Abstract
Activation of procaspase-9 by Apaf-1 in the cytochrome c/dATP-dependent pathway requires proteolytic cleavage to generate the mature caspase molecule. To elucidate the mechanism of activation of procaspase-9 by Apaf-1, we designed an in vitro Apaf-1-procaspase-9 activation system using recombinant components. Here, we show that deletion of the Apaf-1 WD-40 repeats makes Apaf-1 constitutively active and capable of processing procaspase-9 independent of cytochrome c an dATP. Apaf-1-mediated processing of procaspase-9 occurs at Asp-315 by an intrinsic autocatalytic activity of procaspase-9 itself. We provide evidence that Apaf-1 can form oligomers and may facilitate procaspase-9 autoactivation by oligomerizing its precursor molecules. Once activated, caspase-9 can initiate a caspase cascade involving the downstream executioners caspase-3, -6, and -7.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / metabolism
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Apoptosis / genetics
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Apoptosis / physiology
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Apoptotic Protease-Activating Factor 1
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Binding Sites
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Biopolymers / chemistry
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Biopolymers / metabolism
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Caspase 3
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Caspase 6
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Caspase 7
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Caspase 9
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Caspases*
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Catalysis
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Cysteine Endopeptidases / chemistry
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / metabolism*
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Cytochrome c Group / metabolism
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Dimerization
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Enzyme Activation
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Enzyme Precursors / chemistry
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Enzyme Precursors / genetics
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Enzyme Precursors / metabolism*
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Humans
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Mutation / genetics
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Protein Processing, Post-Translational
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Proteins / chemistry
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Proteins / genetics
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Proteins / pharmacology*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Repetitive Sequences, Nucleic Acid / genetics
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Tumor Cells, Cultured / cytology
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Tumor Cells, Cultured / enzymology
Substances
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APAF1 protein, human
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Apoptotic Protease-Activating Factor 1
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Biopolymers
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Cytochrome c Group
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Enzyme Precursors
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Proteins
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Recombinant Proteins
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Adenosine Triphosphate
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CASP3 protein, human
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CASP6 protein, human
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CASP7 protein, human
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CASP9 protein, human
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Caspase 3
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Caspase 6
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Caspase 7
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Caspase 9
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Caspases
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Cysteine Endopeptidases