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Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2
C Adrain1, E M Creagh, S J Martin
1Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Abstract:
Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV- or gamma-irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro-apoptotic agents, was profoundly inhibited in Bcl-2-overexpressing cells. Thus, in addition to modulating apoptosis-associated mitochondrial cytochrome c release, Bcl-2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress-associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad-spectrum caspase inhibitor. This suggests that apoptosis-associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase-catalysed event that occurs downstream of cytochrome c release.
Insights
The mitochondrial protein Smac/DIABLO is released during apoptosis, but its efflux is caspase-dependent and occurs after cytochrome c release. Bcl-2 overexpression inhibits Smac/DIABLO release, suggesting a shared mitochondrial pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Smac/DIABLO is a mitochondrial protein that promotes apoptosis by inhibiting IAP proteins.
- Smac/DIABLO release from mitochondria into the cytosol is observed during apoptosis induced by various stimuli.
- The role of Bcl-2 and caspases in Smac/DIABLO mitochondrial efflux is not fully understood.
Purpose of the Study:
- To investigate the regulation of Smac/DIABLO release from mitochondria during apoptosis.
- To determine the role of Bcl-2 and caspases in Smac/DIABLO efflux.
- To compare the mechanisms of Smac/DIABLO and cytochrome c release from mitochondria.
Main Methods:
- Induction of apoptosis using cytotoxic drugs, DNA damage, and CD95 ligation.
- Overexpression of Bcl-2 in cells.
- Treatment with a broad-spectrum caspase inhibitor.
- Monitoring of Smac/DIABLO and cytochrome c release from mitochondria.
Main Results:
- Smac/DIABLO release is triggered by cytotoxic drugs, DNA damage, and CD95 ligation.
- Bcl-2 overexpression significantly inhibits Smac/DIABLO efflux from mitochondria.
- Smac/DIABLO release is blocked by caspase inhibitors, indicating a caspase-dependent mechanism.
- Smac/DIABLO release occurs downstream of cytochrome c release and does not share the same mechanism.
Conclusions:
- Bcl-2 regulates Smac/DIABLO release from mitochondria, potentially via the same pathway as cytochrome c.
- Smac/DIABLO efflux is a caspase-catalyzed event occurring downstream of cytochrome c release.
- These findings elucidate the distinct mechanisms governing the release of key apoptotic regulators from mitochondria.