Recruitment, activation and retention of caspases-9 and-3 by Apaf-1 apoptosome and associated XIAP complexes

During apoptosis, release of cytochrome c initiates dATP-dependent oligomer ization of Apaf-1 and formation of the apoptosome. In a cell-free system, w e have addressed the order in which apical and effector caspases, caspases- 9 and -3, respectively, are recruited to, activated and retained within the apoptosome. We propose a multi-step process, whereby catalytically active processed or unprocessed caspase-9 initially binds the Apaf-1 apoptosome in cytochrome c/dATP-activated lysates and consequently recruits caspase-3 vi a an interaction between the active site cysteine (C287) in caspase-9 and a critical aspartate (D175) in caspase-3. We demonstrate that XIAP, an inhib itor-of-apoptosis protein, is normally present in high molecular weight com plexes in unactivated cell lysates, but directly interacts,vith the apoptos ome in cytochrome c/dATP-activated lysates. XIAP associates with oligomeriz ed Apaf-1 and/or processed caspase-9 and influences the activation of caspa se-3, but also binds activated caspase-3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by in hibiting the activation of caspase-3 within the apoptosome and by preventin g release of active caspase-3 from the complex.