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Inhibition of distant caspase homologues by natural caspase inhibitors
S J Snipas1, H R Stennicke, S Riedl
1Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp-->Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.
Insights
Viral caspase inhibitors, like p35, can regulate other proteases in clan CD, not just caspases. This suggests a broader regulatory mechanism for these viral proteins in protease clan CD regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Virology
Background:
- Caspases are key regulators of apoptosis in animal cells.
- Caspase activity is controlled by protease inhibitors, including baculovirus p35 and inhibitor of apoptosis (IAP) proteins, believed to be caspase-specific.
- Caspases belong to protease clan CD, sharing evolutionary links with other proteases like legumains and bacterial gingipains.
Purpose of the Study:
- To investigate if natural caspase inhibitors can also regulate other proteases within protease clan CD.
- To explore the broader regulatory roles of viral protease inhibitors beyond caspases.
Main Methods:
- Tested the inhibitory effects of natural caspase inhibitors (IAP family, CrmA, p35) on various members of protease clan CD, including gingipain-R and gingipain-K.
- Utilized site-directed mutagenesis to map the inhibitory site in p35 responsible for gingipain-K regulation.
Main Results:
- IAP proteins showed minimal inhibition of gingipain-R.
- Cowpox viral cytokine-response modifier A (CrmA) did not inhibit tested proteases, but a CrmA mutant (Asp-->Lys) strongly inhibited gingipain-K.
- Wild-type p35 significantly inhibited gingipain-K, with Lys94 identified as crucial for this interaction, distinct from the caspase-binding site.
Conclusions:
- Virally encoded caspase inhibitors possess a mechanism to regulate diverse proteases within clan CD, not limited to caspases.
- The findings expand the known functions of viral protease inhibitors, highlighting their adaptability in targeting different proteases.