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J. Biol. Chem., Vol. 281, Issue 50, 38781-38790, December 15, 2006

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Cytokine Response Modifier A Inhibition of Initiator Caspases Results in Covalent Complex Formation and Dissociation of the Caspase Tetramer^*

József Dobó¹, Richard Swanson, Guy S. Salvesen^¶, Steven T. Olson, and Peter G. W. Gettins²

From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607, Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, Illinois 60612, and ^¶The Burnham Institute, La Jolla, California 92037

Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10)₂. The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8. Native PAGE showed formation of tight crmA-caspase complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided evidence for a covalent crmA-p20 thioester linkage. SDS-PAGE of isolated complexes showed near complete loss of the p10 subunit from initiator caspases 1 and 8 but not from the executioner caspase-6. This was confirmed for caspase-1 by sequencing and Western blotting. Size exclusion chromatography indicated a size of 60 kDa for complexes with caspases 1 and 8, consistent with a crmA·p20 species, suggesting that the p20-p10 interface and possibly the p10-p10 interface had been disrupted. In contrast, crmA·caspase-6 complex behaved as an equilibrium mixture of crmA₂·(p20-p10)₂ and crmA·(p20-p10). Complex deacylation rates were all slow, suggesting effective kinetic trapping of the covalent thioacyl intermediate. These results suggest a novel serpin inhibition mechanism through which crmA down-regulates apoptosis and inflammation. This involves (i) rapid formation of covalent complex with initiator caspases 8 or 1, (ii) very slow deacylation, and (iii) loss of the caspase p10 subunit for initiator but not for executioner caspases, so that any free p20 formed by deacylation of initiator caspases cannot reassociate to active heterotetramer, thus resulting in irreversible inhibition of apoptosis and inflammation.

Received for publication, May 30, 2006 , and in revised form, September 21, 2006.

^* This work was supported by National Institutes of Health Grants HL79430 (to P. G. W. G.) and HL78827 (to S. T. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary H-1113.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Illinois, 900 S. Ashland, M/C 669, Chicago, IL 60607. Tel.: 312-996-5534; E-mail: pgettins{at}uic.edu.

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