-term goal of this laboratory has been to understand the role of endoplasmic reticulum proteins in the regulation of biosynthesis of Dol-P, the assembly of Glc3Man9GlcNAc2-P-P-dolichol and the transbilayer movement of dolichyl-phospho-saccharide intermediates in mammalian cells. We are currently using biochemical and genetic approaches to isolate and learn more about the structures of ER proteins (flippases) mediating the transverse diffusion of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc2-P-P-Dol using a novel and promising transport assay with water-soluble analogues and sealed ER vesicles. In a related project we have cloned cDNAs from the that the long-chain cis-isoprenyltransferase (cis-IPTase), a key enzyme Contact involved in the de novo biosynthesis of Dol-P in mammalian cells. The cloned [email protected] cDNAs provide coding sequence information and probes to be used to assess (859) 323-6352 developmental changes in the expression of the cis-IPTase and clues to the molecular basis of the association of the cis-IPTase with the ER.

Common techniques used in our laboratory

Tissue culture of mammalian cells; molecular biology and gene expression; isolation and structural characterization of glycoproteins and ; enzymology of membrane- bound enzymes; topological analysis of membrane vesicles.

Recent and representative publications

Rush JS, Gao N, Lehrman MA, and Waechter CJ (2007) Recycling of Dolichyl Monophosphate to the Cytoplasmic Leaflet of the ER after the Cleavage of Dolichyl on the Lumenal Monolayer. J Biol Chem 283, 4087-4093. Shridas P and Waechter CJ (2006) Human Dolichol Kinase: A Polytopic ER Membrane Protein with a Cytoplasmically-Oriented CTP-Binding Site. J Biol Chem 181, 31696-31704. Frank CG, Sanyal S, Rush JS, Waechter CJ, and Menon AK (2008) Does Rft1 Flip and N-glycan Lipid precursor? Nature 454, 7204. Rush JS, Gao N, Lehrman MA, Matveev S, and Waechter CJ (2009) Suppression of Rft1 Expression Does Not Impair the Transbilayer Movement of Man5GlNAc2-P-P-Dolichol in Sealed from Yeast. J Biol Chem 284, 19835-19842. Rush JS, Alaimo C, Robbiani R, Wacker M, and Waechter CJ (2010) A Novel Epimerase that Converts GlcNAc-P-P- Undecaprenol to GalNAc-P-P-Undecaprenol in Escherichia coli O157. J Biol Chem 285, 1671-1680. Rush, JS., Matveev, S, Zigiang, G, Raetz, CRH, and Waechter, CJ (2010) Expression of Functional Bacterial undecaprenyl Pyrophosphate synthase in the yeast rer2 Mutant and CHO Cells. Glycobiology, 20, 1585-1593. Harrison, KD., Park, EJ., Gao, N, Rush, JS., Waechter, CJ., Lehrman, MA., and Sessa, WC. Nogo-B Receptor is Necessary for Cellular Dolichol Biosynthesis and Protein N-Glycosylation. (2011) Embo J. 1-11.

Qingjun Wang

Autophagy is a lysosomal degradation pathway that plays important roles in a variety of essential cellular processes and many devastating human diseases including cancer, infection, liver disease, myopathy, and neurodegeneration. Upon autophagy induction, portions of the containing proteins and organelles is sequestered into de novo generated double-membraned vesicular structures called . Autophagosomes are subsequently delivered to , forming autolysosomes where the contents are degraded. Despite the clear importance of autophagy in health and disease, we are yet to discover the molecular machinery comprising the mammalian Dr. Wang, second from right. autophagy pathway, the molecular mechanism of

how this machinery functions, and the roles of Contact [email protected], (859) 323-5335 autophagy in health and disease. We take an integrated approach that combines mouse genetics, proteomics, and cell biology to investigate the mammalian autophagy pathway and its role in normal physiology and pathological states. We first identify novel protein-protein interactions in the mammalian autophagy pathway directly from living animals and subsequently determine the functions of these novel interactors. Ongoing research in this laboratory targets (i) all key steps of the mammalian autophagy pathway including signaling and regulation, (ii) roles of autophagy in the healthy brain and the brain that undergoes neurodegeneration, (iii) roles of autophagy in other human diseases such as cancer, and (iv) roles of autophagy in aging.

