The Lodge @ Geneva-on-the-lake

November 18-19, 2010

RetreatRetreatRetreat 201020102010 PharmacologyPharmacologyPharmacology

“Somewhere, something incredible is waiting to be known”

—Dr. Carl Sagan

Retreat Program Retreat Program Retreat Program

Thursday, November 18, 2010Thursday, November 18, 2010Thursday, November 18, 2010

8:30-9:00 Registration Registration Registration Lake Erie Ballroom

9:00-9:15 Welcome by Dr. Leslie T. Webster, Jr.

Molecular Pharmacology and Cell Regulation Molecular Pharmacology and Cell Regulation Molecular Pharmacology and Cell Regulation Directors: George Dubyak, Monica MontanoDirectors: George Dubyak, Monica MontanoDirectors: George Dubyak, Monica Montano ProvocateursProvocateursProvocateurs: John Feng, Alan Levine, Irina Pikuleva, Ruth Siegel, Johannes von Lintig: John Feng, Alan Levine, Irina Pikuleva, Ruth Siegel, Johannes von Lintig: John Feng, Alan Levine, Irina Pikuleva, Ruth Siegel, Johannes von Lintig

9:15-9:30 Overview and Session Leader: Dr. Monica Montano 9:30-9:45 Charlotte Chung, Grad Student (Levine Lab) Molecular Definition of Intestinal Barrier Function Loss in HIV infection. 9:45-10:00 Luke Bury, Grad Student (Sabo Lab) Coordination of synaptic vesicle and active zone protein trafficking in developing cortical neurons. 10:00-10:15 Andrea Boyd-Tressler, Grad Student (Dubyak lab) ATP Release from Apoptotic Tumor Cells via Panx1 Hemichannels.

10:15-10:30 Break

10:30-10:45 Meghana Gupta, Grad Student (Manor Lab) Regulation of Rac1 and its upstream activator Dbl, by reversible tyrosine phosphorylation. 10:45-11:00 Casey Charvet, Grad Student (Pikuleva lab) Post-translational Modification of CYP27A1 by Oxidized Arachidonic Acid Seems to Occur in the Retina. 11:00-11:15 Shuiliang Yu, Postdoc (Noy lab) The retinoic acid-activated FABP5/PPARδ pathway represses neuronal differentiation by up-regulation of SIRT1 and Ajuba. 11:15-11:30 Amanda C. Vreeland, Grad Student (Noy lab) Retinoic Acid-Independent Pro-Apoptotic Activities of Cellular Retinoic Acid-Binding Protein II.

11:30-11:45 Break

11:45-12:00 Jaume Amengual, Postdoc (von Lintig lab) β-carotene decreases Peroxisome proliferator receptor γ activity and reduces adiposity in a β,β-Carotene Oxygenase 1-dependent manner. 12:00-12:15 I-Ju Yeh, Grad Student (Montano lab) Mechanistic basis and functional consequences of HEXIM1 downregulation of HIF1 protein levels.

12:15-12:30 Wannarasmi Ketchart, Grad Student (Montano lab) Induced HEXIM1 expression leads to the inhibition of breast cancer metastasis.

12:30-1:30 LunchLunchLunch Lake Erie Room A (see Menu)

Translational TherapeuticsTranslational TherapeuticsTranslational Therapeutics Directors: Tony Berdis, Robert BonomoDirectors: Tony Berdis, Robert BonomoDirectors: Tony Berdis, Robert Bonomo Provocateurs: Charles Hoppel, Mariana Rosca, Tim Kern, Marvin Nieman, Jen YoriProvocateurs: Charles Hoppel, Mariana Rosca, Tim Kern, Marvin Nieman, Jen YoriProvocateurs: Charles Hoppel, Mariana Rosca, Tim Kern, Marvin Nieman, Jen Yori

1:30-1:45 Overview and Session Leader: Dr. Robert Bonomo 1:45-2:00 Alex Veenstra, Grad Student (Kern Lab) Marrow-derived cells regulate the development of diabetic retinopathy in mice. 2:00-2:15 Guilian Tian, Postdoc (Imanishi Lab) Characterization of Clarin1-enriched microdomain proteome. 2:15-2:30 Tonibelle Gatbonton-Schwager, Grad Student (Tochtrop and Letterio Labs) The sprouting of triterpenoids that modulate the inflammatory and the anti-oxidant Phase-II response. 2:30-2:45 Amar Desai, Grad Student (Gerson Lab). The Role of Exo1 in DNA Mismatch and Double Strand Break Repair: Implications for Improved Radiotherapy in Cancer?

2:45-3:15 Chair’s Address and Departmental Awards Ceremony

3:15-3:30 Departmental Photo Main Foyer

3:30-6:00 Afternoon Break Relay Races in Lake Erie Room B

6:00-7:00 DinnerDinnerDinner Lake Erie Room A (see menu)

7:00-9:00 Poster Session Western Reserve Room and Harbor Room

9:00-12:00 Reception: featuring magic by Dr. Yoshi Imanishi! Main Foyer

Friday, November 19, 2010Friday, November 19, 2010Friday, November 19, 2010 7:30-8:30 Breakfast BuffetBreakfast BuffetBreakfast Buffet Horizons Restaurant and Lounge

Membrane Structural Biology and PharmacologyMembrane Structural Biology and PharmacologyMembrane Structural Biology and Pharmacology Directors: Derek Taylor, Chris DealwisDirectors: Derek Taylor, Chris DealwisDirectors: Derek Taylor, Chris Dealwis Provocateurs: Vera MoiseenkovaProvocateurs: Vera MoiseenkovaProvocateurs: Vera Moiseenkova---Bell, Mike Maguire, Masaru Miyagi, Jason Mears, Andrea MoomawBell, Mike Maguire, Masaru Miyagi, Jason Mears, Andrea MoomawBell, Mike Maguire, Masaru Miyagi, Jason Mears, Andrea Moomaw

8:30-8:45 Overview and Session Leader: Dr. Derek Taylor 8:45-9:00 Brian M. Kevany, Postdoc (Palczewski lab) Phagocytosis of Retinal Photoreceptors. 9:00-9:15 Debarshi Mustafi, Grad Student (Palczewski lab) Dissection of the visual signaling network in cone cells.

9:15-9:30 Marcin Golczak, Research Associate (Palczewski Lab) An acyl-covalent enzyme intermediate of lecithin:retinol acyltransferase. 9:30-9:45 Presenter: David Lodowski, Postdoc (Palczewski Lab) Conformational Changes in the G Protein-Coupled Receptor Rhodopsin Revealed by Histidine Hydrogen−Deuterium Exchange.

9:45-10:00 Break

10:00-10:15 Thomas Kowatz, Postdoc (Palczewski Lab) Towards the Structures of Carotenoid Cleaving Enzymes. 10:15-10:30 Yiyuan Yuan, Postdoc (Feng lab) Profiling proteomics of diet restriction and TOR signaling pathways with a novel Stable isotope lysine labeling in C. elegans identifies novel genes regulating C. elegans lifespan. 10:30-10:45 Sanath R. Wijerathna, Grad Student (Dealwis lab) Structural and functional Validation of Nucleotide Induced Oligomerization of the Ribonucleotide Reductase.

10:45-11:00 Break

11:00-12:00 Keynote Address: George Stark, Ph.D. Staff, Distinguished Scientist, Lerner Research Institute, Professor Genetics, CWRU

12:00-1:15 LunchLunchLunch Lake Erie Room A (see Menu)

12:45-1:30 Fireside Chat with Dr. Stark

Cancer TherapeuticsCancer TherapeuticsCancer Therapeutics Directors: Ruth Keri, Bill SchiemannDirectors: Ruth Keri, Bill SchiemannDirectors: Ruth Keri, Bill Schiemann Provocateurs: Noa Noy, Ruth Keri, Bingcheng Wang, Clark Distelhorst, Reema WahdanProvocateurs: Noa Noy, Ruth Keri, Bingcheng Wang, Clark Distelhorst, Reema WahdanProvocateurs: Noa Noy, Ruth Keri, Bingcheng Wang, Clark Distelhorst, Reema Wahdan

1:30-1:45 Overview and Session Leader: Dr. Bill Schiemann 1:45-2:00 Chris Ryder, Grad Student (Distelhorst lab) Acidosis-Sensing TDAG8 Induces Bcl-2 and Prevents Apoptosis Due to Glutamine Starvation. 2:00-2:15 Molly Taylor, Grad Student (Schiemann lab) Extracellular Matrix Rigidity Mediates Mammary Epithelial Cell Response to TGF-β. 2:15-2:30 Brad T. Casali, Grad Student (Keri lab) The neurotransmitter GABA induces proliferation of basal breast cancer cells through endogenous expression of the GABA-receptor pi subunit. 2:30-2:45 Michele Mumaw, Grad Student (Noy Lab) The Role of Transthyretin in STRA6-Mediated Vitamin A Uptake and in Signaling by RBP-retinol.

2:45-3:00 Break

3:00-3:15 Jingna Wang, Postdoc (Zhang lab) Novel insights into the dynamic regulation of the cell cycle checkpoint kinase, Chk1. 3:15-3:30 Steve Sizemore Postdoc (Keri lab)

FOXC1 is required for basal breast cancer viability and promotes invasion through MMP7 expression.

3:30-4:00 Excellence in Research Award Presentation Gina Bernardo, Grad Student (Keri Lab) FOXA1 is an essential determinant of ERa expression and mammary ductal morphogenesis

4:00-4:30 Retreat Awards FACULTY AWARDS

Teaching Service Research 1998 Ruth A. Keri John J. Mieyal Charles L Hoppel 1999 John J. Mieyal Michael E. Maguire Michael E. Maguire 2000 Amy L. Wilson-Delfosse Paul N. MacDonald Himan Sternlicht 2001 Anthony J. Berdis David Danielpour Monica M. Montano 2002 Ruth E. Siegel Daniel E. Davis Ruth A. Keri

OUTSTANDING PERFORMANCE Research Assistant Student Postdoctoral

1998 David W.Starke Minh Lam Not Awarded 1999 Lisa M. Kucharski Algirdas A. Velyvis Not Awarded 2000 Kristen L. Lozada Joan S. Jorgensen Christine Quirk 2001 Richard M. Gibson Colleen Tagliarino Nancy Wang 2002 Darcie Seachrist Erin L. Milliken David Kehres 2003 Tara I. Ellison Philip A. Verhoef Not Awarded 2004 Not Awarded Melissa C. Bentle Not Awarded 2005 Not Awarded Melissa Landis Not Awarded

2006 Not Awarded Vivian Gama Not Awarded 2007 Not Awarded Tara Ellison Not Awarded 2008 Not Awarded Ndiya Ogba Emhonta Johnson 2009 Not Awarded Philip Kiser Marcin Golczak

NATHAN S. GREENFIELD FAMILY TRAVEL AWARD DEDICATION TO MENTORING 2003 Tehnaz Parakh 2000 Brian Fitch 2004 Molly M. Gallogly 2001 Anthony Berdis 2005 Jodi Thomson 2002 John Mieyal and Edmunds Reineks 2006 Elizabeth Sabens 2003 Melissa Bentle, Payal Gandhi, Jonathan 2007 Vivian Gama Mosley, Melissa Shelton, Krisztina Papp 2008 Gina Bernardo 2004 Paul N. MacDonald 2009 Chris Ryder EXCELLENCE IN RESEARCH 2008 Akiko Maeda 2009 Philip Kiser

Molecular Pharmacology and Cell Regulation — Oral session Presenter: Charlotte Chung, Grad Student (Levine lab) Title: Molecular Definition of Intestinal Barrier Function Loss in HIV infection HIV (Human Immunodeficiency Virus) infection, when untreated, will progress to Acquired Immunodeficiency Syndrome (AIDS), as measured by the decline of blood CD4+ T cells. Viral infection, while directly mediating T cell death, cannot account for this vast T cell depletion. Alternatively, virus-induced systemic immune activation, a better predictor of disease progression than plasma viral load, is implicated as a major contributor to T cell death, through the physiological response of activation-induced T cell death. Evidence for the trigger of HIV-induced immune activation has emerged. Within weeks of infection, there is a profound depletion of gut CD4+ T cells from which recovery is incomplete. We propose that this compromised intestinal immune response, or the virus directly, may disrupt the barrier function of the intestinal epithelium to luminal microbiota, thus allowing for translocation of micro-bial products. Circulating microbial products have been detected in HIV-infected individuals, the level of which correlates with im-mune activation. We hypothesize that HIV infection contributes to a loss of intestinal epithelial tight junction (TJ) integrity, leading to increased intestinal permeability that allows for microbial translocation and immune activation. Through measuring the fractional excretion of various saccharide probes, we have localized increased permeability in HIV-infected patients, uncorrected by anti-retroviral therapies, to the small and large intestines. Current efforts are focused on defining the precise molecular dysfunction of TJs underlying this permeability defect, through confocal imaging of TJ proteins occludin and ZO-1 in intestinal biopsies. These results will be complemented by an in vitro model of HIV-induced intestinal permeability, through which the biochemical, functional, and structural integrity of epithelial cell monolayers will be evaluated in the presence of cell-free HIV, HIV-infected T cells, and anti-retroviral drugs. Presenter: Luke Bury, Grad Student (Sabo Lab) Title: Coordination of synaptic vesicle and active zone protein trafficking in developing cortical neurons Transport of proteins from the cell body to developing pre-synaptic terminals in the axon is crucial for proper development of syn-apses. However, whether transport and recruitment of these proteins to pre-synaptic sites are coordinated is not understood. Pro-teins that form the active zone are transported in dense core vesicles deemed piccolo transport vesicles (PTVs), which are distin-guished by the presence of active zone proteins, such as piccolo, syntaxin, and bassoon. Likewise, proteins required for synaptic vesicle formation are transported in vesicles known as synaptic vesicle protein transport vesicles (STVs), and are distinguished by the presence of synaptic vesicle proteins, such as VAMP2/synaptobrevin and synaptophysin. During transport, both STVs and PTVs pause at multiple sites along the axon. We have previously shown that STV pause sites are places where stabilization of a pre-synaptic terminal is likely to occur. Here, we have tested the hypothesis that the pausing of STVs and PTVs is coordinated.. We have also tested whether recruitment of active zone proteins to a pause site along the axon is required for STV recruitment and po-tential pre-synaptic terminal formation. To quantify STV and PTV movement and pausing, we transfected rat cortical neurons with GFP-bassoon and synaptophysin-mcherry to label PTVs and STVs, respectively. Movement and pausing of the two different vesi-cles was then captured via time-lapse confocal microscopy and quantified. To determine whether STV recruitment to a pause site is mediated by active zone proteins, PTV transport was blocked using a dominant-negative corresponding to the syntaxin binding do-main of syntabulin. Overall, our results highlight the importance of STV and PTV pausing during the recruitment of proteins for pre-synaptic terminal formation. Presenter: Andrea Boyd Tressler, Grad Student (Dubyak lab) Title: ATP Release from Apoptotic Tumor Cells via Panx1 Hemichannels Chemotherapeutics such as oxaliplatin or cisplatin are important primary therapies used to combat cancer. Treatment of tumors with these agents causes cell death through DNA damage. In addition recent data have shown that an immune response is initiated which enhances the anti-tumor actions of the primary chemotherapeutic agent. The mechanism underlying this immune component of the anti-tumor response remains unclear. One component of this mechanism may involve the recently reported ability of apoptotically induced tumor cells to release ATP, which acts as a “find me”, signal for dendridic cells (DC) and macrophages to phagocytize dying cancer cell. DCs act as tumor antigen presenting cells to activate T-cells that specifically mount an immune response against the can-cer. Although many stimuli, such as mechanical stress, cause ATP to be released from cells, the mechanism by which apoptotic in-duction triggers ATP release from tumor cells is not understood. This study examines the role of pannexin 1(panx1) hemichannels in the release of ATP from apoptotic cancer cells. Rat glioma C6 cells, which do not express panx1, and C6 cells stably transfected panx1 cDNA were treated with staurosporine to induce the intrinsic pathway of apoptosis. ATP release was assayed by luciferase-based assay. Our results indicate that C6 cells which expressed panx1 are characterized by a large increase in ATP release in re-sponse to both apoptotic induction and mechanical stimulation. Thus, panx1 hemichannels may play an important role in ATP re-lease from apoptotic tumor cells. Presenter: Meghana B. Gupta, Grad Student (Manor lab)

Title: Regulation of Rac1 and its upstream activator Dbl, by reversible tyrosine phosphorylation Mitogenic signals are initiated by the binding of growth factors to their receptors and culminate in a variety of cellular responses including alterations in gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic pathways. Many mitogens exert their effects by stimulating the activity of Guanine Nucleotide Exchange Factors (GEFs), which in turn activate small GTPases of the Rho family (Rho, Rac and Cdc42). Perturbations in the signaling of Rho GTPase and their GEFs are often associated with several types of cancers. For instance, the Dbl GEF is over-expressed in neuroectodermal tumors and Ewing sarcomas, and aberrant

expression of Rac1 is observed in certain types of liver, breast, and colon cancers. Given the important roles that Rac1 and Dbl play in mitogenic signaling, it is not surprising that the activities of these proteins are tightly regulated. We have found that in response to mitogenic signals, both Rac1 and Dbl are subject to phosphorylation on tyrosine residues. Additionally, in both instances phos-phorylation occurs through the action of a src-like kinase family member. Ongoing studies aim to elucidate how phosphorylation of tyrosine residues affects the biochemical and physiological activities of Rac1 and Dbl. Taken together, our findings uncover a novel layer of complexity in the potential regulation of these signaling pathways.

Presenter: Casey Charvet, Grad Student (Pikuleva lab) Title: Post-translational Modification of CYP27A1 by Oxidized Arachidonic Acid Seems to Occur in the Retina. Accumulating evidence indicates a link between cholesterol and age-related macular degeneration. Cytochrome P450 27A1 (CYP27A1) plays a major role in cholesterol degradation and homeostasis in peripheral tissues and is expressed in the human retina. High inter-individual variability in CYP27A1 product formation in the retina hinted at post-translational modification. Arachidonic acid, abundant in the retina, undergoes free-radical mediated transformation into highly reactive -ketoaldehydes known as isole-vuglandins. Isolevuglandins react with proteins’ lysyl residues to form pyrrole-derived adducts. This process has been demonstrated in oculo in drusen; however, this was not investigated further in the retina. To facilitate in vivo studies, we first reconstituted purified recombinant CYP27A1 into different phospholipid vesicles comprised of phosphatidylcholines (PC) alone, a 4:3 PC:phosphatidylethanolamine mixture, or retinal mitochondrial phospholipids, and then modified the protein with authentic iso[4]levuglandin-E2. We show by mass spectrometry that modification occurs in all three types of vesicles at select lysines with the effect of reduced enzyme activity. By using unmodified 15N-labeled CYP27A1 as an internal standard, we are able to quantify the extent of modification and calculate the propensity of each lysine to form adducts. Three of these lysine modification sites were chosen for further study. Next, we modified 15N-labeled CYP27A1 to use as an internal standard for quantifying adducts in donor retinas by mass spectrometry. We present evidence of CYP27A1 modified in vivo at a location predicted by in vitro studies in all donor retina samples analyzed (n = 4). Further, immunohistochemistry studies reveal colocalized fluorescent signals from antibodies to CYP27A1 and isolevuglandin derived adducts in the retina. Thus, we show for the first time that a protein in the human retina is post-translationally modified at a specific site by isolevuglandins. Presenter: Shuiliang Yu, Postdoc (Noy lab) Title: The retinoic acid-activated FABP5/PPARδ pathway represses neuronal differentiation by up-regulation of SIRT1 and Ajuba Retinoic acid (RA) is involved in regulation of multiple biological processes, including embryogenesis and neuronal differentiation. The transcriptional activities of RA can be mediated by two nuclear receptors which cooperate with two lipid-binding proteins. CRABP-II delivers RA to RAR and FABP5 shuttles RA to PPARδ. Partitioning of RA between these two nuclear receptors is de-pendent on the CRABP-II/FABP5 ratio. While the activation of RAR has been known to be essential for RA induced neuronal differ-entiation, very little is known about the role of FABP5/PPARδ during neuronal differentiation. Using P19 mouse embryonic carci-noma cells as a model, we found that the CRABP-II/FABP5 ratio increased dramatically after RA treatment. Down-regulation of either PPARδ or FABP5 enhanced the expression of the neuronal progenitor cell marker, Nestin, and facilitated RA-induced P19 cell neuronal differentiation. Ectopic expression of PPARδ or FABP5 inhibited Nestin expression and neuronal differentiation. Two co-repressors of RAR, SIRT1 and Ajuba, were identified as target genes of PPARδ in P19 cells. SIRT1 is a NAD-dependent deacetylase which inhibits RAR activity by competing with SKIP, a RAR coactivitor, thus impairing RA-induced P19 cells neuronal differentia-tion. Ajuba is a member of the LIM protein family. It binds RAR directly and represses its transcriptional activity. We also showed that the expression of FABP5 and PPARδ was up-regulated by treatment with the PPARδ agonist, GW0742. These observations suggest the existence of a positive feed-back loop between PPARδ and its binding protein FABP5. In agreement, the neuron makers MAP-2 and NeuN, and the astrocyte maker GFAP displayed higher expression in the cerebral cortex of FABP5 knockout mice. Our data indicate that, like two sides of a coin, the two pathways of RA play reciprocal roles in neuronal differentiation. While CRABP-II/RAR signaling promotes the RA-induced neuronal differentiation, FABP5/PPARδ represses neuronal differentiation by up-regulation of SIRT1 and Ajuba expression. Presenter: Amanda C. Vreeland, Grad Student (Noy lab) Title: Retinoic Acid-Independent Pro-Apoptotic Activities of Cellular Retinoic Acid-Binding Protein II Retinoic acid (RA) displays potent anti-carcinogenic activities. These activities are mediated by retinoic acid receptors (RARs) and by cellular RA-binding protein II (CRABP-II). In the cytoplasm of cells, CRABP-II binds RA. Holo-CRABP-II translocates to the nucleus, forming a complex with RAR through which RA is channeled to RAR. Upon ligand binding, RAR induces the expression of target genes including some that are involved in differentiation, apoptosis and cell cycle control. The presence of CRABP-II in cells markedly enhances the transcriptional activities of RAR, and augments the ability of RA to inhibit the growth of mammary carcinoma cells. Interestingly, it has been reported that CRABP-II also functions independently of RA. Specifically, overexpression of CRABP-II in MCF-7 mammary carcinoma cells grown in media free of retinoids results in cleavage of caspases 7 and 9, an in-crease in the mRNA levels for Apaf-1, the core protein of the apoptosome, and upregulation of mRNAs of other apoptotic proteins. The goal of this work was to understand the basis for the RA-independent pro-apoptotic activities of CRABP-II. We found that upregulation of Apaf-1 by CRABP-II is exerted through stabilization of mRNA for this protein. CRABP-II overex-pression also stabilizes the mRNA of HuR, a well characterized mRNA binding and stabilizing protein. Decreasing the expression level of HuR abrogates the ability of CRABP-II to stabilize Apaf-1 mRNA, suggesting that the effect of CRABP-II may be mediated

by HuR. Additional data show that 1) CRABP-II and HuR bind Apaf-1 mRNA and HuR mRNA and 2) CRABP-II interacts with HuR and the association is disrupted by RA. These observations show that CRABP-II and HuR cooperate to stabilize mRNAs and that RA inhibits these activities. In accordance with the ability of CRABP-II to upregulate the expression of pro-apoptotic genes, overexpression of this protein in mammary carcinoma cells triggers apoptosis and enhances apoptosis induced by established apoptotic stimuli. Presenter: Jaume Amengual, Postdoc (von Lintig lab) Title: β-carotene decreases Peroxisome proliferator receptor γ activity and reduces adiposity in a β,β-Carotene Oxygenase 1-dependent manner Increasing evidence indicates that β-carotene-(BC)-derived apocarotenoid signaling molecules can modulate the activities of nuclear receptors that regulate many aspects of adipocyte physiology. Two BC metabolizing enzymes, the BC-15,15’-monooxygenase (BCMO1) and the BC-9’,10’-dioxygenase (BCDO2) are expressed in adipocytes. BCMO1 catalyzes the conversion of BC into reti-naldehyde and BCDO2 into β-10’-apocarotenal and β-ionone. Here we analyzed the role of these enzymes in adipocyte biology us-ing both adipocyte cell cultures and mice models. In NIH 3T3-L1 cells, BCMO1 mRNA and protein expression are induced during adipogenesis. In mature adipocytes, BC but not all-trans-retinol was metabolized to retinoic acid (RA). RA reduced peroxisome pro-liferator receptor γ (PPARγ) expression and decreased intracellular lipid content. This process was inhibited by the RA receptor an-tagonist LE450, showing that it involves canonical retinoid signaling. To genetically dissect the roles of BCMO1 and BCDO2 in adipocyte biology in vivo, we used wild-type and Bcmo1-/- mice. In wild-type mice, BC was converted into retinoids. In contrast, Bcmo1-/- mice showed increased expression of BCDO2 in adipocytes and β-10’-apocarotenol accumulated as the major BC deriva-tive. In wild-type mice, BC significantly reduced body adiposity (by 28%), leptinemia and adipocyte size. Genome wide microarray analysis of inguinal white adipose tissue revealed a generalized decrease of mRNA expression of PPARγ target genes. Consistently, the expression of this key transcription factor for lipogenesis was significantly reduced both on the mRNA and protein levels. De-spite β-10’- apocarotenoid production, this effect of BC was absent in Bcmo1-/- mice, demonstrating that it was dependent on the BCMO1-mediated production of retinoids. Our study evidences an important role of BC for adipocyte biology controlling body adi-posity in mice, and identifies BCMO1 as critical molecular player for the regulation of PPARγ activity in adipocytes. Presenter: I-Ju Yeh, Grad Student (Montano lab) Title: Mechanistic basis and functional consequences of HEXIM1 downregulation of HIF1 protein levels Tumor metastasis is the primary cause of death in cancer patients. Angiogenesis, or the formation of blood vessels from pre-existing vessels, plays a critical role in metastasis. We previously reported that mice expressing mutant HEXIM1 have a higher incidence of mammary tumors with increased levels of Vascular Endothelial Growth Factor (VEGF) and increased vascularization. We also ob-served that targeted overexpression of HEXIM1 in mammary epithelial cells inhibited metastasis in a mouse model of breast cancer. VEGF, an important mediator of angiogenesis, is modulated by hypoxia-inducible factor 1 (HIF1 ). The critical role HIF-1 in tumor metastasis arises not only from the fact that it is a potent activator of angiogenesis, but also of invasion and metabolic repro-gramming. Under hypoxic condition, HIF1 is deacetylated and its ubiquitination inhibited, resulting in stabilization of HIF1 protein. HIF1 then dimerizes with HIF1 and recruits activators to the hypoxic responsive element (HRE). Our results indicate that under hypoxia conditions, HEXIM1 inhibits the recruitment of HIF1 to the HRE in the VEGF promoter. Increased HEXIM1 expression results in decreased HIF1 protein expression in cell line and mouse models of breast cancer. HEXIM1 did not downregulate HIF1 mRNA levels, suggesting post-transcriptional regulation of HIF1 by HEXIM1. Co-immunoprecipitation experiments indicate that en-dogenous HEXIM1 interacts with endogenous HIF1 in breast cell lines. A direct interaction between HEXIM1 and HIF-1 was observed in in vitro Glutathione-S-transferase pull down assays. Previous reports indicate that histone deacetylase 1 (HDAC1) inter-acts with HIF1 and stabilizes its expression. However we observed that downregulation of HEXIM1 expression using microRNA resulted in enhanced interaction between HDAC1-HIF1 in breast cells. Our data suggest that HEXIM1 decreases HIF1 level by inhibiting the HDAC1-HIF1 interaction under hypoxic condition. Presenter: Wannarasmi Ketchart, Grad Student (Montano lab) Title: Induced HEXIM1 expression leads to the inhibition of breast cancer metastasis. We have previously reported that Hexamethylene inducible gene-1 (HEXIM1) has both antiproliferative and antiangiogenic activity in breast cancer cells. We tested the effectiveness of induced HEXIM1 expression through transgene expression or through the dif-ferentiating agent Hexamethylene-bis-acetamide (HMBA) in inhibiting growth of highly aggressive tumors in the MMTV- Polyoma Middle-T antigen (PyMT) mice. Since HEXIM1 can be upregulated in MCF-7 breast cancer cells by HMBA, HMBA is expected to inhibit tumorigenesis and prevent metastasis in the PyMT mice. Tumor progression in the PyMT mouse is comparable to human tumors and the majority of mice that developed on tumors also developed secondary lung metastasis. However, the maximal thera-peutic dose of HMBA is below its effective dose due to severe adverse effects such as thrombocytopenia, neurotoxicity and acidosis. Local delivery of HMBA may allow the drug to be released directly at target sites, increasing exposure time and reducing toxicity. We are also developing derivatives of HMBA with prolonged half-lives. To achieve prolonged and local delivery of HMBA we used Poly (L-lactide-co-glycolide) (PLGA), a biodegradable polymer. After contact with water, PLGA forms a precipitate and develop into a solid implant that encapsulates HMBA inside. After PLGA localizes to a specific site of drug action, the drug will be released in a controlled manner. PLGA will be degraded and metabolized into biologically molecule and excreted out of the body. Our studies

indicate that HEXIM1 expression is significantly increased even after 2 weeks of HMBA-PLGA treatment. HMBA-PLGA treatment also resulted in the reduction of tumor vascularization and lung metastasis. Moreover, there was no evidence of thrombocytopenia and weight loss in HMBA-PLGA- treated mice when compared to PLGA- treated mice. Therefore, local delivery of HMBA is effec-tive in the inhibition of breast tumor growth, angiogenesis and metastasis with less adverse effects.

