Abstract
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 approximately 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 crmA(2).(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.
Highlights
Caspases are intracellular cysteine proteinases involved in inflammation and apoptosis [1]
To better understand how crmA is used by the cowpox virus to abrogate apoptosis and inflammation, we have characterized the complexes formed between crmA and three caspases by SEC, PAGE, mass spectrometry, Western blotting, sequencing, and kinetic assays
It seems likely that this tetramer disruption is an additional ammonio)-propanesulfonic acid; DTT, dithiothreitol; GSSG, oxidized glutathione; MALDI-TOF, matrix-assisted laser desorption ionization time-offlight mass spectrometry; MES, (2-(N-morpholino)ethanesulfonic acid; RCL, reactive center loop; SEC, size exclusion chromatography; Bicine, N,Nbis(2-hydroxyethyl)glycine; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline; BSA, bovine serum albumin
Summary
Caspases are intracellular cysteine proteinases involved in inflammation and apoptosis [1]. Caspase-1, known as interleukin-1 converting enzyme (ICE), activates the pro-in- Orthopox viruses, such as the cowpox virus, enhance their infectivity through specific inhibition of caspases and consequent abrogation of the inflammatory response and of apoptosis [12]. They do this using an inhibitor that is a member of the serpin family [13]. To better understand how crmA is used by the cowpox virus to abrogate apoptosis and inflammation, we have characterized the complexes formed between crmA and three caspases by SEC, PAGE, mass spectrometry, Western blotting, sequencing, and kinetic assays. Serpin-Caspase Complexes means of ensuring irreversible inhibition of apoptosis initiation or inflammation. The complex with the downstream executioner caspase-6, showed much less perturbation of the interface between the p10 subunits, which resulted in an equilibrium mixture of crmA21⁄7(p20-p10) and crmA1⁄7(p20-p10) species
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
