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

Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes
Analysis of the composition, assembly kinetics and activity of native Apaf-1 apoptosomes
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.
apoptosis, apoptosome, caspase-9, phapi, xiap
0261-4189
2134-45
Hill, Michelle M.
67167bf2-9a92-4e7e-83d1-ca97560f3779
Adrain, Colin
f7950ceb-b788-4d86-a607-b9efc4437142
Duriez, Patrick J.
4cf499bc-007a-43b3-b180-d6e5dc3d151b
Creagh, Emma M.
c5311521-65cc-46df-8f7d-d6416aa05a95
Martin, Seamus J.
3df02649-5634-44da-834d-098937a7ae5b
Hill, Michelle M.
67167bf2-9a92-4e7e-83d1-ca97560f3779
Adrain, Colin
f7950ceb-b788-4d86-a607-b9efc4437142
Duriez, Patrick J.
4cf499bc-007a-43b3-b180-d6e5dc3d151b
Creagh, Emma M.
c5311521-65cc-46df-8f7d-d6416aa05a95
Martin, Seamus J.
3df02649-5634-44da-834d-098937a7ae5b

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. The EMBO Journal, 23 (10), 2134-45. (doi:10.1038/sj.emboj.7600210). (PMID:10400610)

Record type: Article

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.

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More information

Published date: 19 May 2004
Keywords: apoptosis, apoptosome, caspase-9, phapi, xiap

Identifiers

Local EPrints ID: 164803
URI: http://eprints.soton.ac.uk/id/eprint/164803
ISSN: 0261-4189
PURE UUID: 36f08827-0aed-4123-80a2-e0d4247512ae
ORCID for Patrick J. Duriez: ORCID iD orcid.org/0000-0003-1814-2552

Catalogue record

Date deposited: 05 Oct 2010 09:01
Last modified: 14 Mar 2024 02:52

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Contributors

Author: Michelle M. Hill
Author: Colin Adrain
Author: Patrick J. Duriez ORCID iD
Author: Emma M. Creagh
Author: Seamus J. Martin

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