Molecular and Cellular Pharmacology Student Seminars 2007-2008

September 10, 2007
Matt Wagoner
Dr. Avtar Roopra Lab

Regulation of PI3K signaling pathways by Neuronal Restrictive Silencing Factor (NRSF)

The transcription factor neuronal restrictive silencing factor (NRSF) suppresses the expression of neuronal genes in non-neuronal cells. Loss of NRSF has recently been found in a third of all breast and colon cancers, and has been shown to promote adhesion independent growth in a PI3K dependent manner. This and other work from our lab have prompted us to investigate the possible mechanisms by which loss of NRSF may cause an upregulation of the PI3K pathway. Here, we present insulin receptor substrate 1, akt2 and mTOR as potential targets of NRSF mediated suppression and suggest a possible mechanism by which they may alter cell survival and metabolism.

Monday, September 17, 2007
Bryan Glaser
Burgess Lab

Characterizing Inhibitors of the Sigma70-RNA Polymerase Interaction as Potential Antibiotics

Antibiotic resistance is an increasing problem that could pose serious health risks in the future. In order to address this situation, antibiotic candidates utilizing novel mechanisms of action need to be developed. The Escherichia coli RNA polymerase holoenzyme is a desirable target because its role in transcription is critical to cell survival. The E. coli holoenzyme is comprised of core RNA polymerase (a2bb'w) along with one of seven sigma factors. Each sigma factor is responsible for driving transcription of a different set of genes with sigma70 regulating the "house keeping" genes. If the sigma70-core interaction can be prevented, the bacteria can no longer transcribe and grow. We designed a high throughput screen using luminescence resonance energy transfer (LRET) to monitor this protein-protein interaction. Using this assay we screened a library of 16,000 small molecules and identified four lead structures. These four compounds have been further characterized using in vitro assays such as LRET and in vitro transcription in both prokaryotic and eukaryotic systems. In vivo assays such as disc diffusion inhibition, growth curve analysis, and time-kill assays were performed in multiple bacterial strains. Two of the compounds show activity against both gram positive and gram negative strains of bacteria. Finally, structure-activity relationship (SAR) studies are under way to determine ways to improve the activity of the compounds to enable future drug development.

1. Arthur TM: Localization and characterization of a sigma subunit-binding site on the Escherichia coli RNA polymerase core enzyme. Ph.D. thesis University of Wisconsin, Madison 2000
2. Arthur TM, Anthony LC, and Burgess RR: Mutational analysis of beta '260-309, a sigma 70 binding site located on Escherichia coli core RNA polymerase. J Biol Chem 2000;275:23113-23119.
3. Arthur TM and Burgess RR: Localization of a sigma70 binding site on the N terminus of the Escherichia coli RNA polymerase beta' subunit. J Biol Chem 1998;273:31381-31387.
4. Bergendahl V, Heyduk T, and Burgess RR: Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase. Appl Environ Microbiol 2003;69:1492-1498.

Monday, September 24, 2007
Derek Pavelec
Kennedy Lab

Identification of Novel Endogenous RNAi Machinery and Characterization of ERI/Dicer Complex function.

We are interested in understanding the endogenous biological functions of the RNAi machinery. In order to further our understanding of RNAi we conducted genetic screens in the model organism C. elegans to identify components of the endogenous RNAi machinery. Previous genetic screens in the laboratory have identified a series of factors termed ERI-1, RRF-3, ERI-3, and ERI-5 that exist in a protein complex with the catalytic engine of RNAi, the RNase III like enzyme Dicer (DCR-1). This complex has been termed the ERI/DCR complex. Recent screens have identified additional components of an endogenous RNAi pathway, ERI-8(ERGO-1) and ERI-9. Animals with mutations in the ERI/DCR complex fail to accumulate a subset of cellular small RNAs. These siRNAs are also missing in eri-8(-) and eri-9(-) animals. Furthermore, Animals carrying mutations in components of the ERI/DCR complex exhibit an increased occurrence of male progeny (Him phenotype) and a temperature sensitive sperm defect. Finally, we have shown that the ERI/DCR complex mutant animals exhibit a dramatic mis-regulation of several genes thought to be expressed exclusively in sperm. We hypothesize that the ERI/DCR complex is essential for regulating gene expression within the male germline while also interacting with additional RNAi components to regulate genes in the soma.

Monday, October 1, 2007
Felix Yeh
Chapman Lab

The mechanism in which tetanus neurotoxin enters neurons is not well established. Literature suggests two mutually exclusive entry pathways: through recycling synaptic vesicles and through another vesicular pathway. To understand the extreme toxicity of tetanus neurotoxin and discover clues about the receptors for tetanus toxin, it is important to determine the methods in which tetanus toxin travels through victims. Our hypothesis is that tetanus toxin is able to travel from the periphery and arrive at its final destination, the inhibitory interneuron, through two unique protein receptors that direct its transport in the motorneuron and its entry in central nervous system. The study is directed towards elucidating the entry pathways of tetanus neurotoxin and also identifying the two receptors that mediate tetanus neurotoxin's entry into motorneurons and inhibitory interneurons. Our studies on hippocampal neurons have shown that tetanus preferentially binds to inhibitory neurons under conditions that stimulate recycling synaptic vesicles. Furthermore, the cleavage of tetanus' subtrate, synaptobrevin 2, is also faster in inhibitory neurons than in excitatory neurons. Similar experiments performed on dissociated spinal cord cultures, which contain the physiological target of tetanus, also confirm these observations. Finally, to further solidy the idea that tetanus enters through synaptic vesicles, experiemnts utilizing botulinum neurotoxin A and dominant negative dynamin were done. Our data suggests that the ability of tetanus to target inhibitory interneurons is by a unique receptor on the synaptic vesicles of inhibitory neurons.

Matthew Marengo
October 8, 2007
Wassarman Lab

Regulated alternative splicing requires signal-activated factors to communicate with the general splicing machinery. The alternative splicing of Drosophila melanogaster TAF1, which can be regulated by DNA damage signaling pathways, is a model system for understanding how signaling pathways interact with the spliceosome. A TAF1 minigene system was developed that recapitulated upregulation of an alternative splicing isoform in response to treatment of S2 cells with the topoisomerase I poison camptothecin. Upregulation of this isoform requires inclusion of two non-consecutive alternative casette exons, 12a and 13a. By miniTAF1 mutation and complementary U1 snRNA mutation, we show that U1 snRNP recruitment to the unusual exon 13a 5' splice site is sufficient to cause exon inclusion in both miniTAF1 and endogenous TAF1 mRNA, bypassing to camptothecin-induced signaling. Gene expression of the mRpL37 gene, which shares this unusual splice site, is regulated by splicing efficiency. In contrast, U1 snRNP recruitment to the exon 12a 5' splice site upregulates exon inclusion but does not bypass camptothecin-induced signaling. Additionally, a conserved intronic element is important for exon 13a inclusion, and mutation of this event uncouples the upregulation of 12a inclusion from 13a inclusion. This element is important only in the context of the unusual 13a 5' splice site and is a candidate target for regulated splicing factor binding. These data suggest that TAF1 alternative splicing events are regulated by mechanistically distinct and independent pre-mRNA elements.

Brian Torres
Ruoho Lab
October 15, 2007

Essential to the storage and release of monoamines is the activity of the vesicular monoamine transporter 2 (VMAT2). The VMAT2 is a 550 amino acid, twelve transmembrane transporter that concentrates monoamines from the cytoplasm into synaptic vesicles. Recently, we have uncovered evidence that the twenty amino acid cytosolic N-terminus of the VMAT2 exerts profound control over VMAT2 transport activity. Preliminary findings are: first, truncation of the twenty-amino acid N-terminus decreases VMAT2 transport activity, second, a peptide representing the 20 amino acid N-terminus inhibits VMAT2 transport activity, third, the N-terminus undergoes in vitro phosphorylation by protein kinase C at two serines (15 and 18), fourth, mutation of serines 15 and 18 to aspartate, to mimic a phosphorylated state, strongly reduces monoamine transport activity. The talk will cover the potential relevance of VMAT2 research to Parkinsons Disease as well as in situ regulation of VMAT activity. The generation of an antibody tool to monitor the phosphorylation state of serines 15 and 18 on the VMAT2 N-terminus will be described.

October 22, 2007
Delana Hopkins
Greenspan Lab

Examining Functions of BMP-1/TLD-like Proteinases in the Nervous System

The BMP-1/TLD-like subgroup of astacin-like metalloproteinases comprises a family of developmentally important proteinases. These enzymes are known to proteolytically process a number of structural extracellular matrix proteins, members of the TGF_-superfamily and the TGF¾-like BMP signaling antagonist chordin. In this manner BMP-1/TLD-like proteinases serve both to regulate the extracellular matrix and facilitate BMP signaling. BMP signaling has been implicated in cell fate determination, survival, and differentiation of several cell types in the brain at specific time points. Further, several BMP-1/TLD-like substrates, such as growth differentiation factor 11 (GDF11), chordin, and neurogenesin (aka. chordin-like 1), are expressed in the nervous system and known to affect neurogenesis and cell fate decisions. Tolloid-related proteinases are also known to be up-regulated following long term sensitization and play pivotal roles in motor neuron guidance, in Aplysia and Drosophila, respectively. One member of the BMP1/Tld-like proteinases, mammalian Tolloid-like 1 (Tll1), is enriched in the hippocampus, cerebellum, and spinal cord. Recently, increased hippocampal Tll1 levels have been associated with neurogenesis in that structure. Mice lacking Tll1 previously generated in our lab die at 13.5 days post-conception due to gross cardiac abnormalities and heart failure. However, much of rodent nervous system development takes place postnatally. Therefore, in order to examine functional roles for Tll1 in the nervous system, we have generated mice which are conditionally ablated for the Tll1 gene in the central nervous system. Future studies will focus on characterizing functions for BMP-1/TLD-like proteinases in genetically ablated mice.

October 29, 2007
Cynthia Koziol
Bertics Lab

Eosinophils are terminally differentiated innate immune cells that have both advantageous and detrimental functions within the host. It has been reported that eosinophils kill parasites and alert the immune system to presence of a foreign invaders, but eosinophils are also thought to contribute to disordered airway function that is characteristic of asthma. Interleukin-5 (IL-5) strongly affects many aspects of eosinophil biology. Interleukin-5-family cytokines have been shown to prolong survival of peripheral blood eosinophils in vitro, thereby prolonging the lifespan of airway eosinophils. However, the mechanisms by which IL-5-family cytokines mediate eosinophil survival have not been elucidated.

Expression of the survival-promoting gene Pim-1 has been observed following IL-5, GM-CSF (Granulocyte Macrophage - Colony Stimulating Factor), and IL-3 stimulation. We have also demonstrated that Pim-1 is expressed in airway eosinophils following segmental allergen challenge (SBP-Ag). We have been able to attenuate Pim-1 expression in human peripheral blood eosinophils following IL-5 stimulation. Accordingly, the overall hypothesis of these studies is that the eosinophil survival induced by IL-5-family cytokines is mediated through expression of Pim-1 with subsequent effects on the expression/activity of other anti-apoptotic proteins.

November 12, 2007
Amanda Branam
Greenspan Lab

BMP1/TLD-like Proteinase Regulation of Chordin-like Proteins

Bone morphogenetic protein 1 (BMP1) is an astacin-like metalloproteinase. It is the founding member of a small group of metalloproteinases with similar domain structure and function. These proteinases have been shown to be involved in the processing of several extracellular matrix proteins as well as proteins involved in TGF-beta-like BMP signaling, such as chordin. Chordin is an extracellular antagonist of BMP signaling. This antagonism is accomplished by binding to the BMP in the extracellular space and preventing the BMP from binding to its cognitive receptor and initiating signaling. BMP1/TLL1 have been shown to play very important roles in this mechanism in that they are able to process chordin, releasing the BMP from its inactive complex and allowing signaling to commence. It has been shown that this mechanism between BMPs and chordin is important in dorsal-ventral patterning of several organisms. Chordin-like 1 (CHL1) and chordin-like 2 (CHL2) are two proteins with homology to chordin, which have been shown to bind a variety of BMP proteins and are also able to induce dorsalization in xenopus embryos upon injection of their mRNA, similar to the effects shown with chordin. For these reasons, our lab has decided to further study these proteins and determine if they are substrates for BMP1/TLD-like proteinases. We are also further studying the functions of these chordin-like proteins, through overexpression and knockdown experiments using zebrafish as a model organism. For these purposes we have cloned a zebrafish chordin-like gene and have begun characterization of this gene. Our initial characterization efforts have provided exciting results, demonstrating that zCHL may be a very important player during early zebrafish embryogenesis.

