title: Caspase-3 cleaves Apaf-1 into an approximately 30 kDa fragment that associates with an inappropriately oligomerized and biologically inactive approximately 1.4 MDa apoptosome complex.
abstract: Cytochrome c and dATP/ATP induce oligomerization of Apaf-1 into two distinct apoptosome complexes: an approximately 700 kDa complex, which recruits and activates caspases-9, -3 and -7, and an approximately 1.4 MDa complex, which recruits and processes caspase-9, but does not efficiently activate effector caspases. While searching for potential inhibitors of the approximately 1.4 MDa apoptosome complex, we observed an approximately 30 kDa Apaf-1 immunoreactive fragment that was associated exclusively with the inactive complex. We subsequently determined that caspase-3 cleaved Apaf-1 within its CED-4 domain (SVTD(271) downward arrowS) in both dATP-activated lysates and apoptotic cells to form a prominent approximately 30 kDa (p30) N-terminal fragment. Purified recombinant Apaf-1 p30 fragment weakly inhibited dATP-dependent activation of caspase-3 in vitro. However, more importantly, prevention of endogenous formation of the p30 fragment did not stimulate latent effector caspase processing activity in the large complex. Similarly, the possibility that XIAP, an inhibitor of apoptosis protein (IAP), was responsible for the inactivity of the approximately 1.4 MDa complex was excluded as immunodepletion of this caspase inhibitor failed to relieve the inhibition. However, selective proteolytic digestion of the approximately 1.4 MDa and approximately 700 kDa complexes showed that Apaf-1 was present in conformationally distinct forms in these two complexes. Therefore, the inability of the approximately 1.4 MDa apoptosome complex to process effector caspases most likely results from inappropriately folded or oligomerized Apaf-1.
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: S B
authors.initials: SB
authors.lastname: Bratton
authors.forename: G
authors.initials: G
authors.lastname: Walker
authors.forename: D L
authors.initials: DL
authors.lastname: Roberts
authors.forename: K
authors.initials: K
authors.lastname: Cain
authors.forename: G M
authors.initials: GM
authors.lastname: Cohen
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein
chemical_list: C506592:APAF1 protein, human; D000906:Antibodies; D053477:Apoptotic Protease-Activating Factor 1; D003838:Deoxyadenine Nucleotides; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; C026600:2'-deoxyadenosine triphosphate