Pore formation in regulated cell death

Review30 October 2020Open Access Pore formation in regulated cell death Hector Flores-Romero orcid.org/0000-0003-0996-5717 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Uris Ros orcid.org/0000-0002-8889-0145 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Ana J Garcia-Saez Corresponding Author [email protected] orcid.org/0000-0002-3894-5945 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Hector Flores-Romero orcid.org/0000-0003-0996-5717 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Uris Ros orcid.org/0000-0002-8889-0145 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, GermanyThese authors contributed equally to this work Search for more papers by this author Ana J Garcia-Saez Corresponding Author [email protected] orcid.org/0000-0002-3894-5945 Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany Search for more papers by this author Author Information Hector Flores-Romero1, Uris Ros1 and Ana J Garcia-Saez *,1 1Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany *Corresponding author. Tel: 49 0221 478 84263; E-mail: [email protected] EMBO J (2020)39:e105753https://doi.org/10.15252/embj.2020105753 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death. Pore formation in membranes is a conserved strategy to kill cells Biological membranes define biochemical environments and are fundamental for the existence of life (Deamer, 2016). Selective transport between these enclosed spaces is highly regulated and sustained disruption in membrane integrity is, as a consequence, a point of no return in cell death (Youle & Strasser, 2008; Kunzelmann, 2016; Broz et al, 2020). Yet, alterations in membrane permeability are relevant for a large number of biological processes including immunity, metabolism, and infection (Kagan, 2012; McCormack et al, 2013). A membrane pore can be defined as any local membrane perturbation that allows the passive flow of molecules (Schwarz & Robert, 1992). Pore-forming proteins (PFPs) represent a large and structurally diverse family of proteins that have the common function of altering membrane permeability by creating pores. They can act exogenously as secreted soluble proteins that permeabilize the plasma membrane of their target cells. This includes most pore-forming toxins (PFTs), which are some of the most potent virulence factors found in nature (Ros & Garcia-Saez, 2015; Dal Peraro & van der Goot, 2016) or perforin, which is released by cytotoxic T cells and Natural Killer cells (Voskoboinik et al, 2015; Prinz et al, 2020). PFPs can also be intracellular executioners as components of cell death signaling pathways (Espiritu et al, 2019; Flores-Romero et al, 2020). For instance, Bcl-2-associated X protein (BAX) and BCL2-antagonist/killer 1 (BAK) form pores that lead to mitochondrial outer membrane permeabilization (MOMP) during intrinsic apoptosis. Gasdermins (GSDMs) execute pyroptosis by a mechanism that culminates with pore opening at the plasma membrane. In necroptosis, mixed lineage kinase domain-like (MLKL) induces plasma membrane permeabilization through a yet unclear mechanism that could also be linked to pore formation (Liu et al, 2016; Cosentino & Garcia-Saez, 2017; Flores-Romero & Garcia-Saez, 2019a). PFPs are usually classified in α or β, depending on the secondary structure of the protein segments forming the pores (Ros & Garcia-Saez, 2015; Cosentino et al, 2016). In addition, PFPs can build different types of pores, as defined by the presence or absence of lipids in the pore structure (Ros & Garcia-Saez, 2015; Gilbert, 2016). They are classified as protein-lined if they are constituted only of proteins, as pure lipid pores, or as protein/lipid pores, when they contain both types of molecules. In protein-lined pores, the lumen is solely covered by transmembrane segments of proteins organized into α- or β-barrel “walls” (Dal Peraro & van der Goot, 2016; Gilbert, 2016). Importantly, in this type of pores, the membrane or certain membrane lipids can play a functional role in protein recruitment, assembly, and folding (Rojko & Anderluh, 2015; Gilbert, 2016). In contrast, membrane lipids together with the amphipathic regions of proteins or peptides form the edge of protein/lipid pores (Ludtke et al, 1996; Matsuzaki et al, 1996). Here, lipids rearrange from their bilayer distribution to a non-lamellar assembly that creates a continuous surface in which the membrane bends at the pore boundary like a torus. Protein/lipid or toroidal pore opening is promoted by proteins or protein fragments that generate membrane tension. PFP accumulation at or next to the pore rim then reduces the line tension and stabilizes the open pore state (Karatekin et al, 2003; Puech et al, 2003) (Fig 1). Pure lipid pores are also toroidal, but in the absence of proteins, their opening probability and lifetime are very low. They mainly occur upon strong membrane perturbations such as mechanical and electrical tension or osmotic swelling (Tieleman et al, 2003; Tieleman & Marrink, 2006). Figure 1. Mechanism of pore formation by cationic amphipathic peptides to exemplify the formation and stabilization of a toroidal pore (i) Pore-forming peptides bind avidly to the accessible interface of the lipid bilayer and occupy a volume only in the interfacial region, which causes asymmetric stretching and membrane thinning (∆h). As a consequence, the membrane is stressed and destabilized, so that defects in the lipid bilayer become more likely and eventually a pore is formed (ii). Once the pore is open, a line tension appears at the pore edge due to the extra energy cost associated with the reorientation of the lipids into a highly curved boundary. This tension increases with the pore perimeter and is therefore a line tension. The initial pore grows quickly as long as the membrane tension dominates. But as the pore size grows, so does the counterbalancing line tension too. Furthermore, with the open pore, the peptides redistribute in the membrane by diffusion through the pore to the other monolayer, which reduces the membrane tension due to asymmetric distribution of the peptides. At a certain moment, the line tension becomes predominant and the pore size starts to decrease. However, if the pore-forming peptides bound near the pore rim are able to reduce the line tension, an equilibrium can be reached with a smaller but stable pore (iii). R = radius, ∆h = change in the thickness of the membrane (absence vs. presence of the protein/peptide mass). Adapted from (Fuertes et al, 2011). Download figure Download PowerPoint In regulated cell death, a diverse repertoire of endogenous pro-death effectors reveals a plethora of strategies evolved to permeabilize cellular membranes. The architecture of the resulting pores is defined by specific protein and lipid compositions, as well as by intramolecular interactions. This determines pore heterogeneity, size, and stability, and thereby the type and extent of molecules that can be released through them. As a consequence, the properties of membrane pores impact the signaling cascades that are activated downstream of membrane permeabilization. BAX and BAK pores in apoptosis: old friends with new habits The toroidal pore of BAX and BAK Currently, it is well established that the BCL2 family members BAX, BAK, and perhaps BOK are the executioners of MOMP and thereby fundamental effectors of the intrinsic apoptotic pathway (Moldoveanu & Czabotar, 2019). In healthy cells, BAX and BAK exist as inactive, monomeric proteins that constitutively shuttle between cytosol and mitochondria, with BAX being mostly cytosolic and BAK mitochondria-associated (Edlich et al, 2011; Schellenberg et al, 2013; Todt et al, 2015; Lauterwasser et al, 2016). Upon apoptosis induction, both proteins accumulate at and insert into the mitochondrial membrane, undergo conformational rearrangements, oligomerize and form pores that release pro-apoptotic factors such as cytochrome c and SMAC/DIABLO (Nechushtan et al, 2001; Rehm et al, 2003; Zhou et al, 2005; Edlich et al, 2011). However, the relative order of events, the structural intermediates involved, as well as the molecular properties of the membrane openings mediated by BAX-type proteins remains controversial (Moldoveanu & Czabotar, 2019; Flores-Romero & Garcia-Saez, 2019a). Despite their functional heterogeneity, the 3D structures of all multidomain BCL2 proteins (including not only BAX, BAK, and BOK, but also the pro-survival family members) present the same globular α-helical fold, with a predominantly hydrophobic central hairpin that is flanked on both sides by pairs of amphipathic α-helices. This peculiar folding is strikingly similar to the pore-forming domain of bacterial α-PFTs such as the colicins and diphtheria toxin (Muchmore et al, 1996; Suzuki et al, 2000; Petros et al, 2004; Moldoveanu et al, 2013; Ke et al, 2018). Considering these structural similarities and their ability to allow currents through artificial membrane systems known as black lipid membranes, it was initially proposed that BAX-type proteins induce MOMP by generating membrane pores or channels (Minn et