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, GSDMDNterm 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 GSDMDNterm assembles into arc-shaped oligomers that can transform into larger slit-shaped and finally into stable ring-shaped oligomers. The mechanism of GSDMDNterm 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. 

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).

 

The reactions catalyzed by gasdermins are:

Solutes (in) → Solutes (out)

Ca2+ (in) → Ca2+ (out)



This family belongs to the Gasdermin Superfamily.

 

References:

Burdette, B.E., A.N. Esparza, H. Zhu, and S. Wang. (2021). Gasdermin D in pyroptosis. Acta Pharm Sin B 11: 2768-2782.

Collin, R.W., E. Kalay, J. Oostrik, R. Caylan, B. Wollnik, S. Arslan, A.I. den Hollander, Y. Birinci, P. Lichtner, T.M. Strom, B. Toraman, L.H. Hoefsloot, C.W. Cremers, H.G. Brunner, F.P. Cremers, A. Karaguzel, and H. Kremer. (2007). Involvement of DFNB59 mutations in autosomal recessive nonsyndromic hearing impairment. Hum Mutat 28: 718-723.

Daskalov, A. and N. Louise Glass. (2021). Gasdermin and gasdermin-like pore-forming proteins in invertebrates, fungi and bacteria. J. Mol. Biol. 167273. [Epub: Ahead of Print]

Delmaghani, S., F.J. del Castillo, V. Michel, M. Leibovici, A. Aghaie, U. Ron, L. Van Laer, N. Ben-Tal, G. Van Camp, D. Weil, F. Langa, M. Lathrop, P. Avan, and C. Petit. (2006). Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy. Nat. Genet. 38: 770-778.

Ding, J., K. Wang, W. Liu, Y. She, Q. Sun, J. Shi, H. Sun, D.C. Wang, and F. Shao. (2016). Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535: 111-116.

Du, G., L.B. Healy, L. David, C. Walker, P. Fontana, Y. Dong, P. Devant, R. Puthenveetil, S.B. Ficarro, A. Banerjee, J.C. Kagan, J. Lieberman, and H. Wu. (2023). ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation. bioRxiv.

Greenwood, C.S., M.A. Wynosky-Dolfi, A.M. Beal, and L.M. Booty. (2023). Gasdermins assemble; recent developments in bacteriology and pharmacology. Front Immunol 14: 1173519.

Hergueta-Redondo, M., D. Sarrió, &.#.1.9.3.;. Molina-Crespo, D. Megias, A. Mota, A. Rojo-Sebastian, P. García-Sanz, S. Morales, S. Abril, A. Cano, H. Peinado, and G. Moreno-Bueno. (2014). Gasdermin-B promotes invasion and metastasis in breast cancer cells. PLoS One 9: e90099.

Hu, Y., B. Wang, S. Li, and S. Yang. (2021). Pyroptosis, and its Role in Central Nervous System Disease. J. Mol. Biol. 167379. [Epub: Ahead of Print]

Imre, G. (2024). Pyroptosis in health and disease. Am. J. Physiol. Cell Physiol. [Epub: Ahead of Print]

Johnson, A.G., T. Wein, M.L. Mayer, B. Duncan-Lowey, E. Yirmiya, Y. Oppenheimer-Shaanan, G. Amitai, R. Sorek, and P.J. Kranzusch. (2022). Bacterial gasdermins reveal an ancient mechanism of cell death. Science 375: 221-225.

Kayagaki, N., I.B. Stowe, B.L. Lee, K. O''Rourke, K. Anderson, S. Warming, T. Cuellar, B. Haley, M. Roose-Girma, Q.T. Phung, P.S. Liu, J.R. Lill, H. Li, J. Wu, S. Kummerfeld, J. Zhang, W.P. Lee, S.J. Snipas, G.S. Salvesen, L.X. Morris, L. Fitzgerald, Y. Zhang, E.M. Bertram, C.C. Goodnow, and V.M. Dixit. (2015). Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526: 666-671.

Kim, M.S., X. Chang, K. Yamashita, J.K. Nagpal, J.H. Baek, G. Wu, B. Trink, E.A. Ratovitski, M. Mori, and D. Sidransky. (2008). Aberrant promoter methylation and tumor suppressive activity of the DFNA5 gene in colorectal carcinoma. Oncogene 27: 3624-3634.

Korn, V. and K. Pluhackova. (2022). Not sorcery after all: