Intrinsic and Extrinsic Modulators of Protein Stability (pilot)

San Francisco State University, San Francisco CA

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Abstract

Treatment of patients suffering from aggressive solid-tumor cancers, which are generally hypoxia tolerant and glycolytic, will be improved by an understanding of how cellular and molecular components critical to the glycolytic phenotype are maintained. Our long-term research goal is to determine how changes in gene sequence, gene expression, post-translational modification, and protein-protein interactions alter cellular and organismal phenotypes to ultimately confer selective advantages. Hypoxia inducible factor 1a (HIF-1a) is considered a master regulator of gene expression that controls hypoxia tolerance, and lactate dehydrogenase (LDH) is the terminal enzyme in glycolysis that permits ATP generation via glycolysis to proceed at the high rate necessary for rapid cell division characteristic of aggressive tumors. During glycolysis, increasing concentrations of lactic acid create an intracellular acidosis, and thus LDH molecules must possess appropriate structural stability in order to maintain their functionality. Although HIF-1a has been the subject of intense study during the past few years, we lack a complete understanding of how the complex suite of proteins from genes activated by HIF-1a may act to enhance hypoxia tolerance, and specifically how these proteins maintain or enhance the glycolytic phenotype by stabilization of key enzymes, such as LDH, during acidosis. Our objectives in this application are to identify variation in primary structure and protein-protein interactions that correlate with increased stability of LDH, and to examine how variation in gene expression during physiological hypoxia is related to stabilization of LDH. Our central hypothesis is that variation in primary structure and in protein-protein interactions that increase LDH stability allow these proteins to function normally during periods of hypoxia-induced acidosis. We will test this central hypothesis in three specific aims: (1) Identify variations in primary structure among LDHs that differ in structural stability using RT-PCR, (2) Identify the specific proteins that interact with LDH to increase its stability using co-immunoprecipitation and peptide mass fingerprinting, and (3) Identify genes that are up-regulated in response to hypoxia using cDNA microarrays. Our rationale for these studies is that if we understand how to block expression of stable LDH isoforms and/or block protein-protein interactions involved in stabilization of LDH we may be able to develop novel therapeutic agents to treat aggressive cancers by disrupting the glycolytic phenotype. Relevance to Public Health: Our ability to treat aggressive cancers will be aided by an understanding of how hypoxia tolerance and the glycolytic phenotype are maintained. These studies are innovative in that the identification of specific protein-protein interactions that confer stabilization of the glycolytic phenotype may lead to novel targets for anti-cancer therapies. Thus this research has a direct relevance to public health, in that the results of the work will have potential for development of novel chemotherapeutic agents.