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Specific cleavage of the large subunit of replication factor C in apoptosis is mediated by CPP32-like protease
1Queensland Institute of Medical Research, University of Queensland, P.O. Royal Brisbane Hospital, Australia. qizhongS@qimr.edu.au
Abstract:
Recent evidence suggests that the growing family of cysteine proteases related to the interleukin-1beta-converting enzyme (ICE) is of central importance in mediating apoptosis. Proteolytic cleavage of a small group of cellular substrates by these enzymes in association with the onset of apoptosis has been reported. In the present study, we searched a protein data base for potential death substrates possessing the CPP32 cleavage site, DEVD, and identified several candidates including RFC140, the large subunit of replication factor C, which we subsequently demonstrated to be specifically cleaved in a variety of cell types undergoing apoptosis in response to different cytotoxic agents, whereas no degradation is observed in a cell line resistant to etoposide-induced apoptosis. The abrogation of RFC140 cleavage in apoptotic extracts by Ac-DEVD-CHO, a potent inhibitor of CPP32, together with the finding that a CPP32 consensus cleavage sequence, DEVD, exists in RFC140, suggests that CPP32 or a close relative is responsible for RFC140 degradation in apoptosis.
Insights
Caspase-activated DNase (CAD) is a key enzyme in apoptosis. This study identifies RFC140 as a novel substrate cleaved by CPP32, a cysteine protease involved in programmed cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspase-activated DNase (CAD) is a cysteine protease family crucial for apoptosis.
- Specific cellular substrates are proteolytically cleaved during apoptosis.
- The interleukin-1beta-converting enzyme (ICE) family plays a central role in apoptosis.
Purpose of the Study:
- To identify novel substrates of CPP32, a key apoptotic protease.
- To investigate the role of RFC140 cleavage in apoptosis.
Main Methods:
- Searched protein databases for potential CPP32 substrates with the DEVD cleavage site.
- Validated RFC140 as a substrate cleaved during apoptosis.
- Utilized Ac-DEVD-CHO, a CPP32 inhibitor, to confirm cleavage mechanism.
Main Results:
- Identified RFC140, a subunit of replication factor C, as a potential CPP32 substrate.
- Demonstrated specific cleavage of RFC140 in various cell types undergoing apoptosis.
- Observed no RFC140 degradation in etoposide-resistant cell lines.
- Confirmed that CPP32 or a related protease mediates RFC140 degradation.
Conclusions:
- RFC140 is a novel death substrate cleaved by CPP32 or a related protease during apoptosis.
- RFC140 cleavage is a specific event during programmed cell death.
- This finding contributes to understanding the molecular mechanisms of apoptosis.