Programmed cell death (PCD) often occurs via apoptosis, a physiological form of cellular demise common during embryogenesis, turnover of tissues, and selection of cell populations. Several genes have been identified that activate, execute, or inhibit the cell death pathway. The execution phase of PCD is implemented by a conserved family of death proteases called caspases. In Caeneorhabditis elegans, activation of a critical caspase, CED-3, is promoted by the adaptor molecule CED-4 and inhibited by CED-9 homologous to mammalian Bcl-2 and Bcl-XL. We have performed preliminary studies that show that a mammalian homologue of CED-4, Apaf-1, can associate with a central death protease, caspase-9, a mammalian homologue of CED-3. The interaction with caspase-9 was mediated by the amino-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the proteolytic activation and killing activity of caspase-9. Pro-survival Bcl-XL physically interacts with Apaf-1 and caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from E. coli or insect cells inhibited Apaf-1 -dependent processing of caspase-9. These interactions suggest that the death machinery and its regulation are evolutionarily conserved from nematodes to humans. The main hypothesis to be tested in this proposal is that Bcl-2 family members regulate apoptosis by regulating caspase activation through physical associations with Apaf-1. The overall goal of this proposal is to gain understanding of the interaction of prosurvival Bcl-XL with caspases through Apaf-1 and the resulting caspase activation by pro-apoptotic members of the Bcl-2 family.
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