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 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.
Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.
S B
SB
Bratton
G
G
Walker
S M
SM
Srinivasula
X M
XM
Sun
M
M
Butterworth
E S
ES
Alnemri
G M
GM
Cohen
D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D001224:Aspartic Acid / Q000378:metabolism; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D046911:Macromolecular Substances; D008954:Models, Biological; D011233:Precipitin Tests; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003838:Deoxyadenine Nucleotides; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D001224:Aspartic Acid; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate