Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their b1-b2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, -acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1β release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

Background: Myocardial apoptosis is heavily implicated in the myocardial damage caused by ischemia-reperfusion (I/R). Toll-like receptor 4 (TLR4) is a potent inducer of these apoptotic cascades. In contrast, the radioprotective 105 kDa protein (RP105) is a specific negative regulator of TLR4 signaling pathways. However, the precise mechanisms by which RP105 inhibits myocardium apoptosis via TLR4-associated pathways during I/R is not fully understood. Methods: We utilized a rat model of myocardial ischemic reperfusion injury (MIRI). Animals were pre-treated with Ad-EGFP adenovirus, Ad-EGFP-RP105 adenovirus, saline, or nothing (sham). After three days, rats underwent a 30min left anterior descending coronary artery occlusion and a 4h reperfusion. Mycardial tissue was assessed by immunohistochemistry, TUNEL-staining, Western blot, quantitative RT-PCR, and a morphometric assay. Results: RP105 overexpression resulted in a reduction in infarct size, fewer TUNEL-positive cardiomyocytes, and a reduction in mitochondrial-associated apoptosis cascade activity. Further, RP105 overexpression repressed I/R-induced myocardial injury by attenuating myocardial apoptosis. This was mediated by inhibiting TLR4 activation and the phosphorylation of P38MAPK and the downstream transcription factor AP-1. Conclusion: RP105 overexpression leads to the de-activation of TLR4, P38MAPK, and AP-1 signaling pathways, and subsequently represses apoptotic cascades and ensuing damage of myocardial ischemic reperfusion. These findings may become the basis of a novel therapeutic approach for reducing of cardiac damage caused by MIRI.

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