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Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA
A J Darmon1, T J Ley, D W Nicholson
1Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Abstract:
Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.
Insights
Granzyme B in cytotoxic T lymphocytes (CTLs) activates CPP32, a protease crucial for inducing target cell DNA fragmentation during apoptosis. This pathway is essential for programmed cell death, distinct from membrane damage.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Cytotoxic T lymphocytes (CTLs) induce target cell death via granule- or Fas-mediated pathways.
- Apoptotic cell death mechanisms, particularly protease involvement, remain incompletely understood.
- Interleukin-1beta converting enzyme (ICE)-related cysteine proteases are implicated in apoptosis induction.
Purpose of the Study:
- To elucidate the role of granzyme B in CPP32 activation and its contribution to CTL-induced apoptosis.
- To confirm granzyme B's nonredundant function in activating CPP32.
- To investigate the specific role of CPP32 activation in target cell DNA fragmentation.
Main Methods:
- Utilized cytolytic cells from granzyme B-deficient mice.
- Assessed CPP32 cleavage and activation.
- Employed a peptide inhibitor of CPP32-like proteases.
- Measured 51Cr release as an indicator of cell lysis.
Main Results:
- Cleavage and activation of CPP32 by granzyme B were confirmed as a nonredundant function.
- Granzyme B-mediated CPP32 activation plays a significant role in inducing target cell DNA fragmentation.
- Inhibition of CPP32-like proteases confirmed their function in DNA fragmentation.
- 51Cr release was not suppressed, indicating granzyme B/CPP32 pathway is not primarily involved in membrane damage.
Conclusions:
- Granzyme B's cleavage and activation of CPP32 is a critical, nonredundant step in inducing DNA fragmentation during CTL-mediated apoptosis.
- This pathway is specifically linked to DNA fragmentation, not membrane damage.
- Cysteine proteases like CPP32 are key mediators of granzyme B's apoptotic function.