Seminar Series

The Eph receptor tyrosine kinases (RTKs) and their ligands, the ephrins, have captured the attention of many researchers as they play a wide variety of roles in development, including axon guidance, synaptic transmission, blood vessel formation and the regulation of cancer. C. elegans mutants that are defective in Eph RTK signaling have a variety of developmental defects including, morphogenesis defects, axon guidance defects, and germline defects. I will present some of our recent research on the molecular, biochemical and genetic approaches we use to investigate Eph RTK signaling in C. elegans. We have identified some key players that function with Eph RTK signaling during development. These genes encode a Robo receptor, the adaptor molecule NCK-1, and the ortholog of the human tumor suppressor PTEN. The Eph receptors are promising therapeutic targets, because of their roles in cancer, neurodegenerative and cardiovascular diseases. However, before drugs are developed we must understand how these molecules signal during development. Using C. elegans to identify genes that function with Eph RTK signaling will provide insight on how Eph RTK signaling functions in more complex organisms including humans.

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My research program focuses on unraveling the basic molecular mechanisms involved in the regulation of G protein-coupled receptor (GPCR) activity. My laboratory utilizes a combination of molecular pharmacological and cell biological techniques to examine the role of small G proteins, GRKs, b-arrestins and a wide variety of other receptor interacting proteins in the signaling, endocytosis and intracellular trafficking of GPCRs. The purpose of these studies is gain a better understanding of differences and similarities in the molecular mechanisms involved in signaling, desensitization and resensitization of distinct GPCRs. Consequently, our efforts are currently focus on four model GPCRs: metabotropic glutamate (mGluRs), 5-HT corticotropin releasing factor (CRF) receptors in the central nervous system and thes angiotensin II type 1A receptor (AT1AR) in the cardiovascular system. Our understanding of the complicated array of GPCR regulatory mechanisms has evolved rapidly as the consequence of our studies of protein expression in heterologous cell culture systems. Although these systems are extremely important for the description and characterization of novel signal transduction mechanisms and protein-protein interactions, the relative physiological consequence of these pathways and interactions is certain to depend upon cellular context and environment. Consequently, we have expanded our studies to primary neuronal cell culture systems and have begun the development of transgenic and knockout mouse models to study receptor regulation in vivo.

Michelle Chan - Stability assessment of a microanalytical system (i-STAT cartridge) at room temperature

Jennifer Bo Wang - Establishing a system for in vivo studies of reverse transcription initiation

Benjamin Yeung - Using glycan array screening to gain insight into binding specificity of Clostridium perfringens glycoside hydrolases 

The posters will be on display in the Craine Room, Botterell Hall Rm. 660.

Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. In previous work, we demonstrated that XMAP215 is a microtubule polymerase that executes multiple rounds of tubulin subunit addition. The most conserved features of the XMAP215 family are the N-terminal TOG domains; XMAP215 has five TOG domains in series. An individual TOG domain has been shown to bind tubulin, although the mechanism by which these domains function in microtubule polymerization remains poorly understood. In order to determine which domains of XMAP215 contribute to its polymerase activity, we generated a range of constructs in which the ability of each TOG domain to bind tubulin was abolished by alanine-substitution, as well as deletion constructs lacking key domains. Surprisingly, only TOG1 + TOG2 are necessary for polymerase activity, although a minimal construct of TOG1 + TOG2 alone is not sufficient to promote growth. Further, we show that the C-terminus of XMAP215 is not involved in the polymerase reaction in vitro and that XMAP215’s lattice diffusion arises from a basic helix between TOG4 and TOG5. Our results demonstrate that XMAP215 requires the tubulin-binding ability of the TOG domains to act as a processive polymerase.

Structure-Function Studies on the  Novel Alpha Kinase Family

Functional and Structural Characterization of a Putative HeterodimericKinesin

Implementing the Bimolecular Fluorescence Complementation Assay to Study Protein Interactions in the Cell Cycle Checkpoint Response

Self-assembly of monomeric units into organized polymers are currently the subject of much attention, particularly in the areas of nanotechnology and biomaterials. One biological example of a protein polymer with a capacity for self-organization is elastin. Elastin is an insoluble, polymeric, extracellular matrix protein that provides tissues, including large arteries, lungs and skin, with the physiologically important properties of extensibility and elastic recoil. Using recombinant elastin-like polypeptides that mimic both self-assembly and elastomeric properties of elastin, our laboratory is working to understand how sequence/structure and domain arrangements in elastin determine its ability to self-assemble into organized matrices and lead to the unusual elastomeric properties of these polymers.

Dr. Greenbaum's laboratory is developing and exploiting new technologies at the interface between biology and chemistry to study protease function. The lab uses a variety of techniques including the synthesis of small molecule inhibitors, quantitative proteomics, recombinant protein expression and molecular genetics in order to better understand proteolytic systems. Although these tools are useful to study any biological system, the laboratory is concentrating on understanding host and parasite proteases that are important for P.falciparum, the causative agent of malaria.

Malaria is a devastating global disease causing at least 500 million clinical cases and more than 1 million deaths each year. Currently quinolines and anti-folates are the most commonly used drugs for disease prevention and treatment. However, multi-drug resistant Plasmodium falciparum has become a major problem. Therefore, discovery and investigation of known and/or novel targets for anti-malarial compounds is essential to develop new ways to combat this disease.

Dr. Greenbaum has developed universal chemical-based proteomics tools to functionally analyze the role of proteases in a variety of biological systems. He also adapted these chemical tools to allow screening of small molecule libraries for specific inhibitors and drug design. His lab will continue to develop new chemical proteomic tools and small molecule libraries to facilitate protease drug target discovery, characterization and therapeutic design with a particular interest in malaria.

The chemical probes that Dr. Greenbaum is developing led to the recent discovery that parasites such as plasmodium and toxoplasma hijack host calpains in order to facilitate their escape (Chandramonanadas et al, Science 324:794-7; 2009). This surprising finding suggests the concept of targeting host pathways for chemotherapy, a strategy that might limit the emergence of drug-resistance.

Our understanding of the central role of the actin protein in a myriad of physiological processes has been hampered by the inability to produce and study a wide range of important mutants. Those mutations that result in loss-of-function have been impossible to produce and there have been limitations on the kind of actin isoforms that could be studied.
We have developed the baculovirus expression system for the production of any actin isoform or mutant actin protein desired, opening the doors to a treasure trove of biochemical, biophysical and structural studies centered on actin. In this seminar, I will present our work with actin mutants, explaining the different systems that have been used and how the baculovirus system works for actin expression. I will touch on two elements of the research in our lab that employ actin mutants: the development of short F-actin complexes for structural work, and the study of human cardiac actin mutations related to the development of disease.

Rhinovirus, responsible for majority of the common colds, promotes secondary bacterial infection. However, the underlying mechanisms are not well understood. We found that rhinovirus induces generation of reactive oxygen species by stimulating expression and activity of NADPH oxidase 1, a homologue of gp97phox in infected airway epithelial cells. This in turn disrupts epithelial barrier function and facilitates transcellular migration and possibly dissemination of bacteria. We speculate that, this is one of the mechanisms by which rhinovirus promotes secondary bacterial infection.

 Src and Src-activatable Stat3 are two important oncogenes implicated often with invasive/metastatic progression of various cancers. On the other hand, two legendary tumor suppressors, p53 and p53-inducible PTEN have been shown to act against the evolution of such behavior in cancer cells. However, very little is known about the roles of these signaling giants in regulation of cytoskeletal events leading to the migratory and invasive phenotypes. We have studied the novel effects of Stat3/p53/PTEN in modulating the Src-induced invasive podosome/invadopodial structures. Our findings provide new evidence for the existence of complex interplays between the Src-Stat3 and p53-PTEN axes, and have demonstrated that mutual antagonism between these two opposing forces plays a critical role in determining the outcome of Src-induced invasive phenotypes.

The Eph receptor tyrosine kinases (RTKs) and their ligands, the ephrins, have captured the attention of many researchers as they play a wide variety of roles in development, including axon guidance, synaptic transmission, blood vessel formation and the regulation of cancer. C. elegans mutants that are defective in Eph RTK signaling have a variety of developmental defects including, morphogenesis defects, axon guidance defects, and germline defects. I will present some of our recent research on the molecular, biochemical and genetic approaches we use to investigate Eph RTK signaling in C. elegans. We have identified some key players that function with Eph RTK signaling during development. These genes encode a Robo receptor, the adaptor molecule NCK-1, and the ortholog of the human tumor suppressor PTEN. The Eph receptors are promising therapeutic targets, because of their roles in cancer, neurodegenerative and cardiovascular diseases. However, before drugs are developed we must understand how these molecules signal during development. Using C. elegans to identify genes that function with Eph RTK signaling will provide insight on how Eph RTK signaling functions in more complex organisms including humans.

Candida albicans is the most commonly isolated human fungal pathogen, causing both debilitating mucosal infections as well as life- threatening systemic infections. Until recently, it was thought that C. albicans was an asexual yeast, with no potential for sexual mating. We now know that C. albicans has a highly elaborate mating cycle, however, that has presumably evolved to help it survive and adapt to growth in the mammalian host.

The serpins form a widely distributed family of chiefly extracellular protease inhibitors. Their “suicide” inhibitory mechanism involves a drastic conformational change that translocates cognate proteases from one pole of the serpin to the other, trapping them in a covalent inhibitory complex. My laboratory has focused on plasma serpins that inhibit thrombin, the effector protease of coagulation, such as antithrombin (AT) and heparin cofactor II (HCII). Another serpin, a1-proteinase inhibitor (a1-PI), ordinarily inhibits neutrophil elastase, but becomes a reasonable thrombin and activated protein C (APC) inhibitor when mutated in its reactive centre loop (M358R). My laboratory and others have used this mutant as a starting point for further alterations aimed at maximizing thrombin inhibition and minimizing anti-APC activity. The seminar will review the results from our published mutagenesis strategies, both inside and outside the reactive centre loop, as well as more recent work in which we have examined the binding of a serpin domain to a thrombin exosite by surface plasmon resonance, and converted several serpins into integral membrane proteins, with variable retention of function.

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