al, 1997; Sattler et al, 1997; Schlesinger et al, 1997; Basanez et al, 1999). Different observations have since then supported the hypothesis that active BAX and BAK form long-lived toroidal pores. In vitro, BAX decreases the lifetime of planar membranes and forms pores with variable conductance (Basanez et al, 1999) that are affected by the physical properties of the membrane and the presence of lipids with intrinsic monolayer curvature (Basanez et al, 2002; Terrones et al, 2004). A peptide derived from helix α5 of BAX forms membrane pores with lipid molecules in the lumen as demonstrated by X-ray diffraction (Qian et al, 2008) and conductance experiments in black lipid membranes (Garcia-Saez et al, 2005). This is in agreement with the transbilayer lipid movement coupled with membrane permeabilization induced by BAX (Epand et al, 2003; Garcia-Saez et al, 2006) (Fig 2A and B). These lines of evidence converge into the current view of a protein/lipid pore of tunable size constituted by BAX/BAK homodimers, where the size of BAX and BAK pores is not constant, but it evolves with time and depends on protein concentration. In this model, the central hairpin of helices α5 and α6 lies on the membrane surface at the edge of the pore (Qian et al, 2008; Basanez et al, 2012; Bleicken et al, 2013; Mandal et al, 2016; Cosentino & Garcia-Saez, 2017; Bleicken et al, 2018) (Fig 2A and B). Not only BAX/BAK molecules, but also the mechanical properties of the membrane play a role in the size and stability of the pores (Karatekin et al, 2003). Figure 2. BAX/BAK toroidal pore(A) Schematic representations of the protein/lipid model shown as a 3D view cut through the membrane pore. Gray layers represent lipid headgroups of the bilayer, the acyl chains are shown in red and protein helices by dark cylinders (top). The corresponding normalized electron density distributions of acyl chains in lipid bilayers containing BAX α5 (bottom). Note that, unlike in a protein channel, in a toroidal pore: (i) the surface of the pore is lined by lipid headgroups, (ii) membrane monolayers are bent at the pore edge, and (iii) the two leaflets of the bilayer become continuous. Taken from (Qian et al, 2008). (B) Structural representation of membrane-embedded BAX in the context of a toroidal pore based on (Bleicken et al, 2014; Bleicken et al, 2018). BAX is represented with nine cylinders corresponding to its nine helices. BH3 domain and C-terminal/tail anchoring domain are depicted in orange and green, respectively. One monomer is shown in gray (1–9) and the other is depicted in white (1ʹ–9ʹ). The relative orientation of the helices α9 remains unresolved. (C) BAX oligomers are organized into line, arc, and rings. Each panel shows the schematic representation (left) and the AFM images (right) of BAX assemblies. Both arcs and rings but not lines, reveal a circular depression (black) that spans the lipid membrane (dark orange). BAX molecules around the pore rim (yellow) protrude above the membrane plane, as confirmed by the height cross-sections shown below each image (corresponding to the white line in the AFM images). The topography of the arc structure reveals a pore only partially surrounded by BAX molecules, while lipids alone form the rest of the pore rim. Based on (Salvador-Gallego et al, 2016). (D) Model for the temporal control of content release during MOMP. Upon apoptotic stimuli, BAX and BAK permeabilize the MOM and induce the release of apoptotic factors, for example, cyt C. The consequent MIM permeabilization and the widening of BAX/BAK pores induce the release of mtDNA in the cytosol. In absence of caspase activity, this leads to the activation of the cGAS/STING signaling pathway. Based on (Cosentino & Garcia-Saez, 2018). Download figure Download PowerPoint According to the toroidal pore model, the initial asymmetric insertion of BAX α-helices into the cytosolic leaflet of the MOM stresses the membrane and generates membrane tension. Once a threshold tension is reached (which may be locally enhanced by protein concentration or oligomerization), the energy required to reorganize lipids out of the bilayer structure becomes thermally accessible and a pore opens, which dissipates membrane tension (Lee et al, 2004). Lipids reassemble into a torus around the membrane pore to avoid exposure of the hydrophobic acyl chains to the water environment. The two membrane monolayers form a continuous surface at the pore edge with negative curvature in the plane of the membrane and positive curvature in the plane perpendicular to the membrane. The