Common techniques used in our laboratory

Molecular cloning; mouse genetics; siRNA/shRNA; protein affinity purification; mass spectrometry; confocal microscopy; Western blot; and immunohistochemistry.

Recent and representative publications

Funderburk SF, Wang QJ, and Yue Z (2010) The Beclin 1–VPS34 complex – at the crossroads of autophagy and beyond. Trends Cell Biol 20, 355-362. Zhong Y, Wang QJ, Li X, Yan Y, Backer JM, Chait BT, Heintz N, and Yue Z (2009) Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat Cell Biol 11, 468-476. Yue Z, Holstein GR, Chait BT, and Wang QJ (2009) ―Using genetic mouse models to study the biology and pathology of autophagy in the central nervous system.‖ In D Klionsky, ed., Methods Enzymol 453 ―Autophagy: Higher Eukaryotes mammalian and clinical,‖ p.159-180. Komatsu M, Wang QJ, Holstein GR, Friedrich VL Jr, Iwata JI, Kominami E, Chait BT, Tanaka K, and Yue Z (2007) Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration. Proc Natl Acad Sci USA 104, 14489-14494.

Web Site http://www.mc.uky.edu/biochemistry/labs/wang/

Dave Watt

Our laboratory collaborates with investigators under the aegis of the Organic Synthesis Core to provide consultation and subsequent access to novel ―small molecules‖ for the study of biological problems. The core provides for the synthesis and characterization of these molecules including, but not limited to, polymer-bound compounds, fluorescent or radiolabeled derivatives of drugs or natural products, photoreactive reagents for crosslinking studies, deuterated reagents for mass spectrometric analyses, modified natural products, and unnatural peptides or peptide analogs. Some of the target Dr. Watt in middle. structures recently completed in our laboratory are shown below. Students in this laboratory undertake a research Contact problem in which another faculty member and myself [email protected], (859) 257-3945 serve as thesis advisors.

Common techniques used in our laboratory

Organic synthesis techniques; mass spectrometry; and NMR spectroscopy.

Recent and representative publications

Sumanasekera C, Watt DS, and Stamm S (2008) Substances that can change alternative splice-site selection. Biochem Soc Trans 36, 483-90. Wendlandt AE, Yelton SM, Lou D, Watt DS, and Noonan DJ (2010) Synthesis and functional analysis of novel bivalent estrogens. Steroids 75, 825-833.

Sidney Whiteheart

Our laboratory is engaged in several major research projects under the common theme of molecular signaling and cellular communication. One project is focused on the identification and characterization of key regulators of platelet function. Platelets are first responders to vascular damage and they secrete a host of molecules that are important for hemostasis and wound repair. As part of this work, the group has identified over 50 proteins that could function in platelet secretion. The present focus Dr. Whiteheart back row, third from left. is on the syntaxin regulators, Munc18, DOC2, Munc13, and Tomosyn. As part of this work we Contact use genetically altered mice and we examine [email protected], (859) 257-4882 platelet function in patients with genetic bleeding disorders. We have discovered that platelet function assays may prove to be a useful diagnostic tool for Familial Hemophagocytic Lymphohistiocytosis (FHL). The overall goal is to understand the molecular machinery that connects platelet activation to secretion so that we can produce drugs that control hemostasis. We are also interested in the pathogenic role of platelet secretion in vivo. Our group has shown that VAMP- 8-mediated secretion is important for thrombogenesis, artherosclerosis (using the ApoE-/- model) and cardiac hypertrophy. The group is developing other mouse strains that can be used to probe the roles and effects of platelet secretion in normal processes such as angiogenesis and pathogenic processes such as vascular inflammation. Additionally, we are studying the role of Arf6 in platelet activation. Arf6 is a small GTPase critical for platelet function. We showed that Arf6 is present in platelets, transitions from GTP- to GDP-bound states upon activation, and is important for Rho family activation and cytoskeletal remodeling. This opens a new window into platelet signaling. A second major project involves the structure/function analysis of NSF, a homohexameric ATPase required for all secretory pathways. It serves as a SNARE chaperone, using ATP hydrolysis to disassemble spent SNARE complexes. Our work has defined specific roles to its three domains (N, D1, and D2) and the relative movements of the domains during an ATPase-driven catalytic cycle. We also study epilepsy. With Dr. John Slevin (UK Neurology), we are examining the secretory machinery in hippocampal synapses from rats that have been rendered epileptic using the kindling paradigm of electrical stimulation. Specifically we are trying to determine if either the levels or the activity of specific SNAREs and SNARE regulators are altered in epileptic animals.

