Analysis of Proteins that Modulate Apoptosis

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DESCRIPTION (provided by applicant): Apoptosis is the morphologically defined and evolutionary conserved process by which most programmed cell deaths occur in metazoans. Cell death by apoptosis is essential to the development and normal physiology of metazoans. Deregulation of the tightly controlled apoptotic program contributes to the pathogenesis of human diseases such as cancer, neurodegenerative and autoimmune diseases, stroke, and cardiac arrest damage. The Bcl-2 protein is a powerful repressor of apoptotic cell death and, despite intense study, many questions remain regarding its molecular mechanisms of action and regulation. To gain further insight into the function and modulation of Bcl-2, a search of Bcl-2 binding proteins was performed, which resulted in the identification of BMRP as a novel Bcl-2 interacting protein. Current knowledge suggests that BMRP is an integral part of the apoptotic machinery. Our long-term goal is to enhance our understanding of the molecular mechanisms that mediate Bcl-2 anti-apoptotic activity and its regulation. Our hypothesis is that BMRP is a pro-apoptotic protein whose ability to stimulate apoptosis is regulated by binding with Bcl-2. An increased understanding of the mechanistic basis of BMRP and Bcl-2 function and modulation will enhance our knowledge of apoptotic pathways, which will facilitate the design of novel drugs to prevent and/or treat human diseases characterized by abnormal levels of apoptosis. Two specific aims are proposed to test our central hypothesis. First, deletion and alanine scanning mutagenesis will be carried out to identify domains and amino acid residues that are essential to the pro-apoptotic activity of BMRP. These studies will utilize PCR site-directed mutagenesis, followed by apoptosis assays. Additionally, the ability of these mutants to bind to Bcl-2 will be tested by yeast two-hybrid assays. Second, the fate of BMRP during apoptosis and the role that it plays in apoptosis signaling pathways will be analyzed by subcellular fractionation, and studies of the cellular effects of over-expression or down-regulation of BMRP in the absence and presence of other over-expressed apoptosis regulators. The proposed research is innovative, as it will establish unknown aspects of BMRP function and regulation, and BMRP's relationship to the anti-apoptotic activity of Bcl-2. This knowledge is relevant to public health, since it should provide novel targets for therapeutic intervention for diseases whose pathogenesis involves deregulated apoptosis, such as cancer.