Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11
2020; Elsevier BV; Volume: 295; Issue: 32 Linguagem: Inglês
10.1074/jbc.ra120.014259
ISSN1083-351X
AutoresBetsaida Bibo‐Verdugo, Scott J. Snipas, Sonia Kołt, Marcin Poręba, Guy S. Salvesen,
Tópico(s)Streptococcal Infections and Treatments
ResumoPyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1′–P4′, the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1′–P4′ region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1′–P4′ region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1′–P4′-based inhibitors could be exploited to specifically target inflammatory caspases. Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1′–P4′, the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1′–P4′ region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1′–P4′ region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1′–P4′-based inhibitors could be exploited to specifically target inflammatory caspases. Caspases constitute a family of cysteine proteases that are obligate dimers in their active conformations (1Pop C. Salvesen G.S. Human caspases: activation, specificity, and regulation.J. Biol. Chem. 2009; 284 (19473994): 21777-2178110.1074/jbc.R800084200Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 2Boatright K.M. Salvesen G.S. Mechanisms of caspase activation.Curr. Opin. Cell Biol. 2003; 15 (14644197): 725-73110.1016/j.ceb.2003.10.009Crossref PubMed Scopus (1074) Google Scholar). 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Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar). However, analysis of the human protein substrates revealed a very different picture. Overall, gasdermin D was preferred to the prointerleukins by caspase-4 and caspase-5. However, whereas caspase-4 was more like caspase-1 in its cleavage of gasdermin D and pro-IL18, caspase-5 was more like caspase-11 (Fig. 3). However, caspase-4 does not cleave pro-IL1β, and caspase-5 cleaves it very poorly (Fig. 3). These data revealed that caspases signaling noncanonical pyroptosis prefer gasdermin D as a substrate. Based on the cleavage site sequence of the protein substrates, previous peptidyl substrate library screens have shown that P1–P4 specificity does not determine specificity for protein substrates in these caspases (caspase-4/-5/-11) (6Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1844) Google Scholar, 24Ramirez M.L.G. Poreba M. Snipas S.J. Groborz K. Drag M. Salvesen G.S. Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.J. Biol. Chem. 2018; 293 (29414788): 7058-706710.1074/jbc.RA117.001329Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Extended interactions at the active-site level beyond the P1–P4 sites are a possible mechanism for enhancing activity. An example of this is caspase-2, which has been demonstrated to cleave pentapeptides at rates 10–40 times higher than those of tetrapeptides (42Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. Substrate specificities of caspase family proteases.J. Biol. Chem. 1997; 272 (9092497): 9677-988210.1074/jbc.272.15.9677Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). Given our previous observations that such interactions occur outside the P1–P4 area and the faster processing of gasdermin D by caspase-11 compared with that of the pro-ILs, we hypothesized that interactions occur at positions outside but proximal to the P1–P4 sites. To investigate such potential interactions, we designed and synthesized extended internally quenched fluorescent (IQF) substrates containing different portions of the mouse gasdermin D sequence around the cleavage site within positions P7–P5′ (Table 1). Caspase-1 was far more efficient at cleaving these substrates than caspase-11 (Fig. 4). Like most caspases, caspase-1 is most influenced by P1–P4. In contrast, caspase-11 is more highly influenced by the prime-side amino acids P1′–P4′ (Fig. 4). These observations confirm that the prime side contains specificity determinants in substrate recognition by inflammatory caspases.Table 1Structures and mass spectrometry analysis of IQF substrates Open table in a new tab Figure 4Extended synthetic substrates revealed potential interactions with the prime side of gasdermin D. A, velocity plots of mouse inflammatory caspases toward internally quenched substrates spanning different lengths of the gasdermin D sequence around its cleavage site motif (indicated by an arrow). B, catalytic efficiency of caspase-1 and caspase-11 on these substrates is represented as kcat/Km. Fold change in kcat/Km was calculated relative to the least efficiently cleaved substrate. The table shows the mean and standard deviation from three technical replicates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the influence of the prime-side region of peptidyl substrates on the activity of caspase-11, we asked if these constitute specificity determinants for interaction with gasdermin D. Consequently, we predicted that introducing gasdermin D sequences from this region into pro-IL18 would make this a better substrate for caspase-11. The hypothesis was supported for caspase-11, where we observed a 40-fold increase in cleavage of the mutant containing P1′–P4′ substitution (Fig. 5). In contrast, we observed minimal enhancement in caspase-1 catalysis. We sought to pinpoint which particular subsite has the most influence on
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