bending of the lipids at the pore rim has an energetic cost that is directly proportional to the length of the pore, giving rise to line tension (Ludtke et al, 1996; Matsuzaki et al, 1996). Line tension acts as the driving force for pore closure and opposes membrane tension. As a result, toroidal pores are metastable structures whose lifetime is governed by the balance between membrane tension and line tension (Valcarcel et al, 2001; Yang et al, 2001; Karatekin et al, 2003; Fuertes et al, 2010b). In support of this model, the rim of BAX pores is formed by both lipid headgroups and protein molecules (Kuwana et al, 2016; Salvador-Gallego et al, 2016), with BAX molecules decreasing the line tension to maintain the pore stably open (Basanez et al, 1999; Garcia-Saez et al, 2007; Fuertes et al, 2010a; Fuertes, 2011; Unsay et al, 2017; Bleicken et al, 2018) (Figs 1 and 2B). BAX/BAK membrane topology in the context of the toroidal pore Once in the membrane, activated-BAX/BAK undergoes a rearrangement of their globular fold that implies first the opening of their N-terminal region and then, in the case of BAX, the dislodgment and transmembrane insertion of the tail anchoring domain in helix α9 (Nechushtan et al, 1999; Griffiths et al, 2001). This is followed by exposure of their BH3 domain, which is thought to occur concomitantly with the reorganization of BAX/BAK into two functionally different regions, namely the dimerization/core (helices α2–α5) and the piercing/latch (helices α6–α8) domains (Dewson et al, 2008; Dewson et al, 2012; Czabotar et al, 2013; Moldoveanu et al, 2013; Bleicken et al, 2014; Flores-Romero et al, 2017). Although the structural organization of BAX dimers in the membrane remains controversial (Westphal et al, 2014; Mandal et al, 2016; Cosentino & Garcia-Saez, 2017; Bleicken et al, 2018), some of the models proposed provide an explanation for how BAX/BAK may generate the membrane stress required for pore opening and alleviate the line tension for pore stabilization (Bleicken et al, 2014; Mandal et al, 2016; Fig 2B). Partial opening and insertion of the hairpin of helices α5–α6 in BAX/BAK dimers within the lipid headgroup region of the cytosolic leaflet of the MOM would initially generate positive curvature and membrane stress leading to pore opening (Czabotar et al, 2013; Bleicken et al, 2014). The crescent-like shape of these two α-helices in the context of the pore rim would then act as a scaffolding chaperone that stabilizes the high lipid curvature, decreases the line tension, and maintains the open pore state (Mandal et al, 2016; Bleicken et al, 2018; Fig 2B). The formation of high-order oligomers with such membrane disposition would potentiate the stabilizing effect (Subburaj et al, 2015). In agreement with this, atomic force microscopy (AFM) experiments on supported bilayers, electron microscopy (EM) assays in outer membrane vesicles (OMVs) and in lipid nanodiscs containing BAX confirmed protein enrichment at the rim of membrane pores of variable sizes. Remarkably, the pore wall was not completely covered by protein molecules (Xu et al, 2013; Subburaj et al, 2015; Kuwana et al, 2016; Salvador-Gallego et al, 2016) (Fig 2C). This evidence supports a model for BAX/BAK-mediated MOMP where oligomerization at the MOM induces the formation of heterogeneous toroidal pore structures of tunable size, which are flexible and evolve overtime leading to the release of apoptotic factors. BAX and BAK supramolecular structures and functions beyond MOMP Historically, the segregation of BAX and BAK into discrete puncta at mitochondria, also known as apoptotic foci, has been linked with the apoptotic phenotype (Nechushtan et al, 2001; Karbowski et al, 2002). High-resolution imaging techniques have recently allowed deciphering the riddles of these supramolecular structures, revealing distinct molecular architectures such as lines, arcs, and full rings (Grosse et al, 2016; Salvador-Gallego et al, 2016). Rings and arcs of BAX similar to those found at the MOM perforated the membrane in supported lipid bilayers indicating that these higher-order oligomerization states do not necessarily require other mitochondrial proteins (Fig 2C). BAX seems to oligomerize by subsequent addition of dimers (Subburaj et al, 2015), but the formation and evolution of these oligomers at the supramolecular level are vaguely understood. BAX molecules might first organize into linear and arc-shaped structures, with some of them evolving to complete rings. Alternatively, lines and arcs might correspond to kinetically trapped assemblies in the process of pore formation. Several inter-dimer binding surfaces have been described for BAX-type proteins, including helices α6 and α9 (Dewson et al, 2009; Zhang et al, 2010) or the interface of helices α3/α5 (Mandal et al, 2016), but none of them appears to be indispensable for BAX/BAK oligomerization. Considering that BAX/BAK self-assembly seems to be crucial for reducing line tension and pore stabilization, one could envision that the membrane itself may play a role in the organization and dynamics of BAX/BAK supramolecular arrangements by contributing to the state of minimal energy. In this scenario, the energy cost of the membrane perturbations (perhaps including initial pore opening) induced by the insertion of BAX dimers into the membrane could be reduced by the coalescence of these membrane alterations and associated BAX/BAK molecules. Such a model would explain the higher-order assembly of BAX/BAK dimers via membrane-mediated interactions (Harroun et al, 1999; Reynwar et al, 2007; Shlomovitz & Gov, 2009; Cowan et al, 2020) and provide a mechanistic basis for the pore growth observed during apoptosis (Riley et al, 2018; Flores-Romero & García-Sáez, 2020). Connected with BAX foci, it has been proposed that BAX and BAK pores of hundreds of nanometers in diameter can also induce mitochondrial inner membrane (MIM) permeabilization and extrusion into the cytosol, leading to the release of mitochondrial DNA (mtDNA) and the activation of cGAS/STING inflammatory pathway (McArthur et al, 2018; Riley et al, 2018; Fig 2D). These discoveries have challenged our understanding of the role of BAX/BAK in apoptosis beyond that of inducing MOMP for caspase activation, which now expands to inflammation and cell-to-cell communication. The mechanism how BAX and BAK promote MIM poration and mtDNA release remains unknown. While some studies suggest that monomers or dimers are sufficient to form functional pores in model membranes (Kushnareva et al, 2012; Xu et al, 2013), it seems unlikely that BAX/BAK monomers or dimers would be able to induce the large membrane disruptions required for mtDNA release. One could envision a scenario in which low order oligomers of BAX/BAK induce openings at the MOM that release cytochrome c and other proteins, while large supramolecular structures may enable other cell functions including mtDNA release and inflammatory responses. The continuous growth of BAX pores upon MOMP would thereby allow a controlled release of the mitochondrial components to regulate in time these alternative functions and with them, the inflammatory outcome of apoptosis (Cosentino & Garcia-Saez, 2018; McArthur et al, 2018; Riley et al, 2018). However, it is important to note that additional components are present at BAX/BAK foci at mitochondria, such as Dynamin-related protein 1 (Drp1) and Mitofusins (Karbowski et al, 2002; Ugarte-Uribe & Garcia-Saez, 2017; Ugarte-Uribe et al, 2018; Hertlein et al, 2020), Optic atrophy-1(OPA1)/ Metalloendopeptidase OMA1 (OMA1) (Yamaguchi et al, 2008; Jiang et al, 2014), or Voltage-Dependent Anion Channel (VDAC) (Lauterwasser et al, 2016; Kim et al, 2019), which might contribute to this phenomenon. As MIM permeabilization appears to occur after MOMP, the driving force that makes the mitochondrial interior squeeze out through BAX/BAK macro-pores might involve mechanical (osmotic) forces. This could possibly be due to or happen in combination with additional mitochondrial alterations like the dismantling of mitochondrial cristae. The mitochondrion-specific lipid cardiolipin (CL), which seems to regulate the action of several BCL2 proteins (Terrones et al, 2004; Landeta et al, 2014; Bleicken et al, 2017; Flores-Romero et al, 2019) and is also implicated in mitochondrial functions including organelle ultrastructure (Schlame & Ren, 2009), could also play a role in MOM/MIM permeabilization. Because of its unique structural properties (e.g., two negative charges, a relatively small head group and four acyl chains), CL can form highly curved inverted hexagonal structures (Grijalba et al, 1999; Ortiz et al, 1999; Unsay et al, 2013) and laterally segregate into defined nanodomains in vitro (Kawai et al, 2004; Sorice et al, 2009). These additional elements might modulate the formation, size, shape, and kinetics of BAX/BAK assemblies in apoptotic foci. At the functional level, it is reasonable to argue that the regulation of the extent and kinetics of BAX/BAK-induced MOMP may elicit different scenarios, which could be: (i) genomic instability and cancer, associated with partial release of cytochrome c and minority MOMP (Ichim et al, 2015), (ii) immunologically silent apoptosis, when MOMP, caspase activation and cell removal due to “eat me signals” are fast and complete (Depraetere, 2000; Singh et al, 2019), or (iii) immunologically active apoptosis in case of prolonged apoptotic signaling leading to mtDNA release and to activation of the cGAS/STING and perhaps Mitochondrial antiviral-signaling protein (MAVS) pathways (Cosentino & Garcia-Saez, 2018; McArthur et al, 2018; Riley et al, 2018; Flores-Romero & Garcia-Saez, 2019b). The regulation of these scenarios could be mechanistically related with the structural flexibility and dynamics of assembly of BAX/BAK structures, ranging from active monomer/dimer units to supramolecular structures with different sizes and shapes. Importantly, if the different oligomeric states of BAX and BAK exert different functions in the cell, their specific targeting could expand the application of BAX and BAK for therapy. Gasdermins are potent pore-formers at the core of pyroptosis The GSDM family is a new class of PFPs GSDMs represent a family of proteins that comprises six members in humans: GSDM A, B, C, D, and E (also known as DFNA5), and PJVK (also known as DFNB59), and ten in mice (GSDM A1-3, C1-4, D, E, and PJVK). Some of these proteins have proved to be essential for the highly inflammatory pathway of pyroptosis (Aglietti & Dueber, 2017; Galluzzi et al, 2018; Broz et al, 2020). Pyroptotic cell death is characterized by extensive cell swelling and membrane blebbing in absence of cell detachment (Chen et al, 2016; de Vasconcelos et al, 2019), which resembles the phenotype induced by PFTs during their attack to the plasma membrane of target cells (Garcia-Saez et al, 2011; Ros et al, 2017). This, together with the essential role of GSDMs in pyroptosis, suggested that GSDMs have an intrinsic and potent pore-forming activity that mediates osmotic lysis in pyroptosis. Accordingly, the N-terminal domain (GSDMNT) of GSDMs alone displays pore-forming activity in liposomes, which has been the basis to define GSDMs as the minimal machinery for pyroptosis execution (Aglietti et al, 2016; Ding et al, 2016; Liu et al, 2016). Intense research during the recent years has provided insight into the structure of GSDMs (Fig 3A–D). All GSDMs (except DFNB59) display a two-domain architecture formed by an N-terminal (GSDMNT) and a C-terminal (GSDMCT) domain, separated by a linker region (Fig 3A; Broz et al, 2020). The crystal structure of full-length GSDMA3 (Ding et al, 2016) and GSDMD (Kuang et al, 2017) revealed that the GSDMNT is inhibited by inter-domain interactions with juxtaposed regions of the GSDMCT. The α-helical fold of GSDMCT interacts with the helix α1 and a short β-hairpin located on the concave side of the β-sheet of GSDMNT. Additionally, the short α-helix at the end of the β-sheet of GSDMNT protrudes from the globular structure to interact with GSDMCT (Ding et al, 2016). For many GSDMs, caspase-mediated proteolytic processing induces the dissociation of the GSDMNT from its auto-inhibitory C-domain (Fig 3A; Kuang et al, 2017; Liu et al, 2018; Liu et al, 2019). Free monomers of GSDMNT translocate then to the inner leaflet of the plasma membrane and induce pyroptotic pores. Remarkably, the sequence and 3D-structure of all GSDMs differ significantly from any other known PFP (Ruan et al, 2018; Broz et al, 2020). Therefore, GSDMs have emerged as a new group of PFPs with a common function in pyroptosis. Figure 3. GSDMs pores evolve from toroidal to barrel structures(A) Crystal structure of GSDMA3 in its auto-inhibited form (PDB: 5B5R). The GSDMA3NT and GSDMA3CT domains are colored pink and blue, respectively. Inter-domain interactions between the GSDMA3NT and the GSDMA3CT keep the protein in an auto-inhibited state (Ding et al, 2016). (B) GSMDs involves the form arc-, slit, and ring-shaped GSDMDNT oligomers as imaged using time-resolved AFM (Mulvihill et al, 2018). (C) Cryo-EM structure of the GSDMA3 membrane pore (PDB: 6CB8). Atomic model of the 27-fold symmetric GSDMA3 pore at 3.8 Å resolution (Ruan et al, 2018). (D) Model of pore formation by GSDMs. After cleavage, monomers of GSDMNT translocate to the inner leaflet of the plasma membrane and then self-associate into arcs or slit structures that resemble toroidal pores and later evolve into ring-shape protein-lined pores with a β-barrel configuration. Download figure Download PowerPoint The structure of a GSDMA3NT pore was recently determined by cryo-EM (Fig 3C) (Ruan et al, 2018). Together with th