Molecular mechanisms of gasdermin D autoinhibition and recognition by inflammatory caspases

Abstract

Abstract Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The crystal structures of the human and murine GSDMD C-terminal domains differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis, in agreement with the role of the GSDMD C-terminal domain as an autoinhibition domain. We further demonstrate that the full-length GSDMD and its cleavage site peptide, FLTD, can directly bind the catalytic domains of inflammatory caspases. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, as well as suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes. 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. The present study not only contributes to our understanding of the distinct mode of GSDMD autoinhibition and recognition by inflammatory caspases, but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.