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Caspase-mediated degradation of T-cell receptor zeta-chain
B R Gastman1, D E Johnson, T L Whiteside
1Department of Otolaryngology, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Abstract:
We recently reported an association between loss in T-cell receptor (TcR) zeta-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that zeta-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the zeta-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of zeta-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of zeta-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated zeta-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-AspGlu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated zeta-chain was observed. These results suggest that the loss of TcR zeta-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the zeta-chain by activated caspases. This is the first report of involvement of caspases in degradation of the zeta protein.
Insights
The T-cell receptor zeta-chain is degraded by caspases during T-cell apoptosis. This caspase-mediated degradation of the zeta-chain contributes to tumor-induced immune dysfunction.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Loss of T-cell receptor (TcR) zeta-chain expression is linked to tumor-induced T-lymphocyte apoptosis.
- The precise mechanism of zeta-chain loss during apoptosis requires further investigation.
Purpose of the Study:
- To investigate if the zeta-chain is a direct substrate for activated caspases.
- To elucidate the role of caspases in the degradation of TcR zeta-chain during T-cell apoptosis.
Main Methods:
- Analysis of zeta-chain cleavage in apoptotic Jurkat T lymphocytes using specific caspase inhibitors.
- Overexpression of intracellular caspase inhibitors (Bcl-2, cytokine response-modifier A) to assess their effect on zeta-chain cleavage.
- In vitro cleavage assays using recombinant caspase-3 and caspase-7 with in vitro translated zeta-chain.
Main Results:
- Two DXXD motifs in the zeta-chain, recognized by caspase-3-like proteases, were found to be cleaved in apoptotic Jurkat cells.
- Fas-induced cleavage of zeta-chain was inhibited by caspase inhibitors and by overexpression of Bcl-2 or cytokine response-modifier A.
- Recombinant caspase-3 and caspase-7 directly cleaved in vitro translated zeta-chain in a dose-dependent manner, an effect blocked by a specific caspase inhibitor.
Conclusions:
- The loss of TcR zeta-chain during T-cell apoptosis is mediated by direct degradation by activated caspases.
- This study provides the first evidence of caspase involvement in the degradation of the zeta protein, linking it to tumor-induced immune dysfunction.