Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes

Michelle M Hill, Colin Adrain, Patrick J Duriez, Emma M Creagh, Seamus J Martin

Research output: Contribution to journalArticle

181 Citations (Scopus)

Abstract

The Apaf-1 apoptosome is a multi-subunit caspase-activating scaffold that is assembled in response to diverse forms of cellular stress that culminate in apoptosis. To date, most studies on apoptosome composition and function have used apoptosomes reassembled from recombinant or purified proteins. Thus, the precise composition of native apoptosomes remains unresolved. Here, we have used a one-step immunopurification approach to isolate catalytically active Apaf-1/caspase-9 apoptosomes, and have identified the major constituents of these complexes using mass spectrometry methods. Using this approach, we have also assessed the ability of putative apoptosome regulatory proteins, such as Smac/DIABLO and PHAPI, to regulate the activity of native apoptosomes. We show that Apaf-1, caspase-9, caspase-3 and XIAP are the major constituents of native apoptosomes and that cytochrome c is not stably associated with the active complex. We also demonstrate that the IAP-neutralizing protein Smac/DIABLO and the tumor-suppressor protein PHAPI can enhance the catalytic activity of apoptosome complexes in distinct ways. Surprisingly, PHAPI also enhanced the activity of purified caspase-3, suggesting that it may act as a co-factor for this protease.

Original languageEnglish
Pages (from-to)2134-45
Number of pages12
JournalThe EMBO Journal
Volume23
Issue number10
DOIs
Publication statusPublished - 19 May 2004

Keywords

  • Animals
  • Apoptosis/physiology
  • Apoptotic Protease-Activating Factor 1
  • Carrier Proteins/metabolism
  • Caspase 3
  • Caspase 9
  • Caspases/isolation & purification
  • Cytochromes c/metabolism
  • Electrophoresis, Gel, Two-Dimensional
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Jurkat Cells
  • Mitochondrial Proteins/metabolism
  • Multiprotein Complexes
  • Peptide Mapping
  • Proteins/isolation & purification
  • Recombinant Proteins/chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • X-Linked Inhibitor of Apoptosis Protein

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