Nucleotide Receptor P2RX7 Mediated ATP-Induced CREB Activation and Subsequent AP-1 Protein Expression in Monocytic Cells

Inflammation is a localized response to injury or infection and is mediated by factors released from leukocytes present in the inflammatory microenvironment. Usually accompanying infection or inflammation is the release of a high concentration of extracellular nucleotides, supplying a source of ligands for purinergic receptors that are present on the cell surface of many immune cells. One particular nucleotide receptor that is mainly expressed on leukocytes is the P2X7 receptor (P2X7R). Previous work with P2X7R has linked this receptor to the modulation of macrophage and monocyte (macrophage precursor cell) responsiveness to inflammatory stimuli, such as the production of pro-inflammatory cytokines, reactive oxygen species, and prostaglandins. Interestingly, the promoter for many P2X7R-modulated inflammatory genes (i.e. TNF-alpha, iNOS, IL-1, IL-6, IL-8) contains consensus sites for cyclic-AMP response element binding protein (CREB) binding. Previous studies, along with my preliminary data, suggest that stimulation of mitogen-activated protein kinase (MAPK) cascades, and the control of transcription via CREB-linked pathways, are potentially involved in the nucleotide regulation of macrophage-induced inflammation. To this end, the induction of Activating Protein-1 (AP-1) protein expression by P2X7R agonists was investigated. In both murine RAW 264.7 macrophages and isolated human peripheral blood monocytes P2X7R stimulation can induce formation of the AP-1 proteins FosB and JunB, which likely play a role in modulating monocytic gene expression during inflammation. This is one of only a few reports where P2X7R activation leads to protein expression without having to prime cells with other inflammatory stimuli (e.g., lipopolysaccharide).

November 26, 2007
Yiming Zhu
Bertics Lab

The Crosstalk Between Interleukin-5 (IL-5) - and Chemoattractant/G Protein-Coupled Receptor (GPCR) -mediated Pathways in Human Peripheral Blood Eosinophils

Exposure of human peripheral blood eosinophils to IL-5 family cytokines is well-recognized to prime/potentiate eosinophil responsiveness and extracellular-regulated kinase 1/2 (ERK1/2) activation following stimulation with chemoattractants that bind to GPCRs. In the current study, I tested the hypothesis that the Src family member Lyn is involved in the signaling pathways of priming in blood eosinophils. Two inhibitors SU and PILR are utilized to test the involvement of Lyn in the priming mechanism as a tyrosine kinase and an adaptor protein respectively. My data suggest that the kinase activity and adaptor function of Lyn contribute independently to chemoattractant-induced ERK 1/2 phosphorylation. There is also indication that other Src family members may be involved in the crosstalk between IL-5 and GPCR pathways.

December 3, 2007
Zhen Zhang
Jackson Lab

Exocytosis plays an important role in the communication between neurons

Amperometric recording of single vesicle release events reveals a sequence of steps, beginning with fusion pore opening and proceeding through fusion pore dilation, with the termination step either as fusion pore closure or complete membrane fusion. At the molecular level, these steps of exocytosis must involve protein conformational changes, lipid rearrangement, and diffusion of released substances. By varying temprature, I found that the rate of fusion pore closure and fusion pore dilation is strong temperature dependent. By contrast, flux through an open fusion pore, the spike 50-90% rise time, and spike exponential decay time constant depend weakly on temperature. These weak temperature dependences suggest that efflux through the fusion pore is diffusive or electrodiffusive, and that the time course for the spike is governed by diffusion through an expanding fusion pore. In my second project, by adding phosphatidylserine in cell culture media, I found phosphatidylserine regulates the stability of fusion pores and this regulation is most likely due to the interaction between synaptotagmin and phospatidylserine.

December 10, 2007
Keefe Chan
Huttenlocher Lab

Calpain Cleavage of FAK in Focal Adhesion Turnover

Cell migration requires the coordinated and dynamic regulation of focal adhesions. Previous studies in our laboratory have demonstrated that the limited proteolysis of talin 1 by the calcium-dependent protease calpain 2 plays a critical role in focal adhesion disassembly. We, and others, have identified another calpain substrate-the focal adhesion kinase (FAK)-which has also been shown to be a key component in the modulation of focal adhesion turnover. Using time-lapse video microscopy, we have examined the adhesion dynamics of GFP-talin1 in FAK-deficient HEK 293 cells. Quantification of adhesion assembly and disassembly rates demonstrates that FAK is required for the regulation of talin dynamics. However, the mechanisms by which FAK regulates the adhesion dynamics of talin are not well understood. To begin to address this, we have mapped the calpain cleavage site of FAK and have generated a mutant form of FAK that is resistant to calpain proteolysis. We report that calpain cleavage of FAK is critical for efficient focal assembly and disassembly of talin. Furthermore, we are mapping the site of direct interaction between FAK and calpain 2. We are actively performing studies that will address the relative contribution of the FAK-calpain 2 interaction and cleavage of FAK by calpain to the regulation of adhesion dynamics.

December 17, 2007
Naval P. Shanware
Tibbetts Lab

Coregulated ataxia telangiectasia-mutated and casein kinase sites modulate cAMP-response element-binding protein-coactivator interactions in response to stress

The cyclic AMP-response element-binding protein (CREB) is a bZIP family transcription factor implicated in the regulation of diverse cellular processes ranging from neuronal survival to circadian rhythm entrainment. CREB is activated in response to cellular stimuli, including cAMP and Ca(2+), via phosphorylation of Ser-133, which promotes interaction between the kinase-inducible domain (KID) of CREB and the KID-interacting domain (KIX) of CREB-binding protein (CBP). We have previously demonstrated that the interaction between CREB and CBP is inhibited by DNA-damaging stimuli through a mechanism whereby CREB is phosphorylated by the ataxia telangiectasia-mutated (ATM) protein kinase. We have also recently shown that the ATM phosphorylation sites in CREB are functionally intertwined with a cluster of coregulated casein kinase (CK) sites that play a role in CREB phosphorylation in the presence and absence of DNA damage. The phosphorylation of these CK sites renders CREB permissive for ATM-dependent phosphorylation and leads to the abrogation of CREB-CBP complexes in response to genotoxic stress. A role for the casein kinase sites in the absence of DNA damage has however been elusive. We now present recent data implicating a p38 dependent pathway in the regulation of the DNA-damage independent CREB phosphorylation. In other studies, we provide evidence for regulation of another bZIP transcription factor ATF1 by a similar phosphorylation cascade. Our findings outline a complex mechanism of CREB and ATF1 phosphorylation in which coregulated ATM and CK sites control transactivation potential by modulating CBP-binding affinity. Present studies are aimed at further biochemical characterization of these events and in the identification of specific gene targets for this pathway.

February 4, 2008
Nick Schill
Anderson Lab

Characterization of novel PIPKIg splice variants and their putative roles in trafficking

The spatial and temporal metabolism of phosphoinositides regulates many cell signaling pathways. Phosphatidylinositol 4,5-bisphosphate (PI4,5P2) is a lipid signaling molecule which plays a role in the regulation of endocytosis, actin assembly, cell migration, and the maintenance of cellular adhesion structures. Phosphatidylinositol 4-phosphate 5-kinase type I gamma (PIPKIg) synthesizes PI4,5P2 at specific subcellular sites via interactions with distinct protein binding partners. Variation of the C-terminal region of PIPKIg is the mechanism by which the kinase interacts with these different protein subsets. Four PIPKIg splice variants have been identified in human cells: PIPKIg640, 668, 700, and 707. These splice variants all exhibit kinase activity and distinctive subcellular localization patterns. Importantly, each splice variant seems to interact with a set of specific targeting proteins. PIPKIg668 interacts with both E-cadherin, an adhesion molecule present at cell-cell junctions, and adaptor complexes (AP), which mediate aspects of intracellular trafficking. With PIPKIg668 acting as a bridge between E-cadherin and AP1B, the efficient delivery of E-cadherin to the basolateral membrane is facilitated. Interestingly, we have observed that endogenous PIPKIg668 and PIPKIg707 can be co-immunoprecipitated with E-cadherin. Consistent with a role for PIPKIg in the trafficking of cellular proteins, PIPKIg707 has been found to directly associate with sorting nexin 5 (SNX5) via its unique C-terminal sequence. Members of the SNX family are known to regulate multiple cellular trafficking pathways, including the internalization, recycling, and turnover of several cellular receptors. The PIPKIg707/SNX5 complex has been found to co-localize with Rab11, suggesting that this complex may be involved in the transport of newly-synthesized proteins to the plasma membrane. Given the observed role of PIPKIg668 in E-cadherin trafficking, PIPKIg707 may also regulate aspects of E-cadherin transport via its interaction with the SNX proteins.

February 11, 2008
Dominique Fontanilla
Ruoho Lab

Characterization of the Sigma-1 Receptor Binding Site and Sigma-1 Ligand Pharmacophore-like compounds

The sigma receptor represents a ubiquitously expressed unique binding site in the CNS and is a member of the orphan receptor class for which no endogenous ligand is known. It is known, however, that the sigma receptor binds with high affinity to several classes of chemically unrelated ligands such as neurosteroids, neuroleptics, dextrobenzomorphans, and several psychostimulants (e.g. cocaine, methamphetamine, MDMA, methcathinone). In terms of function, a recent breakthrough has been made showing that the Sigma-1 receptor is a chaperone protein at the ER-mitochondrion interface and is involved with cell survival and ER stress (1). Currently, our interests involve the characterization of the receptor binding site as well as the exploration of naturally occurring Sigma-1 ligand pharmacophore-like N-alkyl amines as candidates for the endogenous ligand.

Structurally, the sigma-1 receptor has been shown to have three hydrophobic regions, two of which, have highly conserved residues between the sigma receptor and the yeast C8-C7 sterol isomerase (ERG2) (2). These regions have thus been termed steroid binding domain-like 1 and 2 (SBDL1 and SBDL2) and are hypothesized to comprise the binding site. Previous photoaffinity labeling work in our lab has implicated Asp 188 in SBDL2 as a ligand-binding target using a 3-iodo-4-azidococaine photoprobe. Our biochemical and pharmacological data using two rigid photoprobes and two flexible photoprobes indicate that, indeed, SBDL1 and SBDL2 make up the binding site and that the two regions are juxtaposed.

Through studies involving our Sigma-1 ligand pharmacophore, we also hypothesize that naturally occurring N-alkyl amines represent endogenous ligands for the sigma-1 receptor. These include N-methylated trace amines such as N, N'-Dimethyltryptamine (DMT), N, N'-Dimethylphenethylamine, and N, N'-dimethyltyramine. Interestingly, recent hypotheses suggest that perhaps endogenous DMT is responsible for an anxiolytic calming-effect on the mental state, thereby decreasing psychosis (3). We present DMT functional data using the hNav1.5 channel, Kv1.4 channel, and whole mouse systems.

February 18, 2008
Jennifer Lamberski
Burgess lab

ERR_-co-activator GRIP1 protein-protein interaction: a target for novel breast cancer therapeutics?

At the time of diagnosis, 30% of all breast tumors grow independently of hormone. These cancers are unlikely to respond to currently available hormonal therapies. Of the 70% of breast tumors that do depend on hormone for growth, only about half of these patients will respond to hormonal therapies, such as aromatase inhibitors and the selective estrogen receptor modulator (SERM) tamoxifen. However, drug resistance often develops after long term treatment as the tumor progresses to a hormone-independent state.
There is a great need for novel therapies for hormone-independent breast cancers. Estrogen-related receptor alpha (ERR±) may be a new therapeutic target because it possesses overlapping sequence and functional similarity to estrogen receptor alpha (ER±), the primary focus of therapy for breast cancers. ERR± binds to estrogen response elements (EREs) in the promoters of a subset of estrogen-responsive genes, such as pS2, PgR, ErbB2, and others which are involved in cell proliferation and anti-apoptosis. But ERR± can act independently of hormones; instead, its transcriptional activity can be enhanced by the presence of co-activators. Moreover, ERR± expression correlates with hormone-independent breast tumors and with decreased disease-free survival of breast cancer patients. These observations strongly suggest that ERR± plays an important role in the progression of a significant subset of breast cancers.

We have made progress towards addressing the hypothesis that disrupting certain ERR±-co-activator interactions with small molecular weight compounds will decrease the transcriptional activation of ERR± target genes. We have identified a truncated form of ERR± that is constitutively active, and we have identified the p160-family member, glucocorticoid receptor interacting protein 1 (GRIP1/SRC-2/TIF2/NcoA-2), as a specific co-activator of ERR±-dependent, ERE-regulated transcription in MCF-7 mammary carcinoma cells. We have developed a primary, cell-based high-throughput screen (HTS) to search for potential inhibitors of GRIP1-mediated activation of truncated ERR±. We have carried out an initial HTS of 9,400 compounds and have identified 34 hits that inhibit luciferase expression by greater than 75% and are not generally toxic to cells.

We are developing a secondary HTS using protein fragment complementation assays (PCAs), which measure protein-protein interactions in vivo. We also plan to optimize our counter screens to help us further characterize hits and discard false-positive hits. Lastly, we will test our most promising hits in a variety of biological assays, such as cytotoxicity, cell proliferation, cell migration, anchorage-independence, and transcriptional profiling. We hope that these compounds will lead to the development of a novel breast cancer therapeutic and help patients with aggressive breast cancers that grow independently of hormone and/or are resistant to currently available therapies.

February 25, 2008
Kelly Christopherson
Czajkowski Lab

Stoichiometry and Alcohol Sensitivity of alpha4-beta2-delta GABA(A) Receptors

The gamma-aminobutyric acid type A, GABA (A), receptor mediates the majority of fast synaptic inhibition in the brain and is a target of many depressants, such as the benzodiazipines, barbiturates, and alcohol.The most prevalent receptor subtype contains alpha, beta, and gamma subunits. However, GABA (A) receptors containing the delta subunit, instead of gamma, display a unique sensitivity to ethanol and may play a key role in mediating the behavioral effects of alcohol at physiologically relevant doses. Evidence also indicates that chronic use of alcohol may cause a rearrangment of receptor subtype levels in the brain, which may ultimately result in increased substance dependence and altered receptor pharmacology.

Alcoholism and alcohol abuse are serious problems that have severe repercussions on physical and mental health as well as family and lifestyle. 13.8 million American adults have problems with drinking, 8.1 million of which are alcoholic. Chronic alcohol abuse causes major damage to the brain as well as other symptoms requiring extensive medical care that in total costs society billions of dollars each year.

Alpha-beta-delta receptors have several important and unique characteristics, including extrasynaptic positioning and modulation by neuroactive steroids and alcohol, making them interesting proteins to study. Furthermore, recent research supports the hypothesis that these receptors may, in fact, be responsible for mediating the physiological effects of moderately intoxicating doses of alcohol in the brain, albeit with some controversy as to the exact subunit composition and concentrations of alcohol required to elicit this effect. Unfortunately, alpha-beta-delta receptors are not as well studied as their alpha-beta-gamma counterparts, and fundamental properties, such as the stoichiometry of these receptors, is unknown.

My research projects aim to characterize delta-containing receptors by determining their subunit stoichiometry, using alpha4-beta2-delta receptors as a model. In addition, I will conduct experiments to examine and confirm the responsiveness of these receptors to low doses of alcohol. In addition, future experiments will aim to map the putative high affinity alcohol binding site in these receptors. The success of this project will provide a better understanding of how GABA(A) receptors mediate the effects of alcohol in the brain and will provide insight that will aid in the development of novel pharmacotherapeutics for the treatment of many symptoms of chronic alcohol abuse.

March 3, 2008
Michelle Perry
Berridge Lab

Cholinergic Modulation of Feeding Behavior and Motivation

Feeding is a complex behavior often considered to have two behaviorally distinct phases, anticipatory/preparatory and consummatory. In the anticipatory/preparatory phase, animals engage in active and flexible goal seeking behaviors to acquire food. Examples of such behaviors are rearing, ambulation, investigatory sniffing, and food hoarding. The consummatory phase begins when the animal interacts with the food. At this point one observes more stereotyped, fixed patterns of behavior such as chewing, swallowing, and licking.

The nucleus accumbens (Acb) is a brain region involved in modulating rewarding behaviors, such as feeding. It has been shown that multiple neurotransmitters located within this region are important for, yet play dissociable roles in, the control of feeding behavior. Literature suggests that the dopamine system is associated with the anticipatory/preparatory phase of feeding while the Acb opioid system appears to regulate the pleasurable or rewarding properties of food during the consummatory phase. A third Acb neurotransmitter system that appears to play a role in the modulation of feeding behavior is the acetylcholine system. Previous work from the laboratory demonstrated that one intra-Acb infusion of the muscarinic antagonist scopolamine decreased feeding up to 24 hours post infusion. It was also shown that the cholinergic system may interact or modulate the Acb opioid system as opioid-induced feeding was reduced with pretreatment with scopolamine.

The present work elaborates on the specific effects muscarinic blockade has on feeding behavior. The two phases of feeding will be thoroughly examined, in addition to exploring pharmacological, anatomical and ingestive specificity of drug treatment. We are specifically interested in identifying the precise behavioral mechanism underlying the reduction in feeding observed with scopolamine treatment. For example, do animals feel 'fuller', do they find eating less pleasurable, or are they over-activated by the drug? Our studies will attempt to address these questions.

March 3, 2008
Chao Qi
Ned Kalin Lab

Developing a Rodent Model of Extreme Behavioral Inhibition

Behavioral inhibition (BI) is an adaptive defensive response to threat that has been evolutionarily conserved across species due to its important role in survival. However, when extreme, BI is associated with dysregulated anxiety-related responses and social dysfunction. In human studies, children with extreme BI have been shown to have prolonged and overly intense inhibitory responses when they are exposed to novel or unfamiliar situations. Extremely inhibited children have increased psychological distress and are at greater risk to develop social anxiety disorder. Because other anxiety disorders, depression and alcohol abuse are highly comorbid with social anxiety disorder, it is likely that extreme BI is a broad risk factor for the development of anxiety-related psychopathology.

The Kalin laboratory has put considerable effort into developing a rodent psychological stress model that uses the threat of a natural predator such as a ferret to induce extreme BI. This significant behavioral alteration has also been shown to be associated with changes in physiological traits important in mediating stress responses, such as increased levels of plasma corticosterone and increased Fos-immunoreactivity in the amygdala. Human and nonhuman primate studies have confirmed involvement of a brain circuit that includes the amygdala in mediating threat-induced freezing and suggests that over activity of the amygdala is associated with extreme BI. Our goal is to further characterize the rodent model of BI in hopes that it can be used to elucidate the molecular mechanisms underlying this response. Longer term outcomes of the proposed studies could lead to new molecular drug targets for anxiety and depressive disorders.

March 10, 2008
Jessica Townsend
Jeff Johnson Lab

Nrf2-ARE mediated neuroprotection is not dependent on increasing glutathione levels in primary cortical neuronal cultures

Many neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's Diseases are believed to be partially induced by decreased levels of the antioxidant glutathione (GSH) in given areas of the brain leading to intraneuronal accumulation of reactive oxygen species. Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor associated with the induction of many genes essential for detoxification and maintenance of a cells reducing potential including those involved in glutathione synthesis- glutamate cysteine ligase catalytic and modulatory (GCLM) subunits. Nrf2 achieves this through the binding and activation of the cis-acting regulatory element known as the antioxidant response element (ARE). In previous work it was shown that overexpression of Nrf2 significantly increases total intracellular GSH as well as secreted levels of GSH. Data also suggest that pretreatment with buthionine sulfoximine, a chemical inhibitor of GSH biosynthesis, reverses Nrf2-mediated neuronal protection from oxidative stress in primary cortical cultures. This concept that GSH is not only sufficient but essential for Nrf2-ARE neuroprotection was tested using primary cultures derived from GCLM knockout mice. GSH levels were not increased in the GCLM -/- cultures when treated with tBHQ, an activator of the Nrf2-ARE pathway known to increase GSH and protect neurons from toxicity. Interestingly, the neuroprotective effects of tBHQ treatment were also observed in GCLM -/- cultures implying an increase in GSH is not solely responsible for the protection of neurons from oxidative stress. Microarray analysis of GCLM -/- cultures demonstrate that the expression of numerous ARE-driven genes were increased in response to tBHQ treatment suggesting a cooperative effort among those changed genes is responsible for making neurons resistant to oxidative stress. Future studies will be necessary in order to elucidate the components required for Nrf2-ARE mediated protection.

March 10, 2008
Uyen Chu
Ruoho Lab

Biochemical and Functional Characterization of the Sigma-1 Receptor

Sigma receptors belong to a unique class of receptors that are found in the central nervous system. Because of their high affinity for common neuroleptics such as cocaine, methamphetamine, and haloperidol, sigma receptors have been associated with central nervous system conditions including schizophrenia, depression, and drug addition. Although the endogenous ligand of the sigma-1 receptor has yet to be identified and its exact function in the cell is unclear, the sigma-1 receptor has been implicated in diverse cellular processes. Among the many functions associated with the sigma-1 receptor are potentiation of calcium release from the ER, modulation of the voltage regulated ion channels, and the recently identified chaperone activity in a complex with BiP/GRP78. Our lab has found that sphingosine and other sphingolipids bind to the sigma-1 receptor suggesting its role in signaling cascade(s) regulated by these lipids. Using chemical synthesis, competitive displacement binding assays against [3H]-(+)-pentazocine, and photoaffinity labeling, the first aim of my work focuses on studying biochemical properties of the sigma-1 receptor interaction with compounds structurally similar to sphingosine, including phenylpropyl- and nitrophenylpropyl-N-alkylamines.

The sigma-1 receptor protein levels are overexpressed in many tumor cells including those from the breast, lung, and the CNS. Sigma-1 receptor ligands displayed anti-tumorigenic activities in these cell lines by activating the apoptotic pathway as measured by caspase activation. Data from cytotoxicity assays indicate that the N-alkylamine compounds which have been synthesized as sigma-1 receptor ligands also inhibit tumor cell growth. The mechanisms that result in cell death induced by sigma-1 receptor ligands are unclear but may involve hypoxia-inducible factor 1Éø, a transcription factor responsible for upregulating a multitude of proteins involved in angiogenesis, anaerobic glycolysis, cell proliferation, and apoptosis. Interestingly, very recent data from our lab showed that the sigma-1 receptor knockout mouse displayed higher HIF-1Éø protein levels in the liver as compared to WT. The mechanism of HIF-1 Éø activation in the absence of the sigma-1 receptor suggests a role for this recepto in mediating hypoxic response. In the second aim, I will provide preliminary data and a hypothesis that may link the sigma-1 receptor to the HIF-1Éø pathway.

March 24, 2008
Shihu Sun
Chapman Lab

Translocation of Botulinum Neurotoxin B

Botulinum neurotoxins (BoNTs) are bacterial toxins produced by Clostridium botulinum. They are synthesized as high molecular proteins consisting of two domains: the heavy chain and the light chain. At the motor nerve terminals, BoNTs cause loss control of muscles by preventing neurotransmitter release from presynaptic neurons, and are considered as the most toxic proteins for human beings. Besides their potential use as biological weapons, the toxins also have emerged as a medical tool to treat various neuromuscular diseases and neurological pains. The broad applications of the toxins require elucidating how they function in cells. There are three major steps in BoNT action at nerve terminals: receptor-mediated endocytosis, translocation of BoNT light chains from endosome to cytosol, and cleavage of proteins involved in vesicle exocytosis by BoNT light chains. Previous research in our lab has discovered receptors for BoNT/A and BoNT/B, which helps to understand the endocytosis process. Other studies in the field identified targets of enzymatic cleavage. Detailed mechanism of translocation, however, remains unknown.

My research aims to address the translocation of BoNTs using BoNT/B as a model, and the preliminary research focuses on studying the role of receptors during translocation, structural changes during translocation, and the driving force for translocation. My research shows that unlike many ligands dissociating from their receptors in low pH endosomes, BoNT/B still binds to its receptors at low pH, indicating a potential role of receptors during toxin translocation. Besides, the low pH triggers a structural change in heavy chain that may favor the channel formation for light chain translocation. Previous studies imply that pH is the driving force of translocation; my research, however, shows that besides pH, the electric potential across the membrane may also be required for translocation. Future study will try to elucidate questions like the actual structure of BoNT/B during translocation, thus providing a detailed model for BoNT translocation.

March 24, 2008
Jessica Heck
Anderson Lab

Characterization of a Novel Set of Interactions: Type I PIP Kinases and Beta-Catenin

E-cadherin-based cell-cell contacts are specialized adhesive structures between adjacent cells required for epithelial cell polarization, tissue integrity, and coordinated cell movements. In epithelial cells, E-cadherin is the core of cell-cell contacts and functions via extracellular interactions with cadherin molecules on adjacent cells and intracellular interactions with a protein complex which includes beta-catenin. In addition to its role in cell adhesion beta-catenin also functions downstream of canonical Wnt signaling to regulate the expression of target genes implicated in cell proliferation and tumor progression. Data from our lab demonstrates that the phosphatidylinositol phosphate kinase type I gamma (PIPKIgamma) directly interacts with E-cadherin and regulates the assembly of E-cadherin-based cell-cell contacts. However new data demonstrates this goes beyond E-cadherin as multiple type I PIP kinase isoforms also directly interact with beta-catenin, independent of E-cadherin. Based on the diverse signaling roles of beta catenin in both cell adhesion and downstream of canonical Wnt signaling, we hypothesize that the PIP kinases are a unique set of regulatory molecules for beta-catenin function. In aim I, the interactions between the PIP kinases and beta-catenin will be characterized and the importance of these interactions in the presence of E-cadherin and in relation to epithelial cell polarization will be assessed. In aim II, we will determine if/how the PIP kinases are able to regulate beta-catenin function in the absence of E-cadherin.

March 31, 2008
Lindsay M. Hill
Bertics Lab

Extracellular nucleotides are present in high concentrations at sites of inflammation, cell lysis and platelet degranulation. This extracellular ATP can act as a ligand for nucleotide receptor P2X7 found on leukocytes such as the macrophage. Activation of P2X7 is involved in LPS-mediated macrophage action. Recently, our lab found that stimulation of P2X7 in LPS-primed macrophages results in a synergistic reactive oxygen species (ROS) production. ROS production is an essential component of macrophage immune function, maintaining roles in bacterial cytotoxicity and macrophage signaling. In my studies I have tested the hypothesis that the capacity of P2X7 to synergize with LPS entails the regulation of the ROS producing enzyme NADPH oxidase. Enhanced ROS production was found in LPS-primed murine macrophages and human primary blood monocytes after P2X7 stimulation but not in P2X7-deffective cells. This enhanced ROS production was attenuated by a NADPH oxidase inhibitor. Additionally, these treatment conditions resulted in enhanced phosphorylation of a NADPH oxidase component. These data support a model wherein P2X7 signaling is critical for the modulation of the macrophage immune response. Additionally, I have found that treatment of macrophages with exogenous ceramide, a sphingolipid that possesses similar structure and signaling pathways to LPS, is able to potentiate P2X7 receptor MAP kinase phosphorylation in a ROS dependent manner, as well as potentiate the expression of a component of the AP-1 transcription factor FosB. Therefore, exogenous lipids, like LPS, are able to potentiate several P2X7 receptor-stimulated signaling pathways, and future studies will entail testing the hypothesis that changes in endogenous ceramide concentration will modulate P2X7 receptor function.

March 31, 2008
Stephanie L. Maiden
Hardin Lab

The Role of HMP-1/a-catenin in Regulating Cell Adhesion in the C. elegans Embryo

April 7, 2008
Corinne R. Vokoun
Basso and Jackson Lab

The basal ganglia (BG) are believed to be involved in the processing that precedes movement initiation. A disruption of the delicate balance between excitation and inhibition signals of the BG can account for a diverse range of clinical symptoms apparent in BG disorders such as Parkinson's disease (PD), Huntington's disease (HD), and possibly psychiatric illness involving the BG such as schizophrenia and obsessive compulsive disorder. Because the BG have only two output nuclei, understanding the influence of BG output on target structures is critical to understanding the physiology of the BG.

One of the three main targets of the BG is the midbrain superior colliculus (SC). The SC plays a critical role in transforming sensory information into movement commands to orient the eyes, head and body. Patients of BG disorders including PD, HD and Tourette syndrome often exhibit clinical deficits in oculomotor function. However, membrane properties of individual neurons determined from in vitro work are inadequate to account for the behaviorally relevant neuronal properties. Thus, to understand the fundamental mechanisms of disease, it is necessary to study the biophysical properties of the entire neuronal circuit. For these reasons, the SC and its well characterized role in eye movements serves as an excellent model for study of the role of BG output in disease. Voltage imaging offers a unique opportunity to investigate synaptic function of the SC in vitro, thus bridging the gap between behavioral and physiological studies.

My central hypothesis is that the pattern of activity across the SC arising from electrical stimulation of the SC can be altered by decreased inhibition of the BG. Electrophysiology experiments are performed on slices of brain tissue through the rat midbrain. Stimulation of different layers of the SC is be used to distinguish patterns of response within the SC. In the future, I will extend these experiments to determine whether the response patterns are altered in a rodent model of PD. In addition, stimulation of the substantia nigra pars reticulata, an output nucleus of the BG, will be tested to cause increased SC inhibition.

The social and economic burden of movement and psychiatric disorders of the BG in the United States is overwhelming, and continues to grow. Current treatment options offer inconsistent and unpredictable outcomes. Therefore, in order to design more effective medical therapies, it is crucial to have a more complete understanding of BG function. These experiments are designed to move us closer to that goal.

April 7, 2008
Ginny Powers
Alarid Lab

Contribution of Chymotryptic Activity of the 26S Proteasome to Estrogen Receptor Éø Signaling

Breast cancer is the most commonly diagnosed cancer in women and the majority of these tumors express estrogen receptor Éø (ERÉø). ERÉø is the most significant biomarker for breast tumors and dictates both outcome and treatment. Standard therapy of ERÉø positive breast tumors works through the inhibition of ERÉø. The 26S proteasome is a target for anti-cancer therapy and ERÉø is a substrate for the proteasome. While the proteasome possesses multiple enzymatic activities, the chymotryptic site is the focus of anti-tumor compounds. We asked if the chymotryptic activity of the 26S proteasome is required for ERÉø signaling. To assess the role of the chymotryptic site in ERÉø signaling, Bortezomib, a chymotryptic specific inhibitor, and MG132, a broad-spectrum proteasome inhibitor, were compared. The IC50 of Bortezomib was determined in cell lines representative of the different subtypes of breast cancer luminal A (MCF-7 and T47D), luminal B (BT474) and basal type (MDA-MB-231) cancers as well as in two ERÉø positive non-breast cancer cell lines. The IC50 in MCF-7 cells is 7 nM and 250 nM for Bortezomib and MG132, respectively. Bortezomib has equal efficacy and greater potency for the inhibition of the chymotryptic activity of the proteasome. MCF-7 cells were treated with17-É¿-estradiol (E2) to signal degradation of ERÉø, and receptor protein levels were assessed by Western blots. When treated with Bortezomib and E2, ERÉø protein is degraded in spite of chymotryptic inhibition. MG132 prevents the E2-induced degradation of ERÉø. Both Bortezomib and MG132 resulted in the induction of p53 protein. The effect of Bortezomib is not cell-type specific or dependent on the localization of ERÉø. Interestingly, chronic Bortezomib treatment of MCF-7 cells in combination with either vehicle or E2 depletes ERÉø protein beyond what is observed with E2-dependent proteolysis. This depletion of ERÉø protein is due to a decrease in transcription of ERÉø. Similar effects were observed on the mRNA levels of ERÉø target gene Progesterone Recepton. In conclusion, the chymotryptic activity of the 26S proteasome is not required for E2-induced degradation of ERÉø but is required for ERÉø mediated transcription at long time points. The implications of these findings suggest that inhibitors of other activities of the proteasome may be more efficacious at inhibiting E2-induced ERÉø degradation.

