Proteolytic processing of the caspase-9 zymogen is required for apoptosome-mediated activation of caspase-9

J Biol Chem. 2013 May 24;288(21):15142-7. doi: 10.1074/jbc.M112.441568. Epub 2013 Apr 9.


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.

Keywords: Apaf-1; Apoptosis; Caspase; Cell Biology; Cell Death; Enzyme Mechanisms.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Apoptosomes / chemistry*
  • Apoptosomes / genetics
  • Apoptosomes / metabolism
  • Apoptotic Protease-Activating Factor 1 / chemistry*
  • Apoptotic Protease-Activating Factor 1 / genetics
  • Apoptotic Protease-Activating Factor 1 / metabolism
  • Caspase 9 / chemistry*
  • Caspase 9 / genetics
  • Caspase 9 / metabolism
  • Enzyme Activation / physiology
  • Enzyme Precursors / chemistry*
  • Enzyme Precursors / genetics
  • Enzyme Precursors / metabolism
  • Humans
  • Mutation, Missense
  • Protein Multimerization / physiology*
  • Proteolysis*


  • APAF1 protein, human
  • Apoptosomes
  • Apoptotic Protease-Activating Factor 1
  • Enzyme Precursors
  • Caspase 9