Abstract
Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1–5 μM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9. Cell Death and Differentiation (2000) 7, 402–407
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Abbreviations
- HHCc:
-
horse heart cytochrome c
- HEPES:
-
N[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
- EDTA:
-
ethylenediaminetetraacetic acid
- PIPES:
-
piperazine-N,N′-bis[2-ethanesulfonic acid]
- DEVDase:
-
caspase activity causing cleavage of Asp-Glu-Val-Asp
- DEVD-AFC:
-
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin
- z-DEVD-pNA:
-
carbobenzoxy-Asp-Glu-Val-p-nitroanilide
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Acknowledgements
We thank Drs FS Alnemri and SM Srinivasula for the Flag CMV2 Apaf-1 (wild-type) and Apaf-1 (530) constructs; C Thompson and A Minn for the pET29bΔC construct, and G Salvesen for the generous gift of recombinant caspase-8. This work was supported by National Institute of Health grants to DR Green (CA69381, AI40646) and DDN (GM50284) and by a fellowship to E Bossy-Wetzel from the Swiss National Science Foundation (823A-046638). This is La Jolla Institute for Allergy and Immunology Publication number 305.
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Newmeyer, D., Bossy-Wetzel, E., Kluck, R. et al. Bcl-xL does not inhibit the function of Apaf-1. Cell Death Differ 7, 402–407 (2000). https://doi.org/10.1038/sj.cdd.4400665
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DOI: https://doi.org/10.1038/sj.cdd.4400665
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