Translational Therapeutics — Oral session Presenter: Alex Veenstra, Grad Student (Kern Lab) Title: Marrow-derived cells regulate the development of diabetic retinopathy in mice Objective: Most studies of the pathogenesis of diabetic complications focus on metabolic abnormalities within the affected tissues themselves. We investigated the hypothesis that bone marrow-derived cells play a critical role in the development of diabetes-induced retinopathy. Research design and Methods: The contribution of leukocyte subtypes to diabetes induced capillary degeneration was evaluated using chimeric mice lacking G-CSFR (receptor for Granulocyte Colony Stimulating Factor) in marrow derived cells, and co-culture of retinal endothelial cells with peripheral blood leukocytes immunodepleted of different leukocyte subtypes. Leukocyte transmigra-tion into the neural retina was evaluated using chimeric mice in which only the marrow derived cells expressed GFP Results: Neutrophils (and monocytes) play a major role in the retinopathy development, because diabetes induced degeneration of retinal capillaries was significantly inhibited in mice lacking G-CSFR in marrow-derived cells only. Immunodepletion of neutrophils or monocytes from purified leukocytes inhibited endothelial death otherwise observed when co-culturing leukocytes from diabetic animals with retinal endothelium. Leukostasis of both monocytes and neutrophils increased with diabetes in the retina however transmigration of these cells into the neural retina was not observed suggesting leukocytes kill endothelial cells from within the vas-culature. The addition of antioxidant (lipoic acid) or anti-FasL antibodies to the co-cultures reduced endothelial death induced by leukocytes from diabetic animals. Conclusions: We conclude that leukocytes, especially granulocytes, play a central role in the development of diabetic retinopathy by killing retinal capillary endothelial. Presenter: Guilian Tian, Postdoc (Imanishi Lab) Title: Characterization of Clarin1-enriched microdomain proteome Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Clarin-1 forms a specific membrane microdo-main, clarin-1-enriched microdomain, by incorporating specific proteins and cholesterol, when expressed in HEK293 cells. Pro-teomics analysis revealed that clarin-1-enriched microdomains contain a number of proteins involved in the regulation of F-actin structure. Clarin-1 is expressed in sensory hair cells where it may interact with other co-existing Usher gene products which are es-sential for the maintenance of these cells. Increasing evidence suggests that the products of Usher type I and II genes form a large network of protein-protein interactions, in which F-actin and PDZ (named after postsynaptic density protein, disc-large, and zo-1) domain proteins play major roles in organizing such network. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains with a PDZ domain-binding motif within the C-terminal tail. A current hypothesis is that the dysfunction of this network existing in the cochlea and the eye cause specific symptoms characterized by deafness and blind-ness. We are currently in a process of investigating the PDZ-domain mediated interactions among Usher types I, II and III. To initiate the studies of Usher interaction network, we characterized PDZ-domain proteins related to Usher syndrome. There are two PDZ domain proteins encoded by Usher causative genes. Among them, Harmonin is encoded by Usher syndrome IC gene, and Whirlin is encoded by Usher syndrome IID gene. We cloned harmonin and whirlin from human retina and expressed these two pro-teins in HEK293 cells stably expressing human clarin-1 (HEK-CLRN). We found a novel whirlin isoform with one extra exon that has not been previously reported. Our PCR analysis suggests that this novel isoform is the most predominant among the splicing variants of whirlin. When expressed in HEK293 cells, whirlin localized predominantly to the centriole. When cells exit the cell cycle from mitosis into a quiescent state, the centriole moves to the peri-plasma membrane area and becomes a basal body, from which the cilia structure nucleates. Some of Usher causative gene products were previously reported to form a complex at the base of the cilia, and whirlin may play a role in such assembly. We are currently studying the possible interactions between Clarin1 and PDZ domain proteins. Presenter: Tonibelle Gatbonton-Schwager, Grad Student (Letterio, Tochtrop Lab), Title: The sprouting of triterpenoids that modulate the inflammatory and the anti-oxidant Phase-II response Triterpenoids are naturally occurring molecules that are ubiquitous in the plant kingdom and have a variety of biological activities. One of the most well studied triterpenoids is the oleanene derived semi-synthetic triterpenoid CDDO and its derivatives. This is a novel class of chemopreventive agents with multifunctional properties that include potent anti-inflammatory, anticancer, and cyto-protective activities. This class of molecules targets multiple signaling pathways including suppressing expression of the inflamma-tory enzymes iNOS and COX-2 and potent up-regulation of the anti-oxidant phase II enzymes through the Nrf2-Keap1 signaling pathway. These diverse activities lead us to investigate the role of the triterpenoid structural scaffold in the observed anti-inflammatory and anti-cancer effects. We hypothesize that the skeletal arrangement of the triterpenoids will control the expression profile of the inflammatory and anti-oxidant response via the Nrf2 transcription factor. We took the triterpenoids – lanosterol and bryonolic acid (BA) and use it as a platform for diversity oriented synthesis (DOS), which aims to rearrange the core carbocyclic structure of the molecule. An important step before the application of this chemistry is to understand the anti-inflammatory proper-

ties of BA. Through our studies we discovered that BA’s most robust phenotype is the induction of the Nrf2-Keap1 target gene, heme-oxygenase 1. Further exploration of this pathway led to the discovery that BA induces several Nrf2-Keap1 downstream target genes. In addition, its activity extends beyond the RAW 264.7 cell culture to primary mouse macrophage and is active in vivo. Evaluation of triterpenoid activity will aid in the determination of the mechanism behind the therapeutic properties of these mole-cules. Successful completion of these studies will advance the development of novel chemopreventive agents targeted specifically at inflammation driven carcinogenesis. Presenter: Amar Desai, Grad Student (Gerson Lab). Title: The Role of Exo1 in DNA Mismatch and Double Strand Break Repair: Implications for Improved Radio therapy in Cancer? Conventional cancer therapies have historically targeted DNA repair proteins whose inhibition leads to tumor growth arrest. Treat-ments such as ionizing radiation (IR) or DNA damaging agents result in the formation of double strand breaks (DSB) that are highly toxic to the cell. Many tumors are able to survive traditional chemotherapeutics via activation of DNA repair pathways that allow for continued replication and survival, thus current efforts in pharmacology focus on the identification of specific DNA repair factors that allow for cancer survival after chemotherapeutic treatment. The enzyme Exonuclease 1 (Exo1) plays a role in multiple DNA repair pathways and thus may be an attractive therapeutic target. Exo1 has a variety of functions in mammals and yeast and has been implicated in DNA Mismatch Repair (MMR), homologous re-combination (HR), somatic hypermutation (SHM), and male and female meiosis. While its role in DNA end resection during ho-mologous recombination is still being elucidated, its potential inhibition for improved radiotherapy in cancer models has yet to be studied. Further, our research suggests that Exo1 is not necessary for functional MMR, which would make it an even more promising therapeutic target because its inhibition would not compromise MMR proficiency and thus cells would retain sensitivity to alkylating agents. We thus hypothesized that (1) Exo1 is not critical for MMR and that another nuclease may be involved allowing for Exo1 independent repair, (2) that inhibition of Exo1 in cancer models will improve radiosensitivity due to a defective HR response, and (3) that this inhibition would retain a high therapeutic index because dividing cells require HR and thus loss in hematopoietic stem cells (HSC) would not result in increased immune toxicity. We show that Exo1mut MEFs are more sensitive than WT MEFs to IR due to an inability to repair DNA double strand breaks. We additionally show that shRNA mediated silencing of Exo1 in a variety of cancer models leads to a significant increase in radiosensi-tivity compared to WT MEFs and that the cancer stem cell (CSC) population also appears to be affected by loss of Exo1. Addition-ally we use drug sensitivity assays, competitive repopulation studies and MMR repair assays to show that loss of Exo1 does not com-promise MMR proficiency and are using immunoprecipitation studies to identify the additional components allowing for Exo1 inde-pendent MMR.

Membrane Structural Biology — Oral session Presenter: Brian M. Kevany, Postdoc (Palczewski lab) Title: Phagocytosis of Retinal Photoreceptors Diseases of the retina rob millions of their eyesight each year across the globe. The health of the retina requires an important interac-tion between the photoreceptor cells, responsible for collecting visual data, and the retinal pigment epithelium (RPE), a monolayer of cells which lies at the boundary between the eye and the bloodstream. Photoreceptor cells maintain a roughly constant length by con-tinuously generating new outer segments from their base while simultaneously releasing mature outer segments engulfed by the RPE. Thus, post-mitotic RPE cells phagocytose an immense amount of material over a lifetime, disposing of photoreceptor cell waste while retaining useful content. While a number of important players involved in this process have been identified our under-standing of the steps leading to photoreceptor phagocytosis remains poor. The aim of this project is to establish a reproducible sys-tem in our lab to study this process as well as further our understanding of this complex event. Primary cell cultures, coupled with ex vivo phagocytosis assays, will be utilized to identify candidate genes through knock-down and knock-in assays. Ultimately, trans-genic mice will be generated for any promising candidates, allowing an in vivo analysis for any involvement in retinal maintenance. Presenter: Debarshi Mustafi, Grad Student (Palczewski lab) Title: Dissection of the visual signaling network in cone cells Cone photoreceptors comprise only 5% of the photoreceptor population in the human retina, yet their degeneration is the main cause of clinically significant vision loss. This is attributed to the fact that cone photoreceptors are essential for daytime vision, acuity, and color discrimination. Our understanding of these important cells for the design of therapeutics has severely been hindered by lack of appropriate animal models for study. In this work, by studying an animal model of the human condition enhanced S-cone syndrome (ESCS) in which there is an overproduction of cone cells and complementing that with an animal model with a physiological abun-dance of cone cells, we have gained insight into the structural and genetic aspects of cone photoreceptors that mediate their function and the changes that accompany disease. A variety of imaging techniques, including optical coherence tomography, confocal micros-copy, and scanning electron microscopy (SEM) revealed the spatial relationship of the photoreceptors in the retina and the disruption that is induced in the animal model of ESCS. Furthermore, hybrid SEM techniques allowed serial sectioning and three-dimensional reconstructions to study physiological processes in the retina that accompany cone-degenerative diseases. In addition, the enriched cone population allowed isolation of disc elements to reveal the first insight into cone opsin packing in the native membrane environ-ment using atomic force microscopy. Finally, massively parallel RNA-sequencing revealed the genetic elements that give rise to

these structural findings in disease. Altogether, this top down approach provides the first comprehensive signature of cone photore-ceptors that couples their structure to their function as well as highlights the power of this method to examine and decipher the struc-tural and genetic basis of visual disease states. Presenter: Marcin Golczak, Research Associate (Palczewski Lab) Title: An acyl-covalent enzyme intermediate of lecithin:retinol acyltransferase. Synthesis of fatty acid retinyl esters determines systemic vitamin A levels and provides substrate for production of visual chromo-phore (11-cis-retinal) in vertebrates. Lecithin:retinol acyltransferase (LRAT), the main enzyme responsible for retinyl ester forma-tion, catalyzes the transfer of an acyl group from the sn-1 position of phosphatidylcholine to retinol. To delineate the catalytic mechanism of this reaction, we expressed and purified a fully active, soluble form of this enzyme and used it to examine the possible formation of a transient acyl-enzyme intermediate. Detailed mass spectrometry analyses revealed that LRAT undergoes spontaneous, covalent modification upon incubation with a variety of phosphatidylcholine substrates. The addition of an acyl chain occurs at the Cys(161) residue, indicating formation of a thioester intermediate. This observation provides the first direct experimental evidence of thioester intermediate formation that constitutes the initial step in the proposed LRAT catalytic reaction. Additionally, we examined the effect of increasing fatty acyl side chain length in phosphatidylcholine on substrate accessibility in this reaction, which provided insights into the function of the single membrane-spanning domain of LRAT. These observations are critical to understanding the catalytic mechanism of LRAT protein family members as well as other lecithin:acyltransferases wherein Cys residues are required for catalysis. Presenter: David Lodowski, Postdoc (Palczewski Lab) Title: Conformational Changes in the G Protein-Coupled Receptor Rhodopsin Revealed by Histidine Hydrogen−Deuterium Exchange G protein-coupled receptors (GPCRs) are activated by ligand binding, allowing extracellular signals to be efficiently transmitted through the plasma membrane to the recognition site of interacting proteins, including G proteins 40 Å away. How this allosteric information is transmitted through the transmembrane portion of these receptors is unclear and has been the subject of intense inves-tigation. By focusing on intrinsic His residues found spaced throughout the GPCR, rhodopsin, we used His-hydrogen deuterium ex-change (His-HDX) to monitor long timescale structural rearrangements previously inaccessible by other means. In rhodopsin and opsin, transmembrane His152, His211 and His278 did not undergo HDX, most likely because D2O cannot penetrate the core of the re-ceptor. The half-lives of His-HDX was shorter for His100 and His195 in rhodopsin than for opsin, whereas the exchange for cytoplas-mic His65 indicates that it is more accessible in opsin. Moreover, the pKa for His195 was significantly affected by binding of Zn2+, which was previously identified to be coordinated in vicinity of this residue. Results with rhodopsin indicate that this methodology can be useful in examining structural rearrangements in native source proteins and difficult to analyze membrane proteins without requiring structural modification. Presenter: Thomas Kowatz, Postdoc (Palczewski Lab) Title: Towards the Structures of Carotenoid Cleaving Enzymes Animal visual pigments consist of an integral transmembrane protein (opsin) and a covalently bound chromophore (11-cis-retinal). These G-coupled receptors mediate phototransduction, the process by which a light stimulus is translated into a photoreceptor’s elec-trical response. Phototransduction depends on a diet-derived chromophore that must be continuously recycled after light exposure to sustain vision. The involved chemical transformations are catalyzed by structurally related proteins belonging to the family of carote-noid cleavage enzymes (CCEs). In vertebrates, oxidative cleavage of carotenoids and isomerization of the retinoid cleavage product is catalyzed by distinct family members, the carotenoid oxygenase CMO1 and the retinoid isomerase RPE65. In insects, carotenoids are directly converted to the cis-chromophore by NinaB. In humans, mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber congenital amaurosis (LCA) to autosomal recessive retinitis pigmentosa (RP). Besides null mutations, RPE65 missense mutations have been described that may affect protein stability, catalytic activity and membrane association. However, our knowledge of the molecular structure of CCEs is scant. Without this information, it is impossible to truly understand functioning of these enzymes and the consequences of their mutations. Such knowledge is essential for designing potentially beneficial interven-tions such as gene replacement and targeted drug therapy for patients afflicted with mutations in RPE65. Comparison between the structures of RPE65, CMO1 and NinaB will allow identification of residues critically related to the oxidative cleavage reaction, isomerase reaction, substrate interaction and membrane association. A breakthrough essential for this endeavor already has been ac-complished by determining the crystal structure of native RPE65 at 2.14 Å resolution. We are now aiming to solve the structures of CMO1 and NinaB. These projects will gather a wealth of atomic information essential to understanding the mechanistic and struc-tural basis of chemical trans-to-cis isomerization and the oxidative cleavage of carotenoids. Presenter: Yiyuan Yuan, Postdoc (Feng lab) Title: Profiling proteomics of diet restriction and TOR signaling pathways with a novel Stable isotope lysine labeling in C. elegans identifies novel genes regulating C. elegans lifespan TOR (target of rapamycin), a critical regulator integrating nutrition and growth signaling, plays a key role in energy metabolism and the maintenance of vitality, affecting lifespan and health in various animal models. While reduced TOR signaling may suppress global protein synthesis, it may also strengthen or suppress activities of specific biological processes and pathways to extend life-span, including contributing to diet restrict (DR)-mediated lifespan extension. Yet, the underlying mechanisms of both are not well

understood. A comprehensive proteomic study at a whole organism level will not only quantify global protein homeostasis but also identify TOR signaling-mediated biological processes and pathways that are not directly related to global protein homeostasis. Although such a study can significantly advance our understanding on TOR signaling and its role in lifespan/health regulation, a large scale quantifi-cation of protein abundance at a whole organism level in response to distorted TOR signaling has not been reported. A key reason is the lack of a sensitive and reliable proteomic experimental system that identifies and quantifies protein abundance at a whole organ-ism. Here, we developed a novel quantitative proteomic method called stable isotope labeling by amino acids in C. elegans (SILACE). Using SILACE, we found that reduced TOR signaling led to global protein synthesis down regulation at a whole organism level. We also identified genes whose expression levels were modulated by DR or reduced TOR signaling, and profiled their functional path-ways. Using RNAi and qPCR, we confirmed some candidates for their roles in mediating longevity and found that histone demethy-lation is involved in DR-mediated longevity. Presenter: Sanath R. Wijerathna, Grad Student (Dealwis lab) Title: Structural and functional Validation of Nucleotide Induced Oligomerization of the Ribonucleotide Reductase Ribonucleotide reductase (RR) catalyzes the de novo biosynthesis of 2’deoxynucleotides from their corresponding ribonucleotides. RR consist of a large á subunit also called R1 containing allosteric and catalytic sites, while the â subunit also called R2 houses a free radical required for catalysis. RR is classified into three classes based on how they generate the free-radical. All known eukaryotic RRs are Class Ia enzymes as they all use a tyrosyl-based free-radical. Eukaryotic RRs are ánân heterooligomers. ATP binds at an allosteric site called the a-site and activates RR while dATP binding at the a-site inhibits activity. Recently, we showed that R1 forms inactive hexamers at physiological concentrations of dATP (Fairman and Wijerathna et al., 2010 Nat. Struct. Mol. Biol.). Therefore, we hypothesize that the formation of dATP induced hexamers is a prerequisite for inhibiting RR. In order to understand the molecular basis of the dATPbased inhibition of RR, we determined the first structure of the dATP-hexamer using Xray crystallography. The X-ray structure reveals two possible packing arrangements of the dATP hexamer called model A and B. To determine which packing arrangement occurs in solution several mutants at the hexamer interface were gener-ated. The mutant D16R completely abolishes the hexamer formation at physiological concentration of dATP (20ìM) while the H2E mutant shifted the dimer–hexamer equilibrium towards dimer formation. Enzyme activity assays were carried out to determine the allosteric inhibitory effect of dATP on these mutants. In the presence or absence of dATP, D16R showed similar enzymatic activity with or without dATP, suggesting that dATP is unable to inhibit D16R. This observation validates model B and our hypothesis, which states that dATP induced hexamerization is a prerequisite for inhibition of RR activity. Since the allosteric activator ATP at its physiological concentration also forms active R1 hexamers, we further hypothesized that R1 forms hexamers with a different packing arrangement to dATP. Therefore, D16R and H2E mutants were tested for their ability to form hexamers in the presence of ATP. The D16R and H2E mutants, which were designed to disrupt dATP hexamerization, retained their ability to form hexamers in the presence of ATP. We also showed that these mutants retained a similar affinity for ATP as wild type R1. Taken together, these results confirm that R1 has different packing interfaces for ATP and dATP induced hexamers. Consistent with published data (Wang et al., 2007, PNAS and Fairman and Wijerthna et al., 2010, Nat. Struct. Mol. Biol.), the ATP induced R1 hexamer will enable an RR quaternary structure to consist of an á6ân (where n=2, 4 or 6) holo complex, while the dATP induced hexamer will only accommodate an á6â2 holo complex. We were able to confirm this by determining the first á6â2 cryo-EM struc-ture of the dATP induced RR holo complex (Fairman and Wijearthna et al., 2010, Nat. Struct. Mol. Biol.) Future work will involve determining the structures of ATP induced RR holo complexes.

Cancer Therapeutics — Oral session Presenter: Chris Ryder, Grad Student (Distelhorst lab) Title: Acidosis-Sensing TDAG8 Induces Bcl-2 and Prevents Apoptosis Due to Glutamine Starvation The aberrant metabolism and proliferation of cancer cells facilitates the development of an acidotic, nutrient starved tumor microen-vironment. In turn, these hallmark features are known to influence cancer cell survival and responsiveness to therapy. Here we ex-plore the interplay between these two characteristic tumor conditions in the WEHI7.2 murine lymphoma cell line and uncover a spe-cific cell survival mechanism involving the upregulation of the anti-apoptotic protein Bcl-2. In order to model nutrient limitation we deprived WEHI7.2 cells of glutamine, the amino acid most readily utilized by cancer cells. Despite its abundance in serum, gluta-mine may become limiting in the tumor microenvironment and glutamine starvation is known to rapidly induce apoptosis in many cancer cells. We confirmed that glutamine starvation causes apoptosis in our model and found that reduction of pH almost com-pletely prevents glutamine-starvation-induced apoptosis, concurrent with upregulation of Bcl-2. We demonstrated the importance of Bcl-2 for the survival of glutamine-starved WEHI7.2 cells by overexpression and knockdown experiments. We hypothesized that TDAG8, an acid-sensing G protein-coupled receptor selectively expressed by lymphoid cells in normal conditions but also shown to be overexpressed in a variety of solid tumors, is required for the upregulation of Bcl-2 observed in acidosis. In fact, siRNA-mediated knockdown of TDAG8 attenuates the induction of Bcl-2 during acidosis. Furthermore, knockdown of TDAG8 prevents the anti-apoptotic effect of acidosis on glutamine starvation. Since acidosis is a characteristic feature of the tumor microenvironment and is also known to correlate with chemotherapeutic resistance, this pathway may represent an important survival mechanism and, as such, a target for improving the killing of cancer cells in vivo.

Presenter: Molly Taylor, Grad Student (Schiemann lab) Title: Extracellular Matrix Rigidity Mediates Mammary Epithelial Cell Response to TGF-β Transforming Growth Factor-b (TGF-b) is a multifunctional cytokine that regulates all stages of mammary gland development, in-cluding maintenance of tissue homeostasis and the suppression of tumorigenesis in mammary epithelial cells (MECs). Interestingly, mammary tumorigenesis converts TGF-b from a tumor suppressor to a tumor promoter through mechanisms that remain incom-pletely understood. Changes in tissue compliance promote the acquisition of malignant phenotypes in MECs in part through the ac-tivity of the extracellular matrix (ECM) crosslinking enzyme lysyl oxidase (LOX). We show that TGF-b stimulates the synthesis and secretion of LOX and that inhibition or knockdown of LOX leads to changes in TGF-b-mediated signaling and impairs TGF-b mediated epithelial-mesenchymal transition. Importantly, the manner in which MECs respond to TGF-b can be regulated by ECM rigidity, resulting in dramatic changes in MEC acinar morphology and growth. Furthermore, mechanotransduction leads to changes in micro-RNA expression induced by TGF-b. Using a microRNA microarray we have identified microRNAs differentially regulated by TGF-b and ECM rigidity. Collectively, our findings suggest that mechanotransduction promotes oncogenic signaling by TGF-b and that measures capable of establishing a normal ECM environment may prove effective in diminishing breast cancer progression stimulated by TGF-b. Presenter: Brad T. Casali , Grad Student (Keri lab) Title: The neurotransmitter GABA induces proliferation of basal breast cancer cells through endogenous expression of the GABA-receptor pi subunit A ‘triple-negative’ phenotype—Estrogen Receptor-α (ER-α), Progesterone Receptor, and HER2 negative is characteristic of basal breast cancer. These tumors are highly aggressive, confer poor prognosis, lack targeted therapies, and metastasize preferentially to the brain. Gene expression profiling studies revealed that GABA-receptor subunit pi (GABRP) mRNA is overexpressed in tumors representing the basal, compared to the luminal, breast cancer subtype. GABRP mRNA is robustly expressed in basal breast cancer cell lines, but not in luminal cell lines. GABRP is a subunit of the GABA(A) receptor (GABAR) that mediates synaptic inhibition in the nervous system in response to the neurotransmitter GABA. However, the impact of selective GABRP expression in basal breast cancer remains unknown. We found that GABA dose-dependently increases cell number and proliferation of basal breast cancer cells HCC1143 and HCC1187 that intrinsically express high levels of GABRP. In contrast, GABA treatment had no impact on growth or proliferation of MCF-7 luminal breast cancer cells which do not express appreciable GABRP. GABAR-selective agonist muscimol increased cell number only in the basal cell lines, while the GABAR-selective antagonist bicuculline abrogated the response to GABA. GABRP silencing with siRNA abolished the GABA-induced growth and proliferative responses in HCC1187, implicating GABRP in mediating the responses to GABA in basal breast cancer cells. HCC1143 and HCC1187 cells also formed more tumor-spheres, a functional stem-cell readout, when treated with GABA; this response was abolished upon GABRP knockdown, indicating that GABRP plays a role in stemness. Basal breast cancers with high GABRP expression that metastasize to the brain may have a selective growth advantage due to high local GABA concentration and increased signaling through GABRP; this may permit devel-opment of GABRP-selective inhibitors that slow the growth of brain metastases in basal breast cancer. Presenter: Michele Mumaw, Grad Student (Noy Lab) Title: The Role of Transthyretin in STRA6-Mediated Vitamin A Uptake and in Signaling by RBP-retinol Retinol, vitamin A, is transported throughout the body in the blood bound to Retinol Binding Protein (RBP). It has been proposed that association of the 21 kDa RBP with transthyretin (TTR), a 55 kDa tetramer, prevents glomerular filtration of the smaller protein. In hepatocytes, RBP binds to retinol and subsequently forms a complex with TTR before secretion into the blood. The TTR-RBP-retinol complex interacts with a transmembrane protein termed Stimulated by Retinoic Acid 6 (STRA6). STRA6 then transports retinol into the cell to be converted to active metabolites. Recently, our lab found that, in addition to transporting retinol, STRA6 is also a signaling protein that is phosphorylated in response to RBP bound retinol. This leads to the activation of a JAK2, phosphory-lation of STAT5, and upregulation of STAT target genes. However, previous experiments were performed utilizing RBP bound reti-nol and not with the complete tertiary complex, TTR-RBP-Retinol, found in the blood. The goal of my project is to evaluate the effect of TTR on RBP-ROH STRA6 signaling. In addition, structure/function relationships that underlie the interactions between TTR, RBP, retinol, and STRA6 will be examined. Preliminary data suggests that TTR may inhibit STRA6 signaling. Presenter: Jingna Wang, Postdoc (Zhang lab) Title: Novel insights into the dynamic regulation of the cell cycle checkpoint kinase, Chk1 The DNA in our cells is under continuous assault by DNA-damaging agents produced either in the external environment or intracel-lularly as by-products of oxidative metabolism. If not fixed, those errors will lead to genetic instability, a hallmark of human cancers. Fortunately, cells have evolved elegant mechanisms to detect and repair DNA damages. These are called cell cycle checkpoints. Key components of cell cycle checkpoints include two protein kinases, the upstream kinase ATR, and its downstream target kinase, Chk1. In response to DNA damage, ATR homes to the damage site and phosphorylates Chk1 at two Ser-Gln sites, Ser-317 and Ser-345. This phosphorylation event triggers activation of Chk1, which, in turn, activates a number of downstream ‘effectors’ to provoke cell-cycle arrest, preserve replication fork viability, and activate DNA repair programs. Thus, Chk1 is essential for the maintenance of cell viability. This is consistent with the fact that loss of CHK1 leads to cell death even in the absence of extrinsic genotoxic agents. However, how exactly Chk1 is phosphorylated and how does that phosphorylation lead to checkpoint activation remains un-clear. Here we show that the Chk1 polypeptide sequence contains critical information regulating Chk1 phosphorylation and subse-quence cell cycle checkpoint activation. The Ser-317 phosphorylation is required for the Ser-345 phosphrylation. A highly conserved

motif at the Chk1 C-terminus not only regulates the nuclear exporting of Chk1 but is also indispensable for Chk1 function. A single mutation at the C-terminus can promote Chk1 nuclear exporting, causing Chk1 protein instability and phosphorylation defect after DNA damage. On the other hand, however, single amino acid substitution at the Chk1 C-terminus can also result in constitutive phosphorylation of Chk1 at the ATR sites. These data revealed the importance of Chk1 C-terminus in its phosphorylation and func-tion, indicating the complexity of Chk1 regulation in cells. Presenter: Steve Sizemore, Postdoc (Keri lab)

Title: FOXC1 is required for basal breast cancer viability and promotes invasion through MMP7 expression. Breast cancer is a heterogeneous disease comprised of at least five distinct subtypes. Among these disparate subtypes the basal group has been reported to have the most aggressive clinical behavior. Basal breast cancers generally do not express estrogen or progester-one receptors and are negative for Her-2 (triple negative) and typically do not respond to the primary forms of targeted therapies used to treat breast cancer. Given the limited treatment options for these aggressive tumors, a better understanding of molecular makeup of the basal subtype is urgently needed. We identified the transcription factor FOXC1 as a potential master regulator of the basal subtype and proposed that study of FOXC1's role in basal cancer will elucidate novel therapeutic targets for the treatment of this disease. We report that FOXC1 is required for viability of the basal breast cancer cell line HCC1187. Knockdown of FOXC1 in HCC1187 resulted in an increased sub_G1 fraction and decreased G2/M fraction as measured by Propidium idodide. Western blot analysis revealed that knockdown of FOXC1 in HCC1187 resulted in increased cleavage of PARP, and caspases 3 and 7, all indica-tors of apoptosis. In addition to identifying a role for FOXC1 in basal breast cancer viability we also report that FOXC1 plays a vital role in controlling the expression of MMP7, a matrix metalloproteinase associated with cancer invasiveness. Knockdown of FOXC1 in HCC1187 resulted in decreased MMP7 expression as measured by qRT-PCR, western blots, zymography and ELISA. In recipro-cal experiments we found that expression of exogenous FOXC1 in the non-transformed cell lines MCF10A and MCF12A resulted in increased cellular invasion as measured by modified Boyden chamber assays and increased expression of MMP7 mRNA and protein. Our findings support an important role for FOXC1 in basal breast cancers and suggest that MMP7 may be a downstream target of FOXC1 and a promising therapeutic target in the treatment of this disease.

