Target Protease Specificity of the Viral Serpin CrmA
ANALYSIS OF FIVE CASPASES*
- From the Burnham Institute, San Diego, California 92037 and the
- ‡ Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109
- § To whom correspondence should be addressed: The Burnham Inst., 10901 N. Torrey Pines Rd., San Diego, CA 92037. Tel.: 619-646-3114; Fax: 619-646-3189; E-mail: gsalvesen{at}ljcrf.edu
Abstract
When ectopically expressed in animal cells, cytokine response modifier A (CrmA), a product of the cowpox virus, prevents programmed cell death initiated by a variety of stimuli. Since CrmA is a proteinase inhibitor, its target is probably a protease that promotes cell death. The identification of this target is crucial in delineating essential regulation points that modulate the apoptotic program. We have compared the kinetics of interaction of CrmA with five proteases that may play a role in apoptosis. Four of the proteases, all members of the caspase family, are inhibited with widely different rates and affinities ranging over 5 orders of magnitude. One is not inhibited at all under the experimental conditions. CrmA is quite selective in its ability to inhibit caspases, showing the highest affinity for interleukin-1β-converting enzyme and the second highest for the caspase FLICE (Ki = 0.95 nM), identified as a component of the intracellular signaling complex recruited by ligation of the death receptor Fas. On the basis of comparative inhibitor kinetics, we propose that CrmA is unlikely to inhibit the caspases Yama, Mch2, or LAP3 in vivo but that its inhibition of FLICE is of a magnitude for this protease to be a key target of CrmA during Fas-mediated apoptosis. Therefore, our results support the hypothesis that FLICE catalyzes a crucial step in the promotion of cell death.
Footnotes
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↵* The research was supported in part by National Institutes of Health Grant HL51399 (to G. S. S.) and a fellowship from the G. Harold and Leila Y. Mathers Charitable Foundation (to Q. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- CrmA
-
cytokine response modifier A
- ICE
-
interleukin-1β-converting enzyme
- PAGE
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polyacrylamide gel electrophoresis
- AFC
-
7-amino-4-trifluoromethyl-coumarin
- Chaps
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- Z
-
benzyloxycarbonyl.
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- Received December 23, 1996.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
