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Bax cleavage is mediated by calpain during drug-induced apoptosis
1Department of Pathology, New York University Medical Center and Kaplan Comprehensive Cancer Center, New York 10016, USA.
Abstract:
The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously defined, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calcium-activated neutral cysteine endopeptidase calpain. Partial purification of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, purified calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the specific calpain inhibitor calpeptin effectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.
Insights
Calpains, not caspases, cleave the pro-apoptotic protein Bax in mitochondria during apoptosis. This calpain activity is calcium-dependent and distinct from caspase pathways, suggesting a novel role in cell death regulation.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- The anti-apoptotic protein Bcl-2 localizes to cellular membranes, while the pro-apoptotic protein Bax is primarily cytosolic, translocating to mitochondria during apoptosis.
- The precise mechanisms regulating Bax activity and its cleavage during apoptosis are not fully understood.
Purpose of the Study:
- To identify and characterize the protease responsible for cleaving the pro-apoptotic protein Bax.
- To investigate the role of this protease in drug-induced apoptosis.
Main Methods:
- Mitochondria-enriched membrane fractions and cytosolic fractions from HL-60 cells were used to assay for Bax protease activity.
- Protease activity was characterized using specific inhibitors (E-64, caspase inhibitors, granzyme B inhibitors, calpain inhibitors).
- Partial purification of the protease activity was performed using column chromatography, followed by characterization of purified calpain enzymes.
- HL-60 cells were pretreated with a calpain inhibitor (calpeptin) to assess its effect on Bax cleavage and apoptosis.
Main Results:
- A Bax protease activity was identified in mitochondria-enriched membrane fractions but not in the cytosolic fraction.
- This protease activity was inhibited by cysteine protease inhibitors (distinguishing it from caspases and granzyme B) and calpain inhibitors.
- Partial purification identified a calpain-like activity as responsible for Bax cleavage.
- Purified calpains cleaved Bax in a calcium-dependent manner.
- Calpeptin treatment blocked Bax cleavage and calpain activation but not PARP cleavage or cell death.
Conclusions:
- Calpains, a distinct class of proteases, cleave the pro-apoptotic protein Bax in a calcium-dependent manner during apoptosis.
- Calpain activation occurs alongside caspase activation during drug-induced apoptosis.
- Calpains may play a role in modulating apoptosis by selectively cleaving substrates like Bax, independent of caspase pathways.