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Isolation of Ich-1S (caspase-2S)-binding protein that partially inhibits caspase activity
1Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Abstract:
Members of the caspase family are essential executors of apoptosis. Caspase-2 has two messenger RNAs generated by alternative splicing, which encode caspase-2L and caspase-2S. Although caspase-2L induces apoptosis, caspase-2S also has the ability to antagonize cell death. Experiments in caspase-2-deficient mice showed that caspase-2 functions to delay cell death in some neuronal populations, suggesting that caspase-2S dominantly acts for cell survival in the brain. However, the mechanism of caspase-2S-mediated anti-apoptotic effect is still unclear. Here, we isolated a protein that interacts with caspase-2S, designated as Ich-1S (caspase-2S)-binding protein (ISBP), by yeast two-hybrid screening using full-length caspase-2S cDNA as a bait. ISBP is identical to the recently isolated calcium and integrin-binding protein, and a small molecule calcium-binding protein with two EF-hand motifs of its C-terminus. In vitro transcribed and translated ISBP interacts specifically with glutathione-S-transferase-fused caspase-2S. Moreover, the interaction between ISBP and caspase-2S was observed in cultured cells. Northern blot analysis indicated that ISBP may be a ubiquitous protein. Interestingly, ISBP can partially inhibit the processing of pro-caspase-2L induced by anti-Fas antibody-treated Jurkat cytosolic lysates. These results suggested that ISBP may be the mediator for the survival function of caspase-2S.
Insights
Caspase-2S, a form of caspase-2, promotes cell survival. Researchers identified ISBP as a binding protein that mediates caspase-2S
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspases are key regulators of apoptosis.
- Caspase-2 exists as two isoforms, caspase-2L and caspase-2S, with opposing roles in cell death.
- The anti-apoptotic mechanism of caspase-2S is not well understood.
Purpose of the Study:
- To identify proteins interacting with caspase-2S.
- To elucidate the mechanism underlying caspase-2S-mediated cell survival.
Main Methods:
- Yeast two-hybrid screening using caspase-2S cDNA.
- In vitro binding assays with purified proteins.
- Co-immunoprecipitation in cultured cells.
- Northern blot analysis for protein expression.
Main Results:
- Identified ISBP (Ich-1S-binding protein) as a caspase-2S interacting protein.
- ISBP is a calcium and integrin-binding protein.
- ISBP interacts with caspase-2S in vitro and in vivo.
- ISBP partially inhibits pro-caspase-2L processing.
Conclusions:
- ISBP is a novel mediator of caspase-2S's anti-apoptotic function.
- ISBP may play a significant role in neuronal cell survival regulated by caspase-2S.