Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis
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J P Medema1, R E Toes, C Scaffidi
1Tumorimmunology Program, German Cancer Research Center, Heidelberg.
Abstract:
Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. We show here that in HeLa target cells induction of apoptosis through the perforin/GrB pathway resulted in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage resulted from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice displayed GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) was directly cleaved by GrB in HeLa cells. We therefore conclude that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis.
Insights
Cytotoxic T lymphocytes induce apoptosis via perforin/granzyme B (GrB). Granzyme B directly activates caspase-8 (FLICE), not primarily caspase-3, in target cells, revealing a novel apoptosis pathway.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Cytotoxic T lymphocytes (CTLs) eliminate target cells via apoptosis.
- Key CTL-mediated apoptosis pathways include CD95(APO-1/Fas) and perforin/granzyme B (GrB).
- The precise in vivo substrates of GrB remain elusive, though caspase activity is essential for GrB-induced apoptosis.
Purpose of the Study:
- To elucidate the specific roles of caspases, particularly caspase-3 and caspase-8 (FLICE), in perforin/GrB-mediated apoptosis.
- To identify the direct substrates of GrB during CTL-induced cell death.
- To compare GrB substrate cleavage with that observed in CD95(APO-1/Fas) signaling.
Main Methods:
- Induction of apoptosis in HeLa target cells using the perforin/GrB pathway.
- Analysis of caspase-3 (CPP32) and caspase-8 (FLICE) cleavage by GrB.
- Assessment of poly(ADP-ribose) polymerase (PARP) cleavage in caspase-3 deficient (caspase-3(-/-)) and wild-type target cells.
Main Results:
- Granzyme B (GrB) showed minor direct cleavage of caspase-3 (CPP32); most caspase-3 activation resulted from upstream caspases.
- Target cells lacking caspase-3 still exhibited significant GrB-induced PARP cleavage, indicating involvement of other PARP-cleaving caspases.
- Granzyme B directly cleaved caspase-8 (FLICE) in HeLa cells.
Conclusions:
- Caspase-8 (FLICE) is a direct substrate of GrB during perforin/GrB-induced apoptosis.
- FLICE plays a crucial role not only in CD95(APO-1/Fas) signaling but also in GrB-mediated cell death.
- The perforin/GrB pathway activates caspases beyond direct caspase-3 cleavage, involving other PARP-cleaving enzymes.