Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death

Abstract

Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd(-/-) cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

“…PI influx was not affected by any of the osmoprotectants. IL‐1β release was also partially affected by PEG treatment (Appendix Fig S1E); consistent with the observation that pyroptosis is required for efficient release of the mature cytokine in BMDMs (Shi et al , 2015). Overall, these experiments suggest that GSDMD‐dependent pyroptosis involves the formation of a plasma membrane pore with an inner diameter of over 3.5 nm, the estimated molecular size of PEG3000 (Scherrer & Gerhardt, 1971).…”

“…Thermal denaturation showed a melting point of 43°C, indicating that the protein is folded and can be thermally denatured. Upon incubation with different human caspases, we confirmed that recombinant GSDMD is cleaved by caspase‐1, but not by the apoptotic caspase‐3 or caspase‐8 (Shi et al , 2015). Recombinant GSDMD is thus a functional substrate of its native enzyme.…”

“…Although the morphological features of pyroptotic cell death indicated the formation of a plasma membrane pore and subsequent lysis of the cell (Lamkanfi, 2011), the molecules involved in pyroptosis induction had remained elusive. The recent identification of GSDMD as an essential mediator of pyroptosis significantly expanded our understanding of pyroptotic cell death (He et al , 2015; Kayagaki et al , 2015; Shi et al , 2015), but whether after cleavage by caspases GSDMD executes pyroptosis directly, or by initiating other signaling events remained unclear. In this work, we have shown that the N‐terminal fragment of GSDMD, GSDMD Nterm , can form pores with average diameters of 21 nm in artificial liposomes, as well as large pores in the plasma membrane of cells (Figs 5 and 6).…”

“…Several landmark studies have recently identified GSDMD (gasdermin D), a member of the gasdermin protein family, as an essential mediator of pyroptosis in human and murine cells (He et al , 2015; Kayagaki et al , 2015; Shi et al , 2015). GSDMD is required for pyroptosis induction after canonical and non‐canonical inflammasome activation and is processed by caspase‐1, caspase‐11, caspase‐4, and caspase‐5, but not by apoptotic caspases.…”

“…GSDMD is required for pyroptosis induction after canonical and non‐canonical inflammasome activation and is processed by caspase‐1, caspase‐11, caspase‐4, and caspase‐5, but not by apoptotic caspases. The N‐terminal fragment of GSDMD (GSDMD Nterm ) was found sufficient to induce cell death with the morphological features of pyroptosis (Kayagaki et al , 2015; Shi et al , 2015), and overexpression of the C‐terminal domain GSDMD Cterm was found to block GSDMD Nterm ‐dependent cell death (Shi et al , 2015). These results gave rise to the hypothesis that caspase‐dependent cleavage releases the N‐terminal domain from an inhibitory interaction with the C‐terminus, allowing GSDMD Nterm to induce cell death by a yet undefined mechanism.…”