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

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Abstract
During apoptosis, release of cytochrome c initiates dATP‐dependent oligomerization of Apaf‐1 and formation of the apoptosome. In a cell‐free system, we 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 via an interaction between the active site cysteine (C287) in caspase‐9 and a critical aspartate (D175) in caspase‐3. We demonstrate that XIAP, an inhibitor‐of‐apoptosis protein, is normally present in high molecular weight complexes in unactivated cell lysates, but directly interacts with the apoptosome in cytochrome c /dATP‐activated lysates. XIAP associates with oligomerized Apaf‐1 and/or processed caspase‐9 and influences the activation of caspase‐3, but also binds activated caspase‐3 produced within the apoptosome and sequesters it within the complex. Thus, XIAP may regulate cell death by inhibiting the activation of caspase‐3 within the apoptosome and by preventing release of active caspase‐3 from the complex.