Apoptosis plays a central role in the development and homeostasis of all multi-cellular organisms. Alterations in apoptotic pathways have been implicated in cancer and autoimmune diseases. The formation of a 1.4 MDa apoptosome holoenzyme, involving Apaf-1, cytochrome c, dATP, and caspase-9, is essential to many important forms of apoptosis in mammals. The work proposed here focuses on the following specific aims: (1) Biochemical analysis of the formation of an apoptosome holoenzyme. The cloning, expression, and purification of all three protein components involved in the formation of an apoptosome holoenzyme have been successfully completed. Preliminary characterization of the apoptosome holoenzyme has been performed, verifying that all recombinant proteins are biochemically active. Systematic characterization of the formation of the apoptosome holoenzyme will be pursued. (2) Determination of the structures of a mini-apoptosome involving the CARD domain of Apaf-1 in complex with the activated caspase- 9. The CARD domain of Apaf-1 mediates the formation of a 440-kDa mini-apoptosome involving active caspase-9. Large amounts of this mini-apoptosome have been assembled and initial crystallization trials have been performed. The determination of the three-dimensional structure of this mini-apoptosome will be pursued. (3) Determination of the structures of the full-length Apaf-1 by itself and in complex with cytochrome c. Apaf-1 by itself exists in an inhibitory conformation. Upon binding to cytochrome c, Apaf-1 undergoes conformational change that allows binding to dATP or ATP. These structures will be determined by multiple isomorphous replacement. (4) Determination of the structure of the complete apoptosome holoenzyme. The feasibility of this project has been demonstrated by the reconstitution of the complete and functional apoptosome holoenzyme in vitro. This 1.4 MDa complex will be crystallized. The structure will be determined by either molecular replacement or by multiple isomorphous replacement.
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