Caspase-3 cleaves Apaf-1 into an ∼ 30 kDa fragment that associates with an inappropriately oligomerized and biologically inactive ∼ 1.4 MDa apoptosome complex

S. B. Bratton, G. Walker, D. L. Roberts, K. Cain, G. M. Cohen

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

Cytochrome c and dATP/ATP induce oligomerization of Apaf-1 into two distinct apoptosome complexes: an ∼700 kDa complex, which recruits and activates caspases-9, -3 and -7, and an ∼1.4 MDa complex, which recruits and processes caspase-9, but does not efficiently activate effector caspases. While searching for potential inhibitors of the ∼1.4 MDa apoptosome complex, we observed an ∼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 (SVTD271↓S) in both dATP-activated lysates and apoptotic cells to form a prominent ∼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 ∼1.4 MDa complex was excluded as immunodepletion of this caspase inhibitor failed to relieve the inhibition. However, selective proteolytic digestion of the ∼1.4 MDa and ∼700 kDa complexes showed that Apaf-1 was present in conformationally distinct forms in these two complexes. Therefore, the inability of the ∼1.4 MDa apoptosome complex to process effector caspases most likely results from inappropriately folded or oligomerized Apaf-1.

Original languageEnglish (US)
Pages (from-to)425-433
Number of pages9
JournalCell death and differentiation
Volume8
Issue number4
DOIs
StatePublished - 2001
Externally publishedYes

Keywords

  • Apaf-1
  • Apoptosome
  • Effector caspases

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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