1.C.123 The Pore-forming Gasdermin (Gasdermin) Family 

Pyroptosis (cell death with inflamation) was long regarded as caspase-1-mediated monocyte death in response to certain bacterial insults. Caspase-1 is activated upon various infectious and immunological challenges through different inflammasomes. The discovery of caspase-11/4/5 function in sensing intracellular lipopolysaccharide expanded the spectrum of pyroptosis mediators and also revealed that pyroptosis is not cell type specific. The gasdermin (GSDM) family consists of gasdermin A (GSDMA), B (GSDMB), C (GSDMC), D (GSDMD), E or DNFA5 (GSDME), and DFNB59 in humans. Expressed in the skin, gastrointestinal tract, and various immune cells, GSDMs mediate homeostasis and inflammation upon activation by caspases and unknown proteases (Xia et al. 2019).The electrostatic influence of IL-1 transport exerted by the GSDMD pore has been documented and reveals extrinsic factors, including lipid and salt, that affect the electrostatic environment (Xie et al. 2022). Gasdermin D  is a critical pore-forming effector protein that mediates pro-inflammatory cytokine secretion via releasing its N terminal fragments to form transmembrane pores (Tang et al. 2022). GSDMD is a novel marker for macrophage activation syndrome (MAS) complications and a promising target for MAS treatment (Tang et al. 2022).

The pyroptosis executioner, gasdermin D (GSDMD), is a substrate of both caspase-1 and caspase-11/4/5 and is in the large gasdermin family bearing membrane pore-forming activity (Shi et al. 2016). Thus, pyroptosis is defined as gasdermin-mediated programmed necrosis.  These proteins are associated with various genetic diseases (Burdette et al. 2021). The primary function of pyroptosis is to induce strong inflammatory responses that defend the host against microbe infection. Excessive pyroptosis, however, leads to several inflammatory diseases, including sepsis and autoimmune disorders. Pyroptosis can be canonical or noncanonical. Upon microbe infection, the canonical pathway responds to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), while the noncanonical pathway responds to intracellular lipopolysaccharides (LPS) of Gram-negative bacteria. The last step of pyroptosis requires the cleavage of gasdermin D (GsdmD) at D275 (numbering after human GSDMD) into N- and C-termini by caspase 1 in the canonical pathway and caspase 4/5/11 (caspase 4/5 in humans, caspase 11 in mice) in the noncanonical pathway. Upon cleavage, the N-terminus of GsdmD (GsdmD-N) forms a transmembrane pore that releases cytokines such as IL-1beta and IL-18 and disturbs the regulation of ions and water, eventually resulting in strong inflammation and cell death. Since GsdmD is the effector of pyroptosis, promising inhibitors of GsdmD have been developed for inflammatory diseases (Burdette et al. 2021).

The N-terminal domain of Gasdermin-D promotes pyroptosis in response to microbial infection and danger signals. The active protein is produced by the cleavage of gasdermin-D by an inflammatory caspase, CASP1 or CASP4, in response to canonical, as well as non-canonical (such as cytosolic LPS) inflammasome activators (Shi et al. 2015; Kayagaki et al. 2015; Sborgi et al. 2016). After cleavage, the product moves to the plasma membrane where it binds to inner leaflet lipids, including monophosphorylated phosphatidylinositols, as well as phosphatidic acid and phosphatidylserine (Ding et al. 2016). Homooligomerization within the membrane generates pores of 10 - 15 nanometers (nm) (inner diameter), allowing the release of mature IL1B and triggering pyroptosis (Sborgi et al. 2016; Ding et al. 2016). It thus exhibits bactericidal activity. The N-terminal domain of Gasdermin-D, released from pyroptotic cells into the extracellular milieu rapidly binds to and kills both Gram-negative and Gram-positive bacteria, without harming neighboring mammalian cells, as it does not disrupt the plasma membrane from the outside due to lipid-binding specificity (Ding et al. 2016). It strongly binds to bacterial and mitochondrial lipids, including cardiolipin but does not bind to unphosphorylated phosphatidylinositol, phosphatidylethanolamine or phosphatidylcholine (Ding et al. 2016).

Once inserted, GSDMD^Nterm assembles arc-, slit-, and ring-shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore forming process is independent of whether GSDMD has been cleaved by caspase-1, caspase-4, or caspase-5. Using time-lapse AFM, Mulvihill et al. 2018 monitored how GSDMD^Nterm assembles into arc-shaped oligomers that can transform into larger slit-shaped and finally into stable ring-shaped oligomers. The mechanism of GSDMD^Nterm transmembrane pore assembly is likely shared with other members of the gasdermin protein family. Granzyme A from cytotoxic lymphocytes cleaves gasdermin B (GSDMB) to trigger pyroptosis in target cells via oligomeric pore formation (Zhou et al. 2020).  Inflammasome-mediated activation of inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11) initiates a cascade of cellular events that lead to proinflammatory cell death, or pyroptosis. Proteolytic cleavage of gasdermin D results in the formation of transmembrane pores that allow the release of mature cytokines IL-1β and IL-18. Gasdermin pores also allow calcium influx through the plasma membrane, triggering the fusion of lysosomal compartments with the cell surface and release of their contents into the extracellular milieu in a process termed lysosome exocytosis (Loomis and Bergsbaken 2023).

The gasdermin family of pore-forming proteins (PFPs) includes key molecular players controlling immune-related cell death in mammals (see above). Characterized mammalian gasdermins are activated through proteolytic cleavage by caspases or serine proteases, which remove an inhibitory carboxy-terminal domain, allowing pore-formation. Processed gasdermins form transmembrane pores, permeabilizing the plasma membrane, which often results in lytic and inflammatory cell death. While gasdermin-dependent cell death (pyroptosis) has been predominantly characterized in mammals, gasdermins also control cell death in early vertebrates (teleost fish) and invertebrate animals such as corals (Cnidaria) as well as fungi and bacteria. Fungal and bacterial gasdermins share many features with mammalian gasdermins including their mode of activation through proteolysis. In some cases the proteolytic activation is executed by evolutionarily related proteases acting downstream of proteins resembling immune receptors controlling necroptosis in mammals. Thus, gasdermins and gasdermin-regulated cell death is an ancient mechanism of cellular suicide. Daskalov and Louise Glass 2021 reviewed the broader gasdermin family, focusing on discoveries in invertebrates, fungi and bacteria.

The discovery of gasdermin D (GSDMD) as the terminal executioner of pyroptosis provided a large piece of the cell death puzzle (Greenwood et al. 2023). In its purest form, GSDMD provides a connection between the innate alarm systems to an explosive, inflammatory form of cell death to jolt the local environment into immunological action. Gasdermins regulate and are regulated by mechanisms such as autophagy, metabolism and NETosis in fighting pathogen and protecting the host. Importantly, activators and interactors of the other gasdermins, not just GSDMD, have been demonstrated.  Greenwood et al. 2023 have reviewed some recently discovered areas in relation to bacterial infection before providing an overview of the pharmacological landscape and the challenges associated with targeting gasdermins. Mitochondrial reactive oxygen species initiate gasdermin D-mediated pyroptosis and contribute to paraquat-induced nephrotoxicity (Chen et al. 2024).

Pyroptosis, characterized by its proinflammatory nature, is driven by the accumulation of large plasma membrane pores comprised of gasdermin family protein subunits. In different contexts of cellular homeostatic perturbations, infections and tissue damage, proteases, such as caspase-1 and caspase-4/5, play pivotal roles in pyroptosis by cleaving gasdermins. Gasdermin-D (GSDMD), the most extensively studied member of the gasdermin protein family, is expressed in various immune cells and certain epithelial cells. Upon cleavage by caspases, GSDMD oligomerizes and forms transmembrane pores in the cell membrane, leading to release of pro-inflammatory cytokines. GSDMD-N, the N-terminal fragment, displays an affinity for specific lipids, contributing to its role in pore formation in pyroptosis (Imre 2024).  Pyroptotic executioner pore-forming protein gasdermin D forms an oligomeric assembly and exhibits amyloid-like attributes that could contribute for its pore-forming function (Chatterjee et al. 2024).

The reactions catalyzed by gasdermins are:

Solutes (in) → Solutes (out)

Ca²⁺ (in) → Ca²⁺ (out)

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