Common techniques used in our laboratory

Protein production; electron microscopy; SDS-PAGE and Western blotting; enzymatic assays; PCR and molecular biology techniques; transgenic mouse analyses and in vivo thrombosis models.

Recent and representative publications

Ren Q, Wimmer C, Chicka M, Ye S, Ren Y, Hughson FM, and Whiteheart SW (2010) Munc13-4 is a limiting factor in the pathway required for platelet release and hemostasis. Blood, 116, 869-877. Moeller, A., Zhao, C., Fried, M.G., Wilson-Kubalek, E.M., Carragher, B., and Whiteheart, S.W. (2012) Nucleotide- Dependent Conformational Changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their Potential Role in SNARE Complex Disassembly. Journal of Structural Biology, 177, 335. Al Hawas, R., Ren, Q., Ye, S., Karim, Z.A., Filipovich, A.H., and Whiteheart, S.W. (2012) Munc18b/STXBP2 Is Required for Platelet Secretion. Blood, 120, 2493. Ye, S., Karim, Z.A., Al Hawas, R., Pessin, J.E., Filipovich, A.H., and Whiteheart, S.W. (2012) Syntaxin-11, but not Syntaxin-2 and Syntaxin-4, Is Required for Platelet Secretion. Blood, 120, 2484.

Binhua Peter Zhou

Breast cancer is the most common form of cancer in women. The clinical symptoms and outcome of breast cancer depend largely on whether it is confined to the breast or has spread to adjacent or distant parts of the body. Despite more than 3 decades of research, approximately 90% of breast cancer deaths are caused by local invasion and distant metastasis of tumor cells, and the average time to live after documentation of metastasis is approximately 2 years. Epithelial-mesenchymal transition (EMT) is a vital process for large-scale cell movement during the at . In this EMT event, cells change from ―static‖ to ―motile‖ in appearance and start to migrate. This is because the cell- molecule E-cadherin is lost, and cells Dr. Zhou in front row, middle. break away from one other and start to migrate and invade the

Contact surrounding tissue. Tumor cells usurp this developmental [email protected], (859) 323-4474 process for their invasion and metastasis. The major challenge in breast cancer research is to identify the factors within the cell and the signals outside the cell that initiate this early event of metastasis. Snail and Slug are zinc-finger transcriptional repressors in controlling EMT during embryogenesis and metastasis. Expression of Snail and Slug correlates with the tumor grade and nodal metastasis of many types of tumor and predicts a poor outcome in patients with metastatic cancer. We are employing biochemical, molecular, and cellular approaches to study the functional regulation of Snail in breast cancer and, by discovery dis-regulations, to identify molecules that may serve as the therapeutic targets for metastasis prevention. One of our studies is to identify the tumor microenvironmental signals that induce EMT and metastasis. EMT is provoked by signals that cells receive from their microenvironment (tumor-stromal boundary). Using macro- phage co-culture experiment and mouse model, we showed that inflammatory microenvironmental signals, particularly TNFα, play an important role in the induction of EMT and metastasis. We are currently characterizing how these microenvironmental signals trigger distinct signaling pathways that lead to EMT and metastasis. Another focus of our studies is to define the epigenetic regulation of EMT at metastasis. EMT is a dynamic and reversible process. When breast cancer cells disseminate to distant sites of the body, they no longer encounter the signals that they experienced in the primary tumor, and they can revert to an epithelial state via a mesenchymal- epithelial transition (MET). This phenomenon suggests that an epigenetic program is involved in controlling EMT at metastasis. We are currently identifying the key chromatin and DNA modifying molecules that associate with Snail in regulating the epigenetic program of EMT.

Common techniques used in our laboratory

PCR/DNA cloning/site-directed mutagenesis; protein expression and purification/tandem-array purification and mass spectrometry analysis; cell culture/gene expression/stable clone generation/ and lentivirus expression; Western blotting and immunoprecipitation/immunofluorescence staining/DNA expression array analysis; enzymatic analysis/chromatin-immuoprecipitation (CHIP)/CHIP-sequencing; and animal models/immunohistochemistry on human breast tumor tissues.

Recent and representative publications

Wu Y, Evers BM, and Zhou BP (2009) Small C-terminal domain phosphatase enhances snail activity through dephosphorylation. J Biol Chem 284, 640-648. Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, and Zhou BP (2009) Stabilization of snail is required for inflammation-induced cell migration and invasion. 15, 416-428. Wu Y and Zhou BP (2009) Inflammation: a driving force speeds cancer metastasis. Cell Cycle 8, 3267-3273. Lin Y, Wu Y, Li J, Dong C, Ye X, Chi Y-I, Evers BM, and Zhou BP (2010) The SNAG domain of snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. EMBO J 29, 1803-1816. Wu Y and Zhou BP (2010) Snail: more than epithelial-mesenchymal transition. Cell Adh Mig 4, 199-203.

Haining Zhu

We use various modern biochemical and cell biology approaches to study neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease). An additional feature of our laboratory is integrated application of mass spectrometry-based proteomic technique in research. ALS is a neurodegenerative disease without a cure, and its etiology remains largely unclear. Familial ALS has been linked to mutations in multiple genes including copper-zinc superoxide dismutase (SOD1), and RNA processing proteins TDP-43 and FUS/TLS. Our Dr. Zhu in back row, second from left. long-term goal is to use the familial ALS gene mutations as models to understand Contact the molecular mechanisms underlying the [email protected], (859) 323-3643 pathogenesis and progression of the

disease. Our current focuses include (1) to determine what causes defects in in ALS and the significance of such defects; (2) to understand the mitochondrial involvement in ALS and the mechanism by which mitochondrial function, trafficking, and dynamics are affected in ALS; (3) to study the relationship among protein misfolding, degradation, and aggregation, as well as their contribution to neurodegenerative diseases; and (4) to uncover the dysfunctional pathways linking mutations in RNA processing proteins to the disease. Mass spectrometry has been a prevailing technology for proteomic studies. We are interested in developing simple and effective mass spectrometric approaches. The current focus is to develop methodology to better characterize membrane proteins, protein post-translational modifications (e.g. phosphorylation, acetylation and methylation), and protein complexes. Mass spectrometry has been extremely valuable in our research identifying the targets of the toxicity of the disease-causing mutant proteins. The discovery capability of proteomics and the in-depth functional analysis are fully integrated in our own research projects as well as the collaborations with other colleagues.

Common techniques used in our laboratory

Mass spectrometry (MS and LC-MS/MS) and proteomics; molecular biology (cloning, gene manipulation including mutagenesis); cell biology (cell culture and primary neurons, immunohistochemistry); confocal microscopy and live cell imaging; Western blotting; immunoprecipitation; analysis of protein degradation and aggregation in cells; and use of transgenic animals.

Recent and representative publications

Gal J, Zhang J, Kwinter D, Zhai J, Jia H, Jia J, and Zhu H (2010) ALS mutations in FUS are clustered in the nuclear localization sequence and induces stress granules. Neurobiol Aging (in press). Shi P, Ström A, Gal J, and Zhu H (2010) Effects of ALS-related mutant SOD1 on - and KIF5-mediated retrograde and anterograde axonal transport. Biochim Biophys Acta 1802, 707–716. Gal J, Ström A, Kwinter D, Kilty R, Zhang J, Shi P, Fu W, Wooten MW, and Zhu H (2009) Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism. J Neurochem 111, 1062-1073. Zhai J, Ström A, Kilty R, Venkatakrishnan P, White J, Everson WV, Smart EJ, and Zhu H (2009) Proteomic characterization of proteins in amyotrophic lateral sclerosis mouse spinal cord. FEBS J 276, 3308-3323. Ström A, Shi P, Zhang F, Gal J, Kilty R, Hayward LJ, and Zhu H (2008) Interaction with functional dynein-mediated retrograde transport system facilitates formation of large aggregates / inclusions of familial ALS SOD1 mutants. J Biol Chem 283, 22795-22805.

Core Facilities

Core facilities provide state-of-the-art equipment and services to support the research of graduate students, postdoctoral fellows, research technicians and the faculty:

Protein Analytical Core Director: Dr. David W. Rodgers

This core is designed to facilitate protein characterization by COBRE investigators. It will make available well-maintained equipment and instrumentation that would not normally be found within individual laboratories, particularly those of junior investigators. In particular, the core will provide equipment for the production and purification of proteins and their characterization by a number of analytical techniques.

Imaging Core Director: Dr. Carole Moncman

The overall goal of this core is to serve as a bridge to higher-end imaging methodologies (i.e. confocal). The core allows investigators a rapid and facile way to evaluate their experiments and to determine if further more extensive analysis is warranted or required.

Proteomics Core Director: Dr. Haining Zhu

This core is designed to provide quantitative and qualitative protein analysis as well as posttranslational modification characterization using a combination of gel electrophoresis, liquid chromatography and mass spectrometry techniques.

Organic Synthesis Core Director: Dr. David Watt

This core provides ―small molecule‖ probes to investigators as components of their proposed projects. Specifically, the core furnishes modified drugs and natural products tailored to include suitable functional groups for linkage to proteins, peptide-nucleic-acid (PNA) mimics, and polymers.

Viral Production Core Director: Dr. Michael D. Mendenhall

This core produces the most frequently used viral vectors for use by COBRE-supported research projects. This includes production of lentivirus, adenovirus, and adeno-associated virus vectors within the core.

Student Life

Student life in the Department of Biochemistry is productive, rewarding, and fun! Biochemistry graduate students are well trained in various specialized research areas and in biochemistry in general. A combination of course work and rotations in various laboratories allows each student to sample research in particular areas and select an advisor who is a good ―fit‖ for his or her interests. Students also prepare and present their research at local conferences and seminars, at national meetings, and certainly in journal publications. Students are also encouraged to attend the annual off-campus departmental retreat. Each year, graduate students present a departmental seminar on an interesting area of research in which they do not directly work. This gives the student the ability to learn more about other fields of research as well as develop presentation skills that are crucial as preparation for the thesis defense and for job interviews. Students can also informally present their own research data to other graduate students in monthly Data Club meetings. This gives a student an opportunity get feedback and advice on their projects from other students. Finally, there are numerous invited speaker seminars and faculty interview seminars that students are encouraged to attend. Each year, students also select and invite one speaker from outside the University and take part in hosting and introducing this speaker. These are great opportunities for students to learn current research in other fields and to make contacts for possible postdoctoral positions and/or industry positions. Biochemistry graduate students are encouraged to collaborate with students in other laboratories within or outside of the department. In addition to academic collaborations, students also participate in many social activities such as ice cream socials, fund-raisers, and food drives. Students and faculty work together on various departmental committees in areas such as new student recruitment and event planning. Overall, the graduate students in Biochemistry enjoy a well-rounded graduate experience that prepares them for successful future careers.

Life in Lexington

Lexington is a wonderful place to live and work. The relatively low cost of living allows for affordable housing (some students even own their own home), shopping, dining, and various other entertainment opportunities. Lexington offers all of the excitement and events of a larger city while still maintaining a small, college-town atmosphere that is affordable, safe, convenient, and enjoyable. There are numerous parades, festivals, and events occurring regularly in Lexington for one to attend, and the Farmer’s Market downtown on Saturdays is not to miss. Sports fans can enjoy attending a UK basketball game at legendary Rupp Arena, a UK football game at Commonwealth Stadium, or a minor league baseball game at Applebee’s Park. The many parks in Lexington (including dog parks!) can be used for jogging, biking, picnics, or a number of other outdoor recreational activities. In addition, students often enjoy exploring many of the scenic state parks such as Red River Gorge, Natural Bridge, and Cumberland Falls, which are a short drive from Lexington and offer spectacular hiking, biking, camping, and canoeing. As Lexington is known as the ―Horse Capital of the World,‖ there are endless horse farms that cover the rolling hills surrounding the city. A highlight of living in Lexington is attending the thoroughbred races at Keeneland, which run every April and October. In addition, Lexington and the surrounding areas are home to numerous vineyards where you can tour a wine-making facility and taste some delicious local wines. Of course, being in Kentucky, a short drive west of Lexington will also afford you the opportunity to visit and tour any number of world-renowned bourbon distilleries. And, if this isn’t enough to keep you busy in Lexington, it is just a short drive to larger cities such as Cincinnati and Louisville for even more shopping, dining, and entertainment options. Image © Pat D. Hemlepp. Used with permission of the photographer.