April 14, 2008
Andrew Hedman
Anderson Lab

Investigating a Novel Interaction Between a  PIPKIγ and SNX5, with Potential Roles in Trafficking Pathways

April 14, 2007
Anthony Trinh
Tibbets Lab

The DNA damage response (DDR) is a tightly regulated cellular process that responds to a vast array of stimuli including DNA lesions and genomic stress through the activation of an array of sensors, adaptors, and effectors. In response to these assaults on DNA, the Phosphoinositide3-kinase related protein kinases, ATM (Ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related), become activated triggering a cascade of events leading to cell cycle arrest, DNA repair, apoptosis, and changes in gene expression. Previous work in our lab identified a potentially important link between the ATM-regulated DNA damage response and cAMP response element binding protein (CREB) CREB is a transcription factor that has been widely implicated in a plethora of cellular and systemic responses regulating a wide range of genes important for metabolism, neuronal development, memory formation, circadian rhythm entrainment, and inflammation. Ionizing irradiation induces ATM-dependent phosphorylation of CREB on serine 111, which creates a cluster of consensus phosphorylation sites for casein kinase 1 and 2 (CKI and CKII). CKI and CKII then phosphorylate CREB on serine 108, serine 114, and serine 117, which renders CREB competent for subsequent ATM-dependent phosphorylation on serine 121. Phosphorylation of CREB by ATM reduces its affinity for the transcriptional co-activator CREB binding protein (CBP). Although preliminary evidence suggests ATM regulates CREB dependent gene expression, this has yet to be experimentally validated. In addition, the physiological relevance of CREB phosphorylation by ATM is unclear.

Here, I propose a genetic approach to study the biological consequence of ATM-dependent CREB phosphorylation in mice. I am creating a knock-in mouse strain encoding a mutant CREB protein containing a point mutation in which serine 111 is mutated to alanine that ablates DNA damage-induced CREB phosphorylation . In doing so I hope to create a physiologically relevant system to study the possible roles ATM and CREB in tumorgenesis, hematopoesis, neuron development, and metabolism and how deregulation of this pathway could lead to disease.

April 21, 2008
Brian Hoffmann
Mosher Lab

Transglutaminase Cross-linking in the ECM and Blood Stream

Transglutaminase enzymes are a family of Ca2+-dependent zymogens found in blood, as well as in a variety of tissues, and are known to catalyze the formation of ε-amino(γ-glutamyl)-lysine isopeptide bonds between a variety of interacting proteins.  These protein-protein cross-linking interactions have been shown to have implications in thrombosis, wound healing, extracellular matrix formation, neurodegenerative diseases, and bacterial infection among other processes.  This research focuses on the complex formation between plasma fibronectin (pFN) and fibrin during blood coagulation.  The pFN-fibrin interaction is stabilized by thrombin-activated Factor XIII (FXIIIa), a member of the transglutaminase family.  This research uses a technique called PARTNER (Proteins Aligned so as to be Reactive to Transglutaminase and Nicked to Enable Recognition) that includes a combination of cross-linking, limited proteolytic digestion, immuno-purification, and mass spectrometry to investigate the exact sites of cross-linking between pFN-fibrin.  This process has given insights into not only the covalent cross-linking interaction between pFN-fibrin, but has also provided insights into other requirements needed for the covalent protein-protein interaction to occur.  Additionally, there will be a brief introduction to other FXIIIa-mediated cross-linking interactions of the pFN N-terminus, such as to cell receptor complexes for signaling processes and to bacterial surface proteins aiding in bacterial infection.

April 21, 2008
Lisa Maurer
Mosher Lab

Platelets adhere to and assemble fibronectin on the N-terminal 70 K region of fibronectin

Thrombotic events leading to coronary artery disease and stroke are a leading cause of mortality.  Recently, it’s been shown that fibronectin (FN), a large multi-modular glycoprotein, is assembled by platelets and contributes to thrombus growth and stability.  The N-terminal 70 K region of FN (70 K FN) binds to platelets is thought to mediate the initial interaction between FN and platelets during FN assembly.  We extend these results to show that platelets also adhere to and assemble FN on 70 K FN.  We hypothesize that the determinants by which 70 K FN acts as a substrate for platelet adhesion are similar to the determinants by which the 70 K region of exogenous FN plays a critical role in platelet FN assembly.  Thus, by dissecting platelet adhesion and FN assembly on 70 K FN, we hope to gain insights into FN assembly by platelets and likewise into thrombus formation.

April 28, 2008
Neil Daily
Martin Lab

CAPS Primes Membrane Fusion through Interactions with the SNARE Protein Core Motif

CAPS (Ca2+ -dependent activator protein for secretion) is a novel 1289 amino acid protein that was discovered by its activity in reconstituting Ca2+-dependent dense-core vesicle exocytosis in permeable neuroendocrine cells.  CAPS is expressed in neural and peptide hormone-secreting endocrine cells and has an essential role in pre-fusion events by conferring competence on docked dense-core vesicles for Ca2+ -triggered fusion.  Vesicle fusion with the plasma membrane is mediated through SNARE protein interactions.  Syntaxin is an essential SNARE protein resident on the plasma membrane that is part of the core machinery (which includes VAMP2 and SNAP25) needed for membrane fusion in exocytosis.  Recent studies have shown that CAPS promotes fusion of liposomes containing VAMP2 with those containing syntaxin and SNAP25 in an in vitro reconstituted liposome fusion assay.  Gradient liposome flotation assays showed that CAPS binds to liposomes containing syntaxin/SNAP25, syntaxin or VAMP2, but not to liposomes lacking protein.  Systematic binding studies with syntaxin-containing liposomes showed that CAPS exhibits a KD ~ 220 nM and stoichiometric binding.  Further studies indicated that CAPS binds to a truncated syntaxin that lacks its amino terminal Habc domain but preserves the membrane-proximal H3 SNARE motif, the motif responsible for the formation of the four-helix SNARE complex which allows membrane fusion to occur.  CAPS was able to promote fusion in the in vitro liposome fusion assay without the N-terminal Habc domain of syntaxin suggesting that CAPS acts on the core motif of SNARE proteins.  These studies indicate that CAPS exhibits high affinity stoichiometric interactions with syntaxin, an essential core component of the membrane fusion machinery and that this interaction may play a key role in CAPS function in dense-core vesicle exocytosis by promoting localization and assembly of SNARE complexes required for fusion.  They also emphasize the need to study transmembrane proteins, especially those involved in membrane fusion and exocytotic pathways, in their native environment in order to accurately analyze their function.  Recent reports have shown that the MUN domain of Munc-13, the synaptic vesicle priming factor which has similar sequence homology to CAPS, is able to bind to syntaxin/SNAP25 liposomes as well.  Further studies may confirm a conserved function in vesicle membrane fusion which involves the CAPS Munc-homology domain and the Munc-13 MUN domain with SNARE proteins that is required for rapid Ca2+-triggered vesicle exocytosis.