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Cell Research (2009) 19:449-457. 2009 IBCB, SIBS, CAS All rights reserved 1001-0602/09 $ 30.00
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ORIGINAL ARTICLE
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Oxidative modication of caspase-9 facilitates its activation via disulde-mediated interaction with Apaf-1
Yong Zuo1, *, Binggang Xiang1, *, Jie Yang1, Xuxu Sun1, Yumei Wang1, Hui Cang1, Jing Yi1
1Department 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
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 modication. 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 disulde 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 disulde-linked complexes. Finally, a point mutation at C403 of caspase-9 impairs both H2O2-promoted caspase-9 activation and interaction with Apaf-1 through the abolition of disulde formation. The association between cytochrome c and the C403S mutant is signicantly weaker than that between cytochrome c and wild-type caspase-9, indicating that oxidative modication of caspase-9 contributes to apoptosome formation under oxidative stress. Taken together, oxidative modication 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.
Keywords: oxidative modication, ROS, caspase-9, apoptosis, disuldeCell Research (2009) 19:449-457. doi: 10.1038/cr.2009.19; published online 24 February 2009
Introduction
Aerobic cells unavoidably produce reactive oxygen species (ROS) as a result of their metabolic activities and are consequently vulnerable to oxidative stress originating from the intra- or extracellular environments. Oxidative
*These two authors contributed equally to this work.
Correspondence: Jing Yia, Jie Yangb
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Abbreviations: b-ME (beta mercaptoethanol); As2O3 (arsenic trioxide);
DTT (dithiothreitol); F5M (fluorescein-5-maleimide); FITC (fluorescein
isothiocyanate); H2O2 (hydrogen peroxide); IP (immunoprecipitation); IB
(immunoblot);...