title: Dimerization and processing of procaspase-9 by redox stress in mitochondria.
abstract: We studied the mechanism of intra-mitochondrial death initiator caspase-9 activation by a redox response, in which hydrogen peroxide (H(2)O(2)) caused a subtle decrease in the inner membrane potential (Deltapsim) with little evidence of cytochrome c release. Initiation of the intra-mitochondrial autocleavage of procaspase-9 preceded the onset of caspase cascade induction in the cytosol. Purified mitochondria demonstrated procaspase-9 processing and releasing abilities when exposed to H(2)O(2). Bcl-2 overexpression caused accumulation of the active form caspase-9 in the mitochondria, rendering the cells resistant to the redox stress. Intriguingly, disulfide-bonded dimers of autoprocessed caspase-9 were generated in the mitochondria in the pre-apoptotic phase. Using a substrate-analog inhibitor, dimer formation of procaspase-9 was also detectable inside the mitochondria. Furthermore, thiol reductant thioredoxin blocked the caspase-9 activation step and the cell death induction. Thus, redox stress-responsive thiol-disulfide converting reactions in the mitochondrion seemed to mediate procaspase-9 assembly that allows autoprocessing. This study offers an explanation for the recent observation that Apaf-1-null cells can execute apoptosis, which can be blocked by Bcl-2, and supports the proposition that the cytochrome c-Apaf-1-procaspase-9 complex functions in the caspase amplification rather than in its initiation.
authors.affiliation: Ikawa Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 350-0198, Japan. kushbgen@mri.tmd.ac.jp
authors.forename: Iyoko
authors.initials: I
authors.lastname: Katoh
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Tomimori
authors.forename: Yoji
authors.initials: Y
authors.lastname: Ikawa
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Kurata
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry* / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004789:Enzyme Activation; D005434:Flow Cytometry; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D010084:Oxidation-Reduction; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry; D013997:Time Factors; D020298:U937 Cells
chemical_list: C506592:APAF1 protein, human; D017304:Annexin A5; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D004396:Coloring Agents; D004220:Disulfides; D011498:Protein Precursors; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013879:Thioredoxins; D045304:Cytochromes c; D006861:Hydrogen Peroxide; C506433:CASP9 protein, human; C506434:Casp9 protein, mouse; D053453:Caspase 9; D020169:Caspases