Molecular mechanisms of gasdermin D pore-forming activity

Abstract

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.

Fig. 1 |. Cleavage of GSDMD by diverse cellular proteases activates its pore-forming activity.

Pathogenic viral or bacterial infections trigger diverse innate immune pathways that activate proteases with the ability to activate GSDMD. Cytosolic LPS, a result of gram-negative bacterial infection, can be sensed by caspase-4, caspase-5 or caspase-11. Inflammasome activation can activate caspase-1. TNF receptor (TNFR) signaling (especially through TNF complex IIb) can activate caspase-8. Upon their activation, these proteases can cleave GSDMD within its interdomain linker connecting its C-terminal and N-terminal domains. In addition, proteases encoded by some infectious agents can similarly cleave and activate GSDMD. NT-GSDMD can then translocate to the plasma membrane, mitochondria, lysosomes and perhaps other membranous organelles and assemble into transmembrane pores. Apoptotic signaling can activate caspase-3 or caspase-7, which cleave GSDMD within its N terminus to inhibit its pore-forming abilities.

Fig. 2 |. Assembly of the GSDMD pore can proceed along various paths.

After proteolytic cleavage, NT-GSDMD monomers translocate and bind to the target membrane. NT-GSDMD monomers can then oligomerize into a membrane-associated ring-shaped pre-pore. Upon concerted insertion of the NT-GSDMD subunits into the membrane, the internal lipid plug will deform into a vesicle or bicelle, which leaves the now fully assembled transmembrane pore. Alternatively, individual NT-GSDMD subunits can insert into the membrane and assemble into smaller oligomers. Oligomers of two or more copies of NT-GSDMD will perforate the membrane and allow for ion fluxes. Smaller oligomers can assemble into arc-shaped pores, which may allow small proteins to traverse the membrane. Arc-shaped pores can collapse into smaller slit-shaped pores as a result of the line tension of the excluded lipid bilayer, or further grow and fuse into a full ring-shaped pore.

Fig. 3 |. Evolutionary history of the GSDM protein family.

a, All GSDMs are likely derived from one common ancestor. This pore-forming proto-GSDM diversified to give rise to all GSDM proteins known today in prokaryotes, fungi and metazoans. GSDME is the most ancient GSDM found in metazoans. Gene duplications in early vertebrates, amniotes and mammals resulted in the genes encoding pejvakin, GSDMA and GSDMB/GSDMC/GSDMD, respectively. GSDMs appear to be absent from plants and ecdysozoa (such as insects and nematodes). b, GSDMs from various kingdoms of life can be activated by proteolysis. GSDMs found in metazoans are typically cleaved by proteases such as caspases or granzymes upon pathogen infection. Fungal GSDMs (such as HET-Q1), which are involved in allorecognition, can be cleaved and activated by subtilisin-like proteases (such as HET-Q2). bGSDMs can be cleaved, for example, by caspase-like CHAT proteases, which are activated in response to phage infection. Proteolytic cleavage of these diverse GSDMs leads to membrane pore formation and cell death.