Structural Studies in Apaf 1 mediated Apoptosis

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Description (applicant's description): Apoptosis plays a central role in the development and homeostasis of metazoans. Alterations in apoptotic pathways have been implicated in cancer and autoimmune diseases. The apoptotic protease-activating factor 1 (Apaf-1) controls the initiation of apoptosis by promoting the activation of procaspase-9 and procaspase-3. The Inhibitor of Apoptosis (IAP) family of proteins suppress cell death by inhibiting these caspases. A recently identified protein, Smac, plays a vital role in apoptosis by relieving the inhibitory effects of IAPs to caspases. The work proposed here focuses on the following specific aims: (1) Determination of a high-resolution structure of Smac. We have obtained diffracting crystals of Smac. The structure is being determined; and important insights have begun to emerge from preliminary structural analysis. (2) Biochemical characterization of mechanisms of apoptotic activation by Smac. In collaboration with Xiaodong Wang at the University of Texas, an in vitro system has been developed to study the mechanisms of apoptotic inhibition by IAPs and activation by Smac. Important findings have started to impact our understanding on activation of apoptosis. (3) Determination of the structure of Smac in complex with an IAP. The relief of IAP inhibition to apoptosis by Smac involves a direct interaction between these two proteins. A binary complex between Smac and a functional domain in human c-IAP1 has been characterized. Small crystals have been obtained. Efforts to improve the crystal morphology are pursued. (4) Determination of the structure of caspase-3 in complex with an IAP. To provide a structural basis of caspase inhibition by IAP, the two subunits of active caspase-3 have been over-expressed, refolded and purified to homogeneity as an active complex. A binary complex between caspase-3 and a functional domain of human c-IAP1 will be reconstituted and crystallized. (5) Determination of the structure of procaspase-9 by itself and in complex with an IAP. IAPs function by targeting both the activated caspases and the inactive procaspase-9. A full-length procaspase-9 (C287A) has been over-expressed and purified to homogeneity. The crystals of procaspase-9 by itself and in complex with an inhibitory c-IAP1 fragment will be generated; these structures will be determined by either molecular replacement or multiple isomorphous replacement.