title: Proteolytic processing of the caspase-9 zymogen is required for apoptosome-mediated activation of caspase-9.
abstract: Maturation of the single-chain caspase-9 zymogen through autoproteolytic processing is mediated by the Apaf-1 apoptosome at the onset of apoptosis. Processed caspase-9 and the apoptosome form a holoenzyme with robust proteolytic activity that is 2-3 orders of magnitude higher than that of free processed caspase-9. An unresolved important question is the role of caspase-9 processing, with some experimental data suggesting its dispensability. In this study, we demonstrate that, in contrast to wild-type caspase-9, the unprocessed single-chain caspase-9 triple mutant E306A/D315A/D330A (Casp9-TM) could no longer be adequately activated by the apoptosome. Compared with the protease activity of wild-type caspase-9, that of Casp9-TM in the presence of the apoptosome was drastically reduced. The crippled protease activity of Casp9-TM in the presence of the apoptosome is likely attributable to a markedly reduced ability of Casp9-TM to form homodimers. These data identify an essential role for the autoproteolytic processing of caspase-9 in its activation.
authors.affiliation: Ministry of Education Key Laboratory for Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, and School of Medicine, Tsinghua University, Beijing 100084, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Hu
authors.forename: Di
authors.initials: D
authors.lastname: Wu
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yan
authors.forename: Yigong
authors.initials: Y
authors.lastname: Shi
mesh_terms: D019943:Amino Acid Substitution; D053478:Apoptosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053477:Apoptotic Protease-Activating Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D055503:Protein Multimerization / Q000502:physiology*; D059748:Proteolysis
chemical_list: C506592:APAF1 protein, human; D053478:Apoptosomes; D053477:Apoptotic Protease-Activating Factor 1; D004792:Enzyme Precursors; D053453:Caspase 9
keywords: Apaf-1; Apoptosis; Caspase; Cell Biology; Cell Death; Enzyme Mechanisms