Excellence In Research Award: Presenter: Gina M. Bernardo, Grad Student (Keri Lab) Track: Cancer Therapeutics Title: FOXA1 is an essential determinant of ERa expression and mammary ductal morphogenesis FOXA1, Estrogen Receptor-a (ERa) and GATA-3 independently predict favorable outcome in breast cancer patients, and their ex-pression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERa and GATA-3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of ter-minal end bud formation and ERa expression. In contrast, Foxa1 null glands maintain GATA-3 expression. Unlike ERa and GATA-3 deficiency, Foxa1 null glands form milk-producing alveoli indicating that the defect is restricted to expansion of the ductal epithe-lium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERa expression, but not GATA-3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERa-positive breast cancers, at least in part, due to its control of ERa expression.

Cancer Therapeutics—Poster Session Presenter: Subrata Haldar (Faculty Member)

Poster #1 Title: MicroRNA-375 and microRNA-221: potential noncoding RNAs associated with antiproliferative activity of benzyl isothiocyanate in pancreatic cancer Non-coding RNAs (ncRNAs) are thought to be important regulators of gene expression in eukaryotes. Among all the ncRNAs, mi-croRNAs (miRNAs) have been extensively studied and the key function of these small double-stranded RNAs is attributed to nega-tively regulate gene expression. More importantly, the deregulated presence or absence of the miRNAs might play an important role in molecular pathways leading to neoplastic transformation. At present, it is also thought that the approaches to interfere miRNA functions should be helpful for developing novel therapeutic opportunities for human cancer. Here we were primarily interested to assess whether the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate miRNAs such as miR-221 and miR-375, known to be abnormally expressed in pancreatic cancer patients. Indeed real-time reverse transcription-PCR (RT-PCR) analysis demonstrates the ability of BITC to diminish the expression of oncogenic miR-221 and increase the level of tumor suppressor miR-375 in pancreatic cancer cells. Moreover, further qRT-PCR analysis indi-cates that the expression of miR-375 is more abundant in nonpathological mouse pancreas tissue than in mouse pancreas with acti-vated KRASG12D allele. To the contrary, the expression of oncogenic miR-221 is significantly elevated in KRASG12D driven pan-creatic intraepithelial neoplasia (PanIN) or ductal carcinoma, when compared to normal mouse pancreas. Thus our data suggest im-portant role of these microRNAs in the progression of pancreatic cancer. Interestingly, ectopic expression of tumor suppressor miR-375 or the enforced silencing of tumor promoter miR-221 in cultured pancreatic cancer cells enhances their sensitivity to BITC by

attenuating cell proliferation. This novel observation implicating miR-375 and miR-221 with the antitumor efficacy of BITC, thus, might provide new insight into non-conventional microRNA replacement therapy against pancreatic cancer. Presenter: Padmamalini Kannan-Thulasiraman, Postdoc (Noy Lab) Poster: #2 Title: Kruppel-like factor 2 sensitizes mammary carcinoma cells to retinoic acid-induced apoptosis Retinoic acid (RA) can inhibit carcinoma cell growth by activating nuclear retinoic acid receptors (RAR) but enhances cell prolifera-tion and survival by activating a different nuclear receptor, namely PPARβ/δ. The hormone is targeted to RAR by cellular retinoic acid binding protein (CRABP-II), and is delivered to PPARβ/δ by fatty acid binding protein 5 (FABP5). Hence, RA exerts anticar-cinogenic activities in cells that express a high CRABP-II/FABP5 ratio but facilitates the growth of carcinoma cells in which this ratio is low. On-going work aims to understand the factors that regulate the expression of the two binding proteins in mammary car-cinomas. Recent observations indicate that RA signalling is regulated by the transcription factor KLF2. The data show that, in the RA-resistant MDA-MB231 mammary carcinoma cells, KLF2 induces the expression of CRABP-II as well as the RA receptor RARγ while, concomitantly, suppresses FABP5 expression. KLF2 directly regulates the transcription of CRABP-II and RARγ through binding to KLF2 response elements in the promoters of these genes. Down-regulation of FABP5 by KLF2 is exerted indirectly. Spe-cifically, the data suggest that KLF2 interferes with the ability of the transcription factor NF-κB to induce FABP5 expression, and that it does so by sequestering the transcriptional coactivator PCAF. Additional observations show that over-expression of KLF2 sensitizes MDA-MB231 cells to RA-induced apoptosis. Hence, KLF2 exerts anti-carcinogenic activities by diverting RA signalling from the proliferative FABP/PPAR pathway to the growth inhibitory CRABP-II/RAR path. Presenter: Melissa D. Shelton, Postdoc (Keri Lab) Poster: #3 Title: LMO4-mediated regulation of estrogen receptor (ER ) in breast cancer cells Of the five distinct breast cancer subtypes, tumors classified as basal and ErbB2/Her2 are highly proliferative and convey the worst prognosis. A hallmark of these tumors is a deficiency in the expression of estrogen receptor (ERα) making them unresponsive to treatments targeted to this receptor and therefore posing a grave therapeutic challenge. Although it is clear that ERα encoding gene (ESR1) becomes epigenetically silenced in such tumors, the factors regulating this process remain unknown. ER negative tumors consistently overexpress the Lim only protein 4 (LMO4) compared to tumors that express ERα. LMO4, deficient of a DNA-binding domain, bridges multiple proteins to form large transcriptional complexes and modulates their corresponding transcriptional activity. Overexpression of LMO4 is known to induce mammary tumors in mice and increase proliferation of breast cancer cells. Notably, LMO4 negatively regulates ER transactivation activity. These data as well as the clear ability of LMO4 to regulate proliferation, suggest that LMO4 may modulate ERα activity through several mechanisms including suppression of ERα expression. Unlike the well established proliferative effects of LMO4 in basal breast cancer cells, we found that two separate luminal breast cancer cell lines, MCF-7 and T47D, with stable overexpression of LMO4 grow at a slower rate compared to control cells expressing empty vec-tor. Moreover, LMO4 overexpression also leads to attenuated ER protein and mRNA levels and concomitant decreases in PS2, a transcriptional target of ER . Hence, LMO4 mediated repression of the ESR1 gene is at least one mechanism underlying the low ERα expression that occurs in aggressive breast cancers. Intriguingly, the diminution in ER appears to be restricted to MCF-7 cells at low confluency, and this observation is currently under further investigation. These data serve as a launchpad for elucidating mechanisms underlying LMO4 -mediated regulation of

Membrane Track—Poster Session Presenter: Matthias Buck (Faculty Member) Poster #4 Title: Studies on the Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor. Plexins are transmembrane receptors for semaphorin ligands that regulate cell migration processes in several settings, including in the developing nervous and cardiovascular system. Upon activation, plexin initiates signaling processes that involve several small GTPases of the Ras and Rho families (R-Ras, Rac1, Rnd1, and RhoD)and that regulate cytoskeletal dynamics and cell adhesion. The receptor’s anti-migratory /cell collapse function can be dysregulated by oncogenic mutations[1] , which are known to contribute to cancer metastasis. Plexins are unique amongst transmembrane receptors because their intracellular domains interact directly with small GTPases. Specifically, plexins include domains with homology to GTPase activating proteins (GAPs) and these are thought to possess GAP activity toward R- and M-Ras. The structure of the RhoGTPase binding domain (RBD) [2] and the recent determina-tion of the x-ray structure of the entire intracellular region of plexin-B1 shed new light on the function and substantiate a possible mechanism of activation[3]. We propose that the receptor protein functions via a conformational shift in a monomer-multimer equi-librium on the intracellular side. Implications for therapeutic interventions, such as a rescue of plexin’s tumor suppressor activity are discussed.

Presenter: Pengxiu Cao, Postdoc (Feng and Palczewski Lab) Poster: #5 Title: Light-controlled Go signaling activation in motor neurons of C. elegans via cell-specific rhodopsin expression modulates GABA neurotransmission resulting in locomotion inhibition Go signaling plays a vital role in many biological processes that mediate development, cell migration, behavior and the effect of volatile anesthetics. Go also is the most abundant G protein in human brain and mediates actions of many neurotransmitters, but its output and underlying signaling mechanisms are poorly understood. C. elegans is a genetic model to study in vivo functions of Go signaling. Similar to its mammalian homologs, the gene encoding worm Go alpha subunit (goa-1) is expressed in almost all cells including the whole nervous system where it affects locomotion, egg-laying and mating. The specific contribution of Go signaling in each individual neuron to regulating these behaviors is poorly understood. Here, we used various promoters to express bovine rhodopsin in worm neurons and found that transient visible light exposure specifically activated Go signaling in motor neurons to inhibit worm locomotion. Because worms lack rhodopsin/transducin homologs, they do not respond to visible light exposure under these conditions. Using this model, we then found that Go signaling modulated GABA neurotransmission through GPB-1, a worm homolog of the G protein beta subunit, possibly at GABAergic motor neurons. GABA is the major inhibitory neurotransmitter in mammalian nervous systems. Thus, we identified a previously unknown modulatory role of Go signaling on GABA neurotransmis-sion in worm locomotion. Presenter: Mark Corriveau, Research Assistant (Taylor lab) Poster: #6 Title: Telomere protection by multiple end-binding proteins Telomeres are complex nucleoprotein structures that protect the ends of eukaryotic chromosomes from degradation and end-to-end fusion events. The DNA component of telomeres consists of hexameric DNA repeats of the sequence 5’-TTAGGG-3’ in vertebrates followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins. The six primary telomere end-binding proteins form a core complex collectively known as shelterin. Two of the shelterin proteins, POT1 (protection of telomeres 1) and TPP1, bind as a heterodimer to two hexameric re-peats of single-stranded telomeric DNA. TPP1 is linked to the two double stranded binding proteins, TRF1 and TRF2, by the versa-tile linker protein TIN2. While structural information is currently available for a few of the individual domains of the shelterin proteins, little is known about the structure of the assembled complex or the significance of the protein-protein interactions. To address these questions, we use a combinatorial approach of electron microscopy and biochemistry. Specifically, we wish to understand how the shelterin proteins interact with each other to protect telomeric DNA and, conversely, how they interact with the specialized enzyme telomerase for telomere extension. Our preliminary data suggests that multiple POT1/TPP1 heterodimers coat the single-stranded DNA to form a compact, globular architecture at the ends of chromosomes. We hypothesize that this superstructure is important for chromosome stability by protecting telomeric DNA from fraying and from interactions with DNA repair enzymes. Presenter: Teresa Cvetkov, Postdoc (Moiseenkova-Bell Lab) Poster: #7 Title: Solving the puzzle of TRPA1 ligand interaction mechanism The transient receptor potential (TRP) family of proteins are non-selective cation channels capable of transducing a diverse range of physical and chemical stimuli into action potentials. In this protein family, the TRPA1 channel is expressed in nociceptive neurons and serves as a sensor for pain, cold temperature and a diversity of chemical ligands, including exogenous and endogenous cysteine-reactive compounds, a variety of non-reactive small molecules, and divalent cations such as Ca2+ and Zn2+. It is currently unknown how the channel accommodates such a diversity of ligands and how these ligands promote channel activation and putative conforma-tional rearrangements in the pore and cytosolic domains of the channel. The relationship between channel structural changes and channel activation presents an intriguing puzzle. In this project, mouse TRPA1 and its mutants were expressed in a protease-deficient strain of S. cerevisiae and were purified using immuno-affinity and size-exclusion chromatography. TRPA1 was reconstituted into vesicles containing intravesicular Ca2+, and channel activation in response to allicin was confirmed using Fura-2 as a fluorescent indicator for changes in extravesicular Ca2+ con-centration. Circular dichroism was used to detect ligand binding and resulting conformational changes in detergent-solubilized TRPA1. We found that Ca2+ induced changes in the far UV CD spectrum of TRPA1, an effect which could be reversed by addition of excess EGTA. Finally, fluorescence anisotropy was used to measure the binding of fluorescently-labeled Calmodulin (CaM) to TRPA1. The anisotropy increased significantly when CaM was in the presence of both TRPA1 and Ca2+, and this effect could be prevented or reversed by EGTA or by introducing non-labeled CaM. Taken together, these results indicate our ability to express and purify functional TRPA1 channels, and that Ca2+ and CaM-Ca2+ can bind directly to detergent-solubilized TRPA1. Presenter: Songqi Gao, Postdoc (Palczewski Lab) Poster: #8 Title: A sustained drug delivery system for the treatment of Leber congenital amaurosis Our aim is to design and apply a drug delivery system for sustained release of 9-cis retinyl acetate for the treatment of Leber congenital amaurosis (LCA), a severe retinal dystrophy. The microparticle/hydrogel combination was designed and used for sustained drug release. The drug is entrapped in the microparticles and released by the degradation of the polymer, while the

hydrogel is exploited as a scaffold. Biodegradable poly(d,l-lactide-co-glycolide) (PLGA) microparticles containing the drug were prepared using emulsion and solvent evaporation method, and then loaded into biocompatible alginate hydrogels by ionic cross-linking. The morphology and size of PLGA microspheres were characterized by scanning electron microscopy (SEM). The loading efficiency and capacity of the microparticles were determined by UV spectroscopy. For in vivo drug release study, the microparticles/hydrogel combination was subcutaneously injected to the Lrat-/- mice which were fed with no vitamin A diets for one month before injection. The drug concentrations in plasma were determined by HPLC. Elec-troretinography (ERG), retinoid concentrations, and histologic analysis were used to evaluate the retinoid effects on visual function and morphology. The experimental results demonstrated that the sizes of microparticles are ranged from 20 µm to 120 µm. The loading efficiency of the microparticles is around 50% after drying under vacuum. In vivo study demonstrated that constant drug release from the microparticle/hydrogel combination lasted three weeks after injection. In comparison with control group, drug concentrations in plasma were high and toxic a few hours after oral administration of the same dose, and dropped sharply one day after injection. Significantly increased ERG response and considerably improved RPE-ROS processes were observed in treatment group. In conclusion, sustained drug release and effective treatment were achieved with low toxicity by using this injectable microparticle/hydrogel drug delivery system. Presenter: Kevin Huynh, Research Assistant (Moiseenkova-Bell lab) Poster: #9 Title: Optimizing purification and crystallization conditions of TRPV2 for structural analysis The TRP (transient receptor potential) family of ion channels are noxious stimuli sensors in specialized sensory neurons that detect tissue damage in the central nervous system. Proteins from TRPV branch of TRP channels display a broad diversity to activation by chemical and physical stimuli; activation by heat is common to several members of this group. Very little is known currently about the structure of TRPV channels and their mechanisms of gating by different stimuli. TRPV2 is perhaps the most enigmatic of all TRPV channels. TRPV2 is a non-selective cation channel activated by heat (at very high temperatures, 52°C and above) and me-chanical stimuli. We will use 2- and 3-dimensional crystal analysis to visualize TRPV2 in different functional states. Expression of mouse TRPV2 in a strain of S. cerevisiae has been optimized and our initial detergent screen indicated that TRPV2 could be solubi-lized and purified in a detergent combination of FOS Choline-12 (FC12) and n-dodecyl-b-D-maltoside (DDM); however, we per-formed a second detergent screen since FC12 is not optimal for crystallography. Among the crystallization friendly detergents DDM, b-octyl-glucoside (OG), n-decyl-b-D-maltoside (DM) and CHAPS, DDM gave relatively high TRPV2 protein yields after solubiliza-tion and purification by immuno-affinity and size-exclusion chromatography. Crystallization trays were set up at 5-8mg/ml of pro-teins using various commercial and grid screens, which gave small preliminary TRPV2 crystals. The growth of any sort of protein crystals indicates that crystallization of this membrane protein is possible, even with detergents present, but more screens will be done to find the optimal conditions for larger crystal growth that can be diffracted for structural determination. Presenter: Beata Jasztrebska, Research Associate (Palczewski Lab) Poster: #10 Title: Role of Phospholipids in Rhodopsin Stability and Function Rhodopsin is a prototypical G protein-coupled receptor (GPCR) – a member of the superfamily that shares a similar structural archi-tecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is em-bedded in the lipid bilayer of specialized disk membranes in retinal rod photoreceptor cells, namely rod outer segments (ROS), where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade that results in a neu-ronal response in the brain. Interestingly, the lipid composition of ROS membranes differs from that of the photoreceptor plasma membrane and thus it must be critical for visual transduction. We have found that disruption of lipid environment by detergents or phospholipase A2 results in slower or even inhibited Gt activation by photoexcietd rhodopsin comparing to rhodopsin in native disks. However, when phospholipids were added back to solubilized rhodopsin rates of Gt activation were similar to those obtained for native membranes, indicating significant role of lipids in activating of visual processes. This observation had essential implica-tion for the formation of the rhodopsin-Gt complex in vitro and its purification for structural studies. In detergent solution rhodopsin-Gt complex was formed modestly; however addition of phospholipids resulted in formation of the more stable complex with proper stoichiometry. Thus, better understanding of the role of phospholipids for GPCRs function and stability may benefit in crystallizing and solving more structures of G protein coupled receptors as well as structures of their complexes with other signaling proteins. Presenter: Philip Kiser, Postdoc (Palczewski Lab) Poster: #11 Title: Structural and Biochemical Characterization of RPE65-Membrane Interactions Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). Lipid membranes are a preferred environment for enzymes that metabolize hydrophobic substrates such as retinoids. Thus, critical enzymatic activities required for visual chromophore regeneration, i.e. formation of retinyl esters by lecithin:retinol acyltransferase (LRAT), retinoid isomerization catalyzed by RPE65, and retinol oxidation facilitated by a family of retinol dehydrogenases, are all associated with the microsomal fraction isolated from the RPE (5, 10, 11). Although the molecular basis of membrane association for LRAT and some of the retinol dehydrogenases has been elucidated, the mechanism by which

RPE65 binds membranes and the role of specific phospholipids in its enzymatic activity are matters of debate. Proposed mechanisms of membrane binding include tethering via post-translational lipid modifications, electrostatic interactions with phospholipid head groups, and hydrophobic interactions between amino acid side chains and the hydrophobic core of the lipid bilayer. Here, we present structural and biochemical evidence that the RPE65 interacts with native microsomal membranes primarily via hydrophobic interac-tions and that the structural integrity of these membranes is critical for the enzymatic activity of the protein. Presenter: Kwangwon Lee, Reseaerch Associate (Hoppel Lab) Poster: #12 Title: Mitochondrial carnitine palmitoyltransferase 1a exists in an outer membrane fatty acid transfer complex The liver mitochondrial outer membrane (MOM) carnitine palmitoyltransferase-1 (CPT1a) catalyzes the regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of the quaternary structure of CPT1a exists as a trimer, which under native conditions could form dimers. To examine the state of CPT-1a in the MOM, in the present study, a com-bined approach was used with sizing by mass and isolation using an immunological method. Blue Native electrophoresis followed by detection with immunoblot and mass spectrometry identified large molecular weight complexes that contained not only CPT1a, but long-chain acyl-CoA synthetase (ACSL) and voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the three proteins revealed a strong interaction between them. Immobilized CPT1a specific antibodies immunocaptured not only CPT1a, but also ACSL and VDAC further strengthening findings with BNE and immunoprecipitation. This study shows strong pro-tein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex represents a transfer system for activated fatty acids through the MOM. Presenter: Sineej Madathil, Postdoc (Palczewski Lab) Poster: #13 Title: Structure and dynamics of G protein-coupled receptors assessed by hydrogen/deuterium exchange mass spectrometry Hydrogen/deuterium exchange (HDX) in conjunction with electrospray ionization mass spectrometry (ESI-MS) is increasingly being used to understand the structure, dynamics and folding of proteins. This technique relies on the fact that exposure of a protein to D2O induces N-H to N-D conversion at amide backbone linkages. Solvent-exposed protein regions not involved in hydrogen bonding undergo rapid exchange whereas exchange of hydrogen-bonded N-H groups can be as much as eight orders of magnitude less. De-spite great success with soluble proteins, the use of mass spectrometry for membrane protein HDX studies remains limited. Such studies are not only hampered by solubility issues, but also by the difficulty of generating proteolytic fragments in sufficiently high yield within a short period at low temperature and pH. We have done these experiments with rhodopsin and its activated state in native membranes and detergent micelles. Because the transmembrane portion of rhodopsin is inaccessible to bulk solvent, it is not surprising that only the cytoplasmic and extracellular regions exchange with D2O. Future experiments should yield a clear footprint of the Gt interaction surface within the Rho*Gt complex, an important advance considering there is yet no 3D structure for this com-plex. Currently we are attempting to optimize mass spectrometry-specific HDX conditions for application to other GPCRs. Presenter: Faiz Mohammed, Postdoc (Dealwis Lab) Poster: #14 Title: Structure and thermodynamic characterization of functionally compromised R293A eukaryotic ribonucleotide reductase. Ribonucleotide reductases (RNRs) in all organisms catalyze the essential reduction step in DNA biosynthesis and hence plays a cen-tral role in maintaining a balanced nucleotide pool for DNA replication & repair. Eukaryotic RNR composes of a and b subunits that form heterooligomers (Fairman et al., NSMB 2010). The a subunit contains both the catalytic and allosteric sites. Previously, we have determined X-ray structures of the eukaryotic a subunit of RNR from Saccharomyces cerevisiae with various substrate and nu-cleotide effectors (Xu et al., PNAS 2006). Interestingly, we found the loop 2 region that spans residues 285 to 296 plays a critical role in communicating with the substrate and specificity binding sites that is essential for catalysis. Based on our structural studies, we have generated the point mutation Arginine 293 to Alanine which is lethal in yeast cells. We report the X-ray structure and iso-thermal calorimetry titration (ITC) of R293A yeast RNR1 in complex with dGTP-ADP. Taken together, our structure and ITC data reveals mutation in the loop region abolish substrate binding and hence R293A is crucial in facilitating the communication between specificity and catalytic sites. In parallel to our study another group has shown that mutation in the loop region cause imbalanced nucleotide pools and R293A has lethal phenotype in Saccharomyces Cerevisiae. Presenter: Marvin T. Nieman (Faculty Member) Poster: #15 Title: Mapping the Interface Between Protease Activated Receptor 1 (PAR1) and PAR4 heterodimers. Antiplatelet therapy is a key component of the treatment regimen for acute coronary syndrome and stroke. A major obstacle in de-veloping antiplatelet agents is for agents to be effective without causing bleeding complications. Protease activated receptor 4 (PAR4) is a potential attractive target for antiplatelet therapies because of its requirement for stable clot formation and limited tissue distribution. The purpose of the current study is to determine how PAR4 interacts with other proteins on the surface of platelets and how these interactions influence activation and signaling of PAR4. We have demonstrated that PAR4 forms homodimers using two techniques, bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC). These inter-

actions are specific, as PAR4 does not interact with two unrelated G-protein coupled receptors (GPCRs) rhodopsin and dopamine D2 receptor. In addition to homodimers, PAR4 forms heterodimers with PAR1. The heterodimers are also specific since PAR1 also does not interact with other GPCRs. PAR1 does not interact with rhodopsin; therefore we generated a series of rhodopsin-PAR4 chimeras to determine the region on PAR4 that interacts with PAR1. We have mapped the region on PAR4 required for het-erodimerization to transmembrane helix 4 (TM4). When the rhodopsin-PAR4 junction is at the beginning of TM2, TM3 or TM4, the chimera interacts with PAR1 in a manner indistinguishable from PAR4-wt. However, when the junction is moved to the beginning of TM5, TM6 or TM7 the chimera does not interact with PAR1-wt. Molecular modeling of PAR1 and PAR4 has identified residues within TM4 that are exposed and are directed away from the other TM helices creating a potential interaction interface. Co-expression of PAR1 with PAR4 increased the efficiency of PAR4 activation by thrombin ~10-fold indicating a functional signifi-cance for PAR1-PAR4 interactions. The results from these studies provide detailed map of the interactions between two GPCRs on the cell surface and may provide insights for developing PAR antagonists. The current strategy of developing PAR antagonists is to target the individual receptors. However our data show that the functional unit is the PAR1-4 heterodimer and, as such, this may the relevant target to disrupt thrombin activation of platelets. Antagonists to PAR1 or PAR4 need to developed and studied in the con-text of these interactions, not in isolation. Presenter: Kiichiro Okano, Postdoc (Palczewski Lab)

Poster: #16 Title: Insights into the pathological Spectrum of photoreceptor cell death in light-induced retinal degeneration Purpose:To study the pathological functions of cone and rod photoreceptor cells in light-induced retinal degeneration due to accumu-lation of all-trans-retinal. Materials & Methods: We prepared rod-rich or cone-rich mouse retina by genetically ablating with a cone-specific diphtheria toxin A transgene (cone-DTA) or the transcription factor Nrl (Nrl-/-) respectively in the Rdh8-/-Abca4-/-mouse to compare the contribution of rods and cones to the light-induced retinal degeneration due to all-trans-retinal accumulation. To com-pare the progression of photoreceptor cell death, spectral-domain optical coherence tomography (SD-OCT), immunohistochemistry (IHC) and scanning laser ophthalmoscope (SLO) were used to assess morphological changes in retina. The physiological and bio-chemical functions in retina were evaluated by electroretinography (ERG) and retinoids content and A2E analyses at age of 6 weeks, 3 months and 6months. To evaluate susceptibility to intense light exposure, these mice were exposed to 10,000 lux illumination for 30 mins and evaluated with same methods above at 7 days after exposure. Results: Mild degeneration was observed in both Nrl-/-

Rdh8-/-Abca4-/- (cone-rich) and cone-DTA Rdh8-/-Abca4-/- (rod-rich) retina at 3 months of age whereas marked retinal degeneration was observed in Rdh8-/-Abca4-/- (cone and rod) retina. The ERG response was significantly decreased in rod-rich retina and retina with rod and cone whereas cone-rich showed no significant change at this age and similar results were obtained at 6 months of age. A2E levels were similar in retina involving rod cells whereas the levels were extremely low in cone-rich retina. After intense light exposure, elevated levels of all-trans-retinal accumulated in retina involving rod cells were severely degenerated whereas no signifi-cant cell death was observed in cone-rich retina. Conclusions: Light induced retinal degeneration was found to be more significantly affected by rod rather than cone cells. This different susceptibility to light exposure and the cone/rod cell population ratio, which differ due to the distance from fovea in the macula of human retina, may be at the root of the progression and heterogeneous pattern of retinal degeneration observed in human age-related macular degeneration. Presenter: Tivadar Orban, Research Associate (Palczewski LaB) Poster: #17 Title: Retinyl ester storage particles (retinosomes) from retinal pigmented epithelium are homologous variants of lipid droplets in other tissues Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in storage depots, termed lipid droplets or bodies that have well-defined structures in the cytoplasm and contain additional components including cholesterol and various proteins. Hydrophobic substances contained in these structures become mobilized in a specific and regulated manner as cellular requirements dictate. Retinal pigmented epithelium (RPE) cells in the eye produce retinyl ester-containing lipid droplets referred to as retinosomes. These retinyl esters are mobilized for production of the visual chromophore, 11-cis-retinal, and their storage permits proper visual function under conditions of low vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or are similar to lipid droplets in other organs/tissues that con-tain substances other than retinyl esters as their main components. Thus, we initially investigated the formation of these lipid droplets in experimental cells lines expressing lecithin:retinol acyltransferase (LRAT), a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize con-comitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine RPE and found that their protein and hydrophobic small-molecular constituents were similar to those of lipid droplets previously reported for other ex-perimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad specificity for the chemical structures of hydrophobic substances being stored. Presenter: David Salom (Polgenix) Poster: #18 Title: Heterelogous expression of 5-HT4 receptor in mouse rod cells for structural studies ABSTRACT: Recent breakthroughs in the solution of X-ray structures for G protein-coupled receptors (GPCRs) with diffusible ligands have employed extensively mutated or recombined receptor fusion proteins heterologously expressed in conventional in vitro

cell-based systems. While these advances now show that crystallization of non-rhodopsin members of this superfamily can be ac-complished, the use of radically modified proteins may limit the relevance of the derived structures for precision-guided drug design. To better enable the study of native GPCR structures, we report here efforts to engineer an in vivo expression system that harnesses the photoreceptor system of the retina to express heterologous GPCRs with native human sequences and pharmacologically func-tional conformation. As an example, we show that the human 5HT4 receptor, when placed under the influence of the mouse opsin promoter and a rod outer segment (ROS) targeting sequence, localized to ROS of transgenic mouse retina. The resulting receptor protein was expressed pharmacologically intact as demonstrated by radioligand binding assays. Upon solubilization, the retinal 5HT4 receptor retained the binding properties of its initial state in the retinal membranes. With the engineered T7 monoclonal epitope se-quence, the solubilized receptor was easily purified by one-step immunoaffinity chromatography and the purified receptor in deter-gent solution preserved its ligand binding properties. Efforts to fully characterize this receptor and to crystallize it are under way. This expression method may prove generally useful for generating functional, high-quality GPCR protein. Presenter: Wenyu Sun, (Polgenics)) Poster: #19 Title: C. elegans as an expression system for active GPCRs production Abstract: C. elegans, as a small organism, has great advantages as an expression system for active GPCRs production. Compare to cell culture system, GPCRs expressed in C. elegans is much less stressed than in modified culture condition and has more homoge-nous glycosylation pattern. And compare to transgenic mouse system, bacteria-feeding C. elegans is more cost-effective with short life span (about 2 weeks), great reproduction rate (300 eggs/hermaphrodite) and ease to adapt to amplifiable liquid culture. Here we demonstrate several GPCRs (bovine rhodopsin (bRho), human adenosine receptor 2a (hAA2AR), human adrenergic receptor 2b (hADRB2), human serotonin receptor 4 (h5HT4R) and turkey adrenergic receptor 1b (tADRB1)) can be expressed successfully in C. elegans under a muscle promoter (pMyo3) and a pan-neuron promoter (pH20) and activity can be detected in vivo by light stimula-tion for bRho or locomotion study with agonist and antagonist for different receptors. Presenter: Yaroslav Tsybovsky, Research Associate (Palczewski Lab) Poster: #20 Title: Purification and characterization of ABCA4, the photoreceptor-specific ABC transporter. ATP-binding cassette transporters (ABC transporters) utilize the energy of ATP hydrolysis to translocate their substrates across cel-lular membranes. ABCA4, also known as ABCR, is a ~250 kDa single-chain ABC transporter localized to the disk margins of verte-brate photoreceptor outer segments. The proposed function of this protein is transport of all-trans-retinal or/and its covalent adduct with phosphatidylethanolamine to the outer side of the disk membrane for further processing, thereby preventing formation of toxic by-products of the visual cycle that cause macular degeneration. Hundreds of mutations in ABCA4 lead to impaired vision and blindness such as in Stargardt disease as well as related disorders. As a step towards structural characterization of ABCA4, we devel-oped efficient methods for purification of this protein from the native source and optimized purification conditions to obtain active, stable and monodisperse preparations suitable for crystallization trials and single particle analysis. Using a variety of techniques, including native electrophoresis, gel filtration chromatography and electron microscopy we established that ABCA4 is a dimer. Mass-spectrometry studies identified several posttranslational modifications, which may regulate the ATPase and transport activities of this protein. Presenter: Xiongying Tu, Postdoc (Palczewski Lab) Poster: #21 Title: Toward Structural Studies of GC1/GCAP1 The visual cycle is composed of two phases: phototransduction, a cascade process converting light signal to neuronal electric signal; and recovery phase, a process recovering the dark state of cone or rod cell. Retinal guanylyl cyclase 1 (GC1) synthesizes cGMP which signalizes the recovery phase. Guanylyl cyclase-activating protein 1 (GCAP1) is a calcium sensor, and activates GC1 at low calcium ion concentration (∼50 nM) that results from phototransduction cascade. Therefore, GC1/GCAP1 is the transduction ma-chinery that integrates these two phases. Malfunction of GC1/GCAP1 is associated with retinal degeneration including cone-rod dystrophy and Leber’s congenital amaurosis. As an effort to study structures of GCAP1 and GC1, wild-type GCAP1 and its several variants (Y98C, E74Q, E110Q, and E151Q) have been successfully expressed and purified. Wild-type GCAP1 has been crystal-lized, and crystallization of its variants including its Y98C mutant, unmyristoylated GCAP1, etc, is undergoing. In addition, GC1 and its domains have been successfully expressed in insect cell, and kinase homology domain and catalytic domain of GC1 have been purified. Some problems of these two domains including oligomerization and solubilization need to be addressed before crys-tallization effort. Presenter: Qun Wan, Postdoc (Dealwis Lab) Poster: #22 Title: X-ray Crystallography and Isothermal Titration Calorimetry Studies of the Salmonella Zinc Transporter ZntB The ZntB Zn2+ efflux system is critical for maintenance of Zn2+ homeostasis in Enterobacteria. We report crystal structures of ZntB cytoplasmic domains from Salmonella enterica serovar Typhimurium (StZntB) in dimeric and physiologically relevant homopen-tameric forms at 2.3 Å and 3.1 Å, respectively. The funnel-like structure is similar to that of the paralogous Thermotoga maritima

CorA Mg2+ channel and a Vibrio parahaemolyticus ZntB (VpZntB) soluble domain structure. However, the central a7 helix forming the inner wall of the StZntB funnel is oriented perpendicular to the membrane instead of the marked angle seen in CorA or VpZntB. Consequently, the StZntB funnel pore is cylindrical, not tapered, which may represent an “open” form of the ZntB soluble domain. Our crystal structures and ITC data indicate that there are three Zn2+ binding sites in the full-length ZntB and two of them might be involved in Zn2+ transport. Presenter: Louie Zhou, Research Assistant (Mears lab) Poster: #23 Title: Structural Studies of Drp1 Examine Its Role in Mitochondrial Division Mitochondria, the “powerhouses” of the cell, play a significant role in regulating energy metabolism and cell death. Mitochondrial dysfunctional is associated with numerous human diseases, including cancer and neurological disorders like Alzheimer’s and Parkin-son’s disease. Mitochondrial health is intimately linked to its morphology, which is regulated by proteins that facilitate mitochondrial fission and fusion. Drp1, a large GTPase protein, regulates mammalian, mitochondrial division. Mutations in Drp1 inhibit mitochon-drial fission and lead to large, perinuclear aggregation of mitochondria. It is believed that Drp1 facilitates mitochondrial division by wrapping around constriction sites and contracting the outer membrane. Using electron microscopy, we are able to demonstrate that Drp1 forms multimeric ring-like structures that could mediate such a process. Our research focuses on studying the structure and function of Drp1 and its role in regulating mitochondrial division using cryo-electron microscopy and other biochemical methods. Presenter: Qubo Zhu, Postdoc (Palczewski Lab) Poster: #24 Title: Sponge transgenic mouse model revealed the important roles of miR-183 cluster in retina MiR-96, miR-182 and miR-183 constitute a polycistronic, sensory organ-specific paralogous miRNA cluster called miR-183 cluster. Real time PCR and in situ hybridization data show that miR-183 cluster highly expressed in photoreceptors and the interneurons in the inner nuclear layer, but not detectable in brain or other tissues. Concentrations of miR-183 cluster were downregulated during dark adaptation and upregulated in light-adapted retinas, with rapid decay and increased transcription being responsible for the re-spective changes. To better understand the roles of miR-183 cluster in retinal development, maintenance and light-adaptation function, we generated a sponge transgenic mouse model to knockdown the expression of these three microRNAs at the same time in retina. After establish-ment of the sponge transgenic mouse model, we characterized the retinal defects of these mice. Although the morphology and func-tion showed no differences between transgenic and wild type mice in normal condition, the sponge transgenic mice got serious reti-nal degeneration after 30 min 10,000 Lux light induction. Histological studies showed that the ONL thickness was dramatically de-creased in the superior site of transgenic mouse retina under 30 min light induction, and both the rod and cone cells were decreased in this condition. To figure out the molecular mechanism, we checked the downstream genes of miR-183 cluster. Real time PCR results in both the sponge transgenic mice model and different microRNA stable cell lines demonstrated that Arrdc3, Neurod4, Casp2 maybe the downstream genes of miR-183 cluster. Luciferase assay data also supported this idea. Our research revealed the important roles of miR-183 cluster in retina degeneration in vivo, and found out several candidate down-stream genes of miR-183 cluster. It will benefit the future studies of the relationship between microRNA and retinal diseases.

Molecular Pharmacology and Cell Regulation Poster Session Presenter: Daniel C. Berry, Grad Student (Noy Lab) Poster: #25 Title: The mechanism of retinoic acid inhibition of adipogenesis Obesity is not only accompanied by increased fat storage in pre-existing adipocytes but also its genesis of new adipocytes. Retinoic acid (RA), the active metabolite of vitamin A, has been previously shown to combat obesity by reducing weight and improve insulin sensitivity in mice fed a high fat diet. Furthermore, retinoic acid can inhibit adipogenesis, the process by which adipocytes are cre-ated. However, it is unclear how retinoic acid blocks adipogenesis and if this inhibition occurs in vivo. The data show that treatment of mice challenged with a high fat diet with RA prevents weight gain despite an increase in food intake. Examination of the white adipose tissue of RA treated mice revealed smaller mature adipocytes and more preadipocytes than control mice. In the classical adipocyte cell model, NIH3T3-L1, retinoic acid directly upregulated three inhibitors of adipogenesis, Pref-1, Sox9 and KLF2. Ad-ditionally, these genes were necessary for RA to inhibit adipogenesis. Interestingly, the transcription factor KLF2 and the RA re-sponsive transcription factor (RARγ) comprise a positive feedback loop that perpetuates the ability of RA to inhibit adipogenesis. In white adipose tissue of the intracellular lipid binding protein, Cellular Retinoic Acid Binding Protein, CRABPII +/- mice, Pref-1, Sox9 and KLF2 are all lower, suggesting altered preadipocyte biology. Furthermore, these adipogenic inhibitors were upregulated in isolated human preadipocytes treated with RA suggesting that RA could be efficacious in fighting human obesity. Taken together, RA upregulates inhibitors of adipogenesis, Pref-1, Sox9 and KLF2 which maintains the preadipocyte phenotype. In vivo RA is able to prevent weight gain by blocking adipogenesis and by promoting lipid oxidation.

Presenter: Lori Hezel, Research Associate (Hoppel Lab) Poster: #26 Title: Isolation and Quantification of Phospholipids and Prostaglandin Compounds on Silica Gel Columns Our lab has previously worked out a method for separating key classes of lipid compounds, including and focusing on phospholipids, from extracted mitochondria. Until now, this method has been cumbersome, tedious, and time consuming. One mitochondrial ex-tract (from 1.5 mg) can take up to 5 days from the time the sample was obtained to final quantification by phosphate chromatogram. We have been working on a method to streamline this process. The procedure is much more sensitive, as it uses a fraction of sample (50 µg) and is accompanied by standard curves . This Mass Spec procedure will make quantification much more reliable. Efforts are being made also, to apply this method to other biological samples in order to ascertain their phospholipid content and also look at other compounds ,such as, isoprostanes and prostaglandins. Presenter: Glenn P. Lobo, Research Associate (von Lintig Lab) Poster: #27 Title: A Mitochondrial Enzyme Degrades Carotenoids and Protects Against Oxidative Stress In mammals, carotenoids are the precursors for the production of vitamin A and are proposed to prevent oxidative damage to cells. Mammalian genomes encode a family of structurally related non-heme iron oxygenases that modify double bonds of these com-pounds by oxidative cleavage and cis-to-trans isomerization. The roles of the family members BCMO1 and RPE65 for, respectively vitamin A production and vision, have been well established. Surprisingly, we found that the third family member BCDO2 is a mito-chondrial carotenoid-oxygenase with broad substrate specificity. In BCDO2-deficient mice, carotenoids accumulated systemically and reduced ADP-dependent respiration rates of mitochondria. This pathology was associated with an induction of MnSOD a key marker for mitochondrial dysfunction and signalling pathways, including HIF1a, related to oxidative stress. Accordingly, administra-tion of carotenoids to human hepatocyte HepG2 cells resulted in a burst production of reactive oxygen species and loss of mitochon-dria membrane potential. In contrast to their well-known protective function in photosynthetic membranes, we show here that carote-noids can harm mitochondrial respiration and induce oxidative stress in cells and tissues. Mammalian cells thus express a mitochon-drial carotenoid-oxygenase to proptect these vital organelles. Polymorphisms in genes encoding key components of carotenoid me-tabolism, including BCDO2, are widespread in the general population and BCDO2 has a relatively low turnover rate for its carote-noid substrates. Therefore, our findings likely explain the reported adverse health effects caused by excessive supplementations with these plant lipids and identify BCDO2 as a key defender against oxidative stress. Presenter: Liraz Levi, Postdoc (Noy Lab)

Poster: #28 Title: Suppression of mammary carcinoma cell growth by inhibition of FABP5 The vitamin A metabolite retinoic acid (RA) is a potent anticarcinogenic agent and is used clinically in therapy of some cancers. These anticarcinogenic activities of RA are mediated by the RA-activated nuclear receptor RAR. However, some cancers not only fail to respond to RA but, paradoxically, their growth is enhanced upon treatment with the compound. Our previous work demon-strated that expression of FABP5 may underlie this detrimental activity. We showed that FABP5 functions to deliver RA to the ligand-activated transcription factor PPAR , thereby activating genes involved in proliferation and survival. Our findings demon-strated that upregulation of FABP5 in tumors results in diversion of RA from the antiproliferative RAR to the pro-proliferative PPAR Inhibition of FABP5 is thus expected to re-direct RA from PPAR to RAR. In the present study, we used the fatty acid stearate (18:0), a natural FABP5 ligand, as a competitive inhibitor for the binding of FABP5 to RA. The effects of stearate on reti-noic acid signaling and on cell growth were characterized using NaF cells, a cell line derived from tumors that arise in MMTV-neu mice. The data demonstrate that treatment of these cells with stearate resulted in downregulation of PPAR target genes and upregu-lation of RAR targets. These effects were enhanced by using Triacsin C, an inhibitor of fatty acids metabolism, indicating that the changes in gene expression is caused by the high amounts of stearate in the cells rather then other metabolites. The increase in ex-pression of RAR target genes was abolished in the presence of an RAR-antagonist, demonstrating that the effect of stearate was me-diated by activation of RAR. Treatment of NaF cells with stearate induced cell death in a dose responsive manner, an effect that was dependent on the presence of RA in the media. Taken together, the data demonstrate that inhibition of FABP5 shifts RA signaling from PPAR to RAR thereby sensitizing carcinoma cells to RA-induced growth inhibition. Presenter: Maneesh Mailankot, Postdoc (Nagaraj Lab)

Poster: #29 Title: High glucose sensitizes human retinal endothelial cells to interferon-γ-mediated apoptosis Recent studies implicate inflammatory pathways in diabetic retinopathy. Several pro-inflammatory cytokines including interferon-γ (IFN-γ) are elevated in the eyes of diabetic individuals. The biochemical mechanisms by which inflammatory cytokines cause dam-age to the diabetic retina are poorly understood. In this study, we have investigated the effect of IFN-γ on human retinal endothelial cells (HREC). Methods: Primary cultures of HREC were cultured with normal (5 mM) or high glucose (HG, 25 mM) and treated with 1-100 U/ml of (for 2 days). JAK-STAT pathway activation was determined by Western blotting using specific antibodies for phospho proteins. Indoleamaine 2,3-dioxygenase (IDO) activity in HREC was measured by a HPLC method. 1-methyl tryptophan and Ro61-8048 were used for inhibition of IDO and kynurenine 3-hydoxylase, respectively. Apoptotic cells were assessed by TUNEL staining. Activities of caspases were measured using specific fluorogenic substrates. Results: High glucose (HG) sensitized HREC to IFN-γ mediated

signaling by upregulating IFN-γ receptor-b. IFN-γ dose-dependently activated JAK-STAT signaling and PKC-d, and upregulated IDO. The IDO-mediated tryptophan oxidation led to the formation of kynurenines, which chemically modified proteins in HREC. These changes were accompanied by production of reactive oxygen species (ROS) and depletion of protein-free thiols. IFN-γ in-duced caspase-3-mediated apoptosis, which was enhanced in the presence of HG. We found that IFN-γ mediated cytotoxicity in HREC was primarily due to ROS generated by 3-hydroxykynurenine. Conclusion: Our results suggest that high glucose sensitizes HREC to deleterious effects of IFN- Presenter: Avijit Majumdar, Postdoc (Noy Lab) Poster: #30 Title: Modulation of the activity of Cellular Retinoic Acid Binding Protein – II by Sumoylation The vitamin A metabolite all-trans-retinoic acid (RA) plays important roles in the modulation of cell growth, differentiation and apoptosis, and it effectively inhibits the growth of mammary carcinoma cells. Many of these biological activities of RA are mediated by retinoic acid receptors (RARs) and cellular retinoic acid binding protein – II (CRABP-II). Binding of RA causes the translocation of CRABP-II from cytoplasm to nucleus where it binds to RAR and releases the ligand to the receptor. CRABP-II thus facilitates the activation of RAR and it markedly augments the biological activities of RA. Following ligand transfer, CRABP-II exits the nucleus. The primary sequence of CRABP-II contains three consensus sumoylation sites around residues (K45, K87, and K102) that may serve as sites for post-translational modification by SUMO proteins. The functional significance of sumoylation of CRABP-II was examined by generating point mutants (K45R, K87R, K102R) of the putative sumoylation sites. Mutation of the K102 abolishes the ability of CRABP-II to translocate into the nucleus in response to RA. In parallel to this finding this mutant abrogates the transcriptional activity of RAR. The observations also suggest that over-expression of Ubc9, an E2 ligase in the sumoylation machinery, results in delayed displacement from the nucleus after RA treatment accompanied by an enhancement in the transcriptional activity of RAR. Conversely, the presence of desumoylase in the sumoyla-tion machinery, SENP abrogates the nuclear localization of CRABP-II. Additional data indicate that CRABP-II interacts with Ubc9. Preliminary data further suggest that sumoylation of CRABP-II is induced by RA. Taken together the observations indicate that RA induced sumoylation of CRABP-II is required for its nuclear translocation. Presenter: Paul Minkler, Research Associate (Hoppel Lab) Poster: #31 Title: Synthesis, Purification, Standardization and Characterization of Malonylcarnitine and d3-Malonylcarnitine: Reference Standard and Internal Standard for Quantification of Malonylcarnitine in Biological Samples Malonyl-CoA decarboxylase deficiency is an uncommon inherited metabolic disease (OMIM 248360). Elevated levels of malonoyl-carnitine occur in patients with this disorder; therefore we want to quantify this acylcarnitine species as a part of our HPLC-MS/MS quantitative acylcarnitine procedure for patient samples. Quantification requires reference material and a suitable internal standard. However, neither malonylcarnitine nor its labeled internal standard (d3-malonylcarnitine), is commercially available. Therefore, we undertook the synthesis of these two compounds using commercially available reagents (Johnson DW. Synthesis of dicarboxylic acylcarnitines. Chem Phys Lipids. 2004 129, 161-171). Characterization was by NMR. Purification consisted of two steps. To re-move unreacted acid(s) from the raw synthetic material, we applied crude synthetic product to an Oasis MCX column and eluted using 90+10+1+2 acetonitrile / water / formic acid / pyridine. The second step used preparative HPLC (Atlantis Prep T3 OBD 5µ, 19x15 mm column and a 5 ml/min flow rate). A gradient of 100% A (0.1% heptafluorobutyric acid in water) to 60% B (95% MeCN) over 60 min was used. Fractions (1 mL) were collected. Those that were UV positive were examined by HPLC-MS/MS following derivatization with pentafluorophenacyl trifluoromethanesulfonate. Standardization of the pure material was performed by multiple analyses for free and total carnitine, with the difference being the determined concentration of the pure acylcarnitine solution. Char-acterization was performed by MS and MS/MS. This reference standard solution was then combined with the other reference stan-dard solutions prepared in a like manner to generate calibration curves and quality control samples. Malonylcarnitine is now one of 65 acylcarnitines that we quantify in patient samples. Presenter: Saida Omarova, Postdoc (Pikuleva lab) Poster: #32 Title: Studies of knockout mice provide insight into the retinal significance of CYP27A1. There is increasing evidence that a link exists between cholesterol and age- related macular degeneration (AMD). Cholesterol is eliminated from different organs by lipoprotein- and enzyme-mediated mechanisms. In the retina, our data indicate that CYP27A1 is important for enzyme-dependent cholesterol elimination. Deficiency of this enzyme in humans leads to a lipid storage disease called cerebrotendinous xanthomatosis, hallmarked by premature atherosclerosis, neurodegeneration, juvenile cataracts, and early retinal senescence. The retinal significance of CYP27A1 has not been clearly established. Through the use of CYP27A1 knockout mice (KO), we assess the role of CYP27A1 in visual function. We utilize electroretinography (ERG) to assess visual function, optical coherence tomogra-phy (OCT) and histochemistry studies for evaluation of retinal morphology, and intense light exposure to evaluate effects on the vis-ual cycle. Two-way repeated-measures ANOVA in 1.5 and 3 months (mo) old mice show that ERG responses are genotype- and gender- specific. We observe statistically significant increases in the amplitude of the photopic b-wave in 3 mo CYP27A1 female KOs as compared to WT littermates. Male CYP27A1 KOs also have statistically significant increases in the photopic b-wave ampli-tude, but starting later, at 6 mo. These ERG changes reflect the activity of bipolar cells postsynaptic to the cone photoreceptors. Ad-

ditionally, at the age of 4 mo, CYP27A1 KO males begin to develop retinal deposits in the inferior retina as revealed by OCT. One 12-mo male KO that we currently have exhibits retinal pathology both in the inferior retina and near the optic nerve starting from the choroid and penetrating through Bruch’s membrane and RPE into the outer retina. Doppler flow OCT reveals abnormal blood flow in this area of pathology, which may indicate the presence of choroidal neovascularization. Further investigation of these lesions via fluorescein angiography and histologic studies is underway. Additionally, when exposed to intense light (10,000 lux) for one hour at 4 mo, male CYP27A1 KO mice do not display subsequent decreases in scotopic a- and scotopic b-wave ERG amplitudes, as do their WT littermates. These results suggest that cholesterol metabolism may have significant effects on retinal function and morphology. Presenter: Jiyeon Yang, Grad Student (Dubyak Lab) Poster: #33 Title: Human beta defensin 3 induces STAT1 phosphorylation in mouse T cells Human β–defensin 3 (hBD-3) is an inducible, epithelial cell-derived antimicrobial peptide produced upon bacterial and cytokine stimulation. hBD-3 overexpression is observed at mucosal sites, such as the skin and lung, during periods of chronic inflammation. Limited studies, however, have analyzed hBD-3 expression in the intestine of healthy and inflammatory bowel disease (IBD) pa-tients. Deficiency of β-defensins is hypothesized to be a contributory cause of IBD; however, our laboratory has found that patients with IBD express elevated levels of hBD-3 in the small intestine. In addition to having antimicrobial properties, hBD-3 also has immunomodulatory properties, primarily regulating innate responses by targeting myeloid cells. We previously demonstrated that hBD-3 can directly modulate adaptive immune responses, by regulating effector/memory human T cells, which maintain the chronic inflammation of IBD patients, signaling through a novel STAT1 pathway. Thus, we hypothesize that differential and coordinated regulation of T cells by hBD-3 is central to maintaining intestinal immune homeostasis and overexpression of hBD-3 may modulate the inflammatory progression of IBD. To investigate this hypothesis, we tested the stimulatory effects of hBD-3 on murine splenic T cells, isolated from wild type and SAMP1/YitFc mice, which develop spontaneous ileitis and thus serve as a model of IBD. Our results show that hBD-3 stimulates immediate and transient tyrosine phosphorylation of 65 kDa proteins and STAT 1. These results demonstrate hBD-3 regulates immune responses across the species barrier and shapes our future objective to generate genetically modified SAMP1/YitFc mice to express hBD-3 in the small intestine. This model will enable us to further investigate hBD-3 medi-ated regulation of T cells in vivo, as well as evaluate its therapeutic potential in treating IBD. Presenter: Wenchao Zheng, Postdoc (Pikuleva lab) Poster: #34 Title: Expression patterns of genes involved in cholesterol homeostasis in human neural retina and retinal pigmented epithelium Very little is currently known about cholesterol homeostasis in the retina. To determine the expression of genes involved in choles-terol metabolism and lipoprotein transport in the retina, PCR arrays, quantitative real-time PCR, and immunofluorescence staining were utilized. A total of 84 genes were analyzed with PCR array. About 69 genes are expressed in human retina as assessed by the threshold cycle number (Ct) of PCR amplification. PCR array and quantitative real-time PCR results show inter-individual variabil-ity in gene expression. Cholesterol-metabolizing Cytochrome P450s CYP27A1, CYP46A1 and CYP11A1 mRNA expressions var-ies up to 17-fold between individuals. Immunofluorescence staining shows that these proteins are expressed in the neural retina with specific layer distribution, but not in the retinal pigmented epithelium (RPE). Cholesterol homeostasis is stringently regulated by the sterol regulatory element binding proteins (SREBPs). Key components of SREBP-mediated feedback pathways, including SREBP1, SREBP2, SCAP, Insig1, Insig2, HMGCR, LDLR, and ABCA1, are detectable on immunofluorescence staining. All of the above genes are expressed more highly in ganglion cells than in other retinal cell types. SREBP2 and ABCA1 immunoreactivities were detectable in neural retina with faint staining of inner segment and outer segment, but not in RPE. SCAP, Insig1, Insig2, and HMGCR were observed in all layers of the neural retina. Insig1 and Insig2 were also found in the RPE and Bruch’s membrane (BM). In summary, our results indicate that the human retina expresses most of the same genes involved in cholesterol metabolism and lipoprotein transport in hepatocytes. Most importantly, our data further demonstrate that complete SREBPs-mediated feedback networks are present in the neural retina, but probably not in RPE and BM, suggesting that regulation of cholesterol homeostasis in neural retina is similar to that in extra-ocular tissues.

Translational Thereaputics—Poster Session Presenter: Sanae Sakami, Postdoc (Palczewski Lab) Poster: #35 Title: Genetic ablation of 11-cis retinal exacerbates photoreceptor degeneration in the P23H knock-in mouse: a new animal model of autosomal dominant retinitis pigmentosa. A proline-23-histidine (P23H) mutation in rhodopsin, the visual pigment mediating night vision, is the most prevalent cause of auto-somal retinitis pigmentosa in the USA. Although cultured cell and transgenic animal models have been developed, they did not fully mimic human phenotype, and the mechanism by which this mutation leads to photoreceptor cell degeneration remains unknown. By generating P23H mutant rhodopsin knock-in mice, we report here that the P23H mutant protein was inadequately glycosylated and its level was between 1 to 10 % that of wild type rhodopsin. P23H protein did not accumulate in the endoplasmic reticulum, but in-stead disrupted the photoreceptor disk. Genetic ablation of the chromophore ligand of rhodopsin, 11-cis-retinal, accelerated photore-ceptor cell degeneration, whereas pharmacological inhibition of chromophore production for one week did not. These results indicate

that most P23H protein is degraded and its cytotoxicity is enhanced by lack of the 11-cis-retinal chromophore. Presenter: Peter Levitt, Grad Student (Bonomo Lab) Poster: #36 Title: Probing the role of the Ω loop in the KPC-2 ß-lactamase Among serine ß -lactamases, those that hydrolyze the carbapenem class of ß -lactams (e.g., imipenem), often the “last line” of de-fense against drug-resistant bacterial infections, presents a critical clinical challenge. Presently, the KPC-2 ß -lactamase is the most widespread carbapenemase in the world and threatens the efficacy of carbapenems. KPC-2 hydrolyzes carbapenems using a 2 step-mechanism. First, the substrate is acylated by S70. Then a water molecule, activated by E166 in the Ω loop, hydrolyzes the acyl-enzyme intermediate and regenerates the enzyme. The flexibility of the Ω loop, a uni-form structural feature of serine ß -lactamases, is governed by a salt bridge between R164 and D179, also conserved amino acids. Naturally occurring substitutions at either R164 or D179 in narrow-spectrum enzymes (e.g., TEM-1 and SHV-1) disrupts the salt bridge, which broadens its hydrolytic profile to include extended-spectrum cephalosporins, but reduces catalytic activity against other substrates, especially carbapenems. Interestingly, substitution of G170 in the enzyme GES-1, another clinically important ß -lactamase, decreases resistance towards cephalosporins, but enhances resistance to carbapenems. Anticipating the substrate profile of KPC-2 with substitutions in important Ω-loop residues is essential to understanding the evolution of substrate specificity and may have implications for the future of ß -lactam or ß -lactamase inhibitor design. To explore the contribution of R164, D179 and N170 residues on the function of KPC-2, we will engineer substitutions at these resi-dues, characterize their phenotype in a uniform genetic background by susceptibility studies, and assess pre-steady and steady state kinetic parameters using cephalosporin and carbapenem substrates containing different side chains. We will identify reaction inter-mediates using mass spectrometry and perform molecular modeling to understand the contribution of these residues to catalysis. Finally, we will examine the impact of the mutations on protein stability and local secondary structure using circular dichroism. These studies will strive to provide novel structure/function insights into catalysis by the KPC-2 ß -lactamase and may serve as the first step in drug design.

Notes:

The Department of Pharmacology, Case Western Reserve

University School of Medicine, gratefully acknowledges the

following for their support:

Greenfield Family In honor of Nathan. S. Greenfield and family

Sponsor of the Greenfield Travel Grant

Dr. Robert Bonomo Associate Professor of Medicine & Pharmacology

Case Western Reserve University Veterans Affairs Medical Center

Location & Directions The Lodge & Conference Center at Geneva State Park, located in Ashtabula County, Ohio, in the northeast corner of the state, is conveniently located near I-90.

Driving Distances Downtown Cleveland: 53 miles

Downtown Youngstown: 72 miles

Downtown Akron: 75 miles

Downtown Pittsburgh, PA: 174 miles

Cleveland Hopkins International Airport: 64 miles

Driving Directions The Lodge is just 50 minutes east of Cleveland. From Inter-state 90, take Exit #218 on to OH-534 North, which is also the exit for Geneva, Geneva-on-the-lake and several wineries. Follow the signs to the lodge, less than 10 minutes away just one mile north of the Geneva State Park entrance.

Geneva onGeneva onGeneva on---thethethe---LakeLakeLake

4888 North Broadway (St. Rt 534)

Geneva-On-The-Lake, OH 44041

Cleveland