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

Hill, Michelle M., Adrain, Colin, Duriez, Patrick J., Creagh, Emma M. and Martin, Seamus J. (2004) Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes. EMBO Journal, 23 10: 2134-2145. doi:10.1038/sj.emboj.7600210


Author Hill, Michelle M.
Adrain, Colin
Duriez, Patrick J.
Creagh, Emma M.
Martin, Seamus J.
Title Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes
Journal name EMBO Journal   Check publisher's open access policy
ISSN 0261-4189
1460-2075
Publication date 2004-05
Sub-type Article (original research)
DOI 10.1038/sj.emboj.7600210
Volume 23
Issue 10
Start page 2134
End page 2145
Total pages 12
Place of publication London, United Kingdom
Publisher Nature Publishing Group
Language eng
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.
Keyword Apoptosis
Apoptosome
Caspase-9
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: UQ Diamantina Institute - Open Access Collection
UQ Diamantina Institute Publications
 
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Created: Mon, 20 Dec 2010, 13:45:17 EST