title: Regulation of the Apaf-1/caspase-9 apoptosome by caspase-3 and XIAP.
abstract: The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.
authors.affiliation: Idun Pharmaceuticals, Inc., San Diego, California 92121, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Zou
authors.forename: Ruomei
authors.initials: R
authors.lastname: Yang
authors.forename: Junshan
authors.initials: J
authors.lastname: Hao
authors.forename: Jean
authors.initials: J
authors.lastname: Wang
authors.forename: Chaohong
authors.initials: C
authors.lastname: Sun
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Fesik
authors.forename: Joe C
authors.initials: JC
authors.lastname: Wu
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Tomaselli
authors.forename: Robert C
authors.initials: RC
authors.lastname: Armstrong
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D011506:Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases