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Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c
D G Kirsch1, A Doseff, B N Chau
1Oncology Center, Pharmacology and Molecular Sciences, and Neurology, Johns Hopkins Schools of Public Health and Medicine, Baltimore, Maryland 21205, USA.
Abstract:
Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.
Insights
Caspase-3 is essential for cleaving the apoptosis-inhibiting Bcl-2 protein during cell death. This cleavage promotes further caspase activation, creating a positive feedback loop for apoptosis execution.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspases are key proteases in apoptosis, but specific caspase substrates remain largely unknown.
- Bcl-2, an apoptosis inhibitor, is cleaved during apoptosis, but the responsible caspase is not fully established.
Purpose of the Study:
- To identify the specific caspase responsible for cleaving the Bcl-2 protein during apoptosis.
- To investigate the functional consequences of Bcl-2 cleavage by caspase-3.
Main Methods:
- Utilized apoptotic cell extracts and caspase inhibitors to assess Bcl-2 and Bax cleavage.
- Employed immunodepletion of caspases (caspase-3 and caspase-7) to determine their roles in Bcl-2 cleavage.
- Transfected caspase-3 into caspase-3-deficient cells to restore Bcl-2 cleavage.
- Investigated the subcellular localization and function of the Bcl-2 cleavage fragment.
Main Results:
- Endogenous caspase-3 was identified as the primary caspase responsible for Bcl-2 cleavage.
- Bcl-2 cleavage was abolished by caspase-3 immunodepletion and absent in caspase-3-deficient cells.
- Bax cleavage was less dependent on caspases, suggesting involvement of other proteases.
- The Bcl-2 cleavage fragment localized to mitochondria and induced cytochrome c release, activating a positive feedback loop.
Conclusions:
- Caspase-3 activation is critical for the specific cleavage of Bcl-2 in apoptotic pathways.
- Caspase-3-mediated Bcl-2 cleavage amplifies apoptosis through a positive feedback mechanism involving mitochondria and cytochrome c release.