Structure-Function Analysis of Pro-Apoptotic BMRP and Its Role as a Tumor Suppressor

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Abstract Cell death by apoptosis is required for the normal development of metazoans, as well as for the maintenance of tissue homeostasis and normal functioning of the adult organism. Abnormal levels of apoptosis have been implicated in the etiology of human diseases including cancer, disorders of the immune system, infertility, neurodegenerative diseases, and cardiac arrest and stroke damage. In the case of cancer, impaired apoptosis is a major cause of resistance to chemo- and radio-therapies. Bcl-2 is an anti- apoptotic protein that plays a major regulatory role in this process of cell demise and, despite intense investigation, several aspects of its mechanisms of action and regulation are still not well understood. To gain further insight into the molecular mechanisms that mediate Bcl-2's anti-apoptotic function and its regulation, we conducted a search of Bcl-2 interacting proteins, which identified BMRP as a novel Bcl-2 binding protein. Current knowledge suggests that BMRP is a pro-apoptotic protein that plays a tumor- suppressor role. Our long-term goal is to enhance our understanding of the molecular mechanisms that mediate Bcl-2's pro-survival activity and its regulation, and to exploit this knowledge for the development of novel therapeutic approaches. Our hypothesis is that BMRP is a relevant component of the molecular apoptotic machinery, that its pro-apoptotic activity counteracts the activity of Bcl-2, and that mechanistic studies will yield novel targets and tools for modulating apoptosis. An increased knowledge of the molecular mechanistic basis of BMRP and Bcl-2 function and modulation will enhance our understanding of the players and signaling in apoptosis pathways, which will contribute to the development of novel drugs to prevent and/or treat human diseases involving deregulated apoptosis. Two specific aims are proposed to test our hypothesis. In the first aim, chimeric and alanine substitution mutants of BMRP will be generated to identify a killer peptide (or peptides) for future development of novel tumor combinatorial therapies, as well as amino acid residues essential to its pro-apoptotic activity. Additionally, the ability of these mutants to bind to Bcl-2 will be tested by yeast Two-Hybrid and co-immunoprecipitation assays. A second aim will investigate the role of BMRP as a tumor suppressor by determining the expression levels of the BMRP protein and mRNA in cancer cell lines, and analyzing the genetic mechanisms responsible for abnormal BMRP expression in these cancer cell lines. The proposed research is innovative, as it will establish unknown molecular aspects of BMRP function and regulation, and BMRP's role as a tumor suppressor. This knowledge is relevant to public health, since it should provide novel targets and tools for the design of advanced therapies for the treatment and/or prevention of human diseases characterized by deregulated apoptosis, such as cancer.