Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with Apaf-1.
Intracellular reactive oxygen species (ROS) are known to regulate apoptosis. Activation of caspase-9, the initial caspase in the mitochondrial apoptotic cascade, is closely associated with ROS, but it is unclear whether ROS regulate caspase-9 via direct oxidative modification. The present study aims to elucidate the molecular mechanisms by which ROS mediate caspase-9 activation. Our results show that the cellular oxidative state facilitates caspase-9 activation. Hydrogen peroxide treatment causes the activation of caspase-9 and apoptosis, and promotes an interaction between caspase-9 and apoptotic protease-activating factor 1 (Apaf-1) via disulfide formation. In addition, in an in vitro mitochondria-free system, the thiol-oxidant diamide promotes auto-cleavage of caspase-9 and the caspase-9/Apaf-1 interaction by facilitating the formation of disulfide-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H(2)O(2)-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulfide formation. The association between cytochrome c and the C403S mutant is significantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modification of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modification of caspase-9 by ROS can mediate its interaction with Apaf-1, and can thus promote its auto-cleavage and activation. This mechanism may facilitate apoptosome formation and caspase-9 activation under oxidative stress.
Zuo
Yong
Y
Department of Cell Biology, Key Laboratory of the Education Ministry for Cell Differentiation and Apoptosis, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Xiang
Binggang
B
Yang
Jie
J
Sun
Xuxu
X
Wang
Yumei
Y
Cang
Hui
H
Yi
Jing
J
D019943:Amino Acid Substitution; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology
D053477:Apoptotic Protease-Activating Factor 1; D004220:Disulfides; D050505:Mutant Proteins; D017382:Reactive Oxygen Species; D003958:Diamide; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D053453:Caspase 9