Molecular mechanisms of gasdermin D pore-forming activity

nature immunology Review article https://doi.org/10.1038/s41590-023-01526-w Molecular mechanisms of gasdermin D pore-forming activity Received: 22 February 2023 Pascal Devant & Jonathan C. Kagan Accepted: 3 May 2023 Published online: 5 June 2023 Check for updates The regulated disruption of the plasma membrane, which can promote cell death, cytokine secretion or both is central to organismal health. The protein gasdermin D (GSDMD) is a key player in this process. GSDMD forms membrane pores that can promote cytolysis and the release of interleukin-1 family cytokines into the extracellular space. Recent discoveries have revealed biochemical and cell biological mechanisms that control GSDMD pore-forming activity and its diverse downstream immunological effects. Here, we review these multifaceted regulatory activities, including mechanisms of GSDMD activation by proteolytic cleavage, dynamics of pore assembly, regulation of GSDMD activities by posttranslational modifications, membrane repair and the interplay of GSDMD and mitochondria. We also address recent insights into the evolution of the gasdermin family and their activities in species across the kingdoms of life. In doing so, we hope to condense recent progress and inform future studies in this rapidly moving field in immunology. Programmed cell death (PCD) has important roles in immune cell development and homeostasis, infection, autoimmunity and cancer. There are different forms of PCD, which can be classified as immunologically silent (such as apoptosis) or immunostimulatory (such as pyroptosis and necroptosis). A key difference between these forms of PCD is whether the plasma membrane of the dying cell becomes permeabilized. In the case of pyroptosis, the plasma membrane is permeabilized by proteins called gasdermins (GSDMs). Humans encode six GSDM proteins (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and pejvakin) with considerable sequence and structural homology. GSDMD is the best-characterized member of this family, and is a key mediator of inflammasome-dependent pyroptotic cell death1–3. GSDMD is activated by proteolytic cleavage, which releases its N-terminal domain (NT-GSDMD) to form membrane pores4–7. These pores can serve as channels to mediate interleukin-1 (IL-1) family cytokine secretion, as well as ion exchange8,9. In instances where the extent of pore formation exceeds the membrane reparative capacity of the cell, lytic cell death (that is, pyroptosis) ensues1,2. General aspects of GSDMD biology, including its role in immune defense to infection and its therapeutic potential have been excellently reviewed elsewhere10–14. This Review is focused on biochemical and cell biological mechanisms that control GSDM activities in species that span the kingdoms of life. Activation of GSDMD by inflammatory caspases In resting human and mouse cells, GSDMD exists as an inactive pro-protein in the cytoplasm. GSDMD consists of two domains: the C-terminal domain (CT-GSDMD) and the NT-GSDMD, connected by a flexible interdomain linker. Binding of CT-GSDMD to NT-GSDMD keeps the protein in an auto-inhibited state that cannot form membrane pores. Pore-forming activity is achieved upon proteolytic cleavage at site Asp275 or Asp276 within the linker domain of human or mouse GSDMD, respectively7,15. Proteolytic activation of GSDMD is carried out primarily by a class of proteases known as inflammatory caspases, including caspase-1, caspase-4 and caspase-5 in humans as well as caspase-11 in mice. Caspase-1 becomes activated upon recruitment into an inflammasome. Inflammasomes are one of a growing number of supramolecular organizing centers (SMOCs), which represent the signaling organelles of the innate immune system16. Like all SMOCs, inflammasomes are not present in resting cells, but they are assembled upon infection or metabolic dysregulation of various sorts17. Caspase-4, caspase-5 and caspase-11 also cleave and activate GSDMD pore-forming activities, but these enzymes are not typically activated upon recruitment into inflammasomes. Rather, these caspases are activated to cleave GSDMD upon recognition of bacterial lipopolysaccharides (LPS) in the cytosol. This process can lead to the downstream Division of Gastroenterology, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA. Nature Immunology | Volume 24 | July 2023 | 1064–1075 e-mail: 1064 Review article activation of the NLRP3 inflammasome through GSDMD-mediated K+ efflux (often referred to as the noncanonical pathway of inflammasome activation)18–20. Dissection of enzyme–substrate interactions has revealed mechanisms of GSDMD cleavage by inflammatory caspases. Upon activation, caspases undergo autoprocessing at several sites (for example, Asp270 and Asp289 in caspase-4, or Asp285 in caspase-11) to remove an inhibitory interdomain linker. Cleavage at these sites generates the small (p10) catalytic subunit and facilitates formation of the enzymatically active caspase dimer21. This dimer can then bind to full-length GSDMD through a protease exosite interaction. X-ray crystallography has shown that the processed L2 loops in the caspase dimer form an intermolecular beta-sheet, harboring a cluster of conserved hydrophobic residues (Trp267 and Val291 in caspase-4 or Trp263 and Val287 in caspase-11), which inserts into a hydrophobic pocket in CT-GSDMD formed by Leu306, Leu310, Val367 and Leu370 (refs. 21,22). Moreover, a structure of caspase-1 bound to full-length GSDMD suggests the presence of a second binding interface between the interdomain linker of GSDMD and the peptide-binding groove near the caspase active site22,23. These interactions position the interdomain linker of GSDMD for cleavage. Although no contacts are observed between NT-GSDMD and the caspase in this structure, it was suggested that a RFWK motif in the β1–β2 loop of NT-GSDMD can bind and inhibit inflammatory caspases after cleavage, acting in a negative feedback loop24. Consistent with the structural evidence, biochemical experiments suggest that the amino acid residues directly downstream of the scissile peptide bond (called ‘prime site’ residues) in GSDMD impact substrate recognition, while cleavage is independent of the residues directly upstream of Asp276/ Asp275 (the ‘tetrapeptide’)21,25. This exosite-mediated mode of substrate recognition appears to be specific to GSDMD, as mutations in the exosite have little effect on the cleavage of another prominent caspase-1 substrate, pro-IL-1β26. Mutations that modulate pro-IL-1β cleavage have little effect on GSDMD processing, suggesting that GSDMD and pro-IL-1β are recognized by distinct regions of caspase-1 (ref. 26). Inflammatory caspase-independent activation In addition to inflammatory caspases, other proteases can cleave GSDMD and activate its pore-forming activities (Fig. 1). For example, cathepsin G and elastase can generate a pore-forming NT-GSDMD fragment in neutrophils27,28. GSDMD activated in this manner is thought to c (...truncated)