Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1.
We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.
Howard Hughes Medical Institute & Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235, USA.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D053477:Apoptotic Protease-Activating Factor 1; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003574:Cytochrome c Group / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014867:Water / Q000378:metabolism
D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D009711:Nucleotides; D011506:Proteins; D011994:Recombinant Proteins; D014867:Water; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D053453:Caspase 9; D020169:Caspases; D019204:GTP-Binding Proteins; C002630:alpha,beta-methyleneadenosine 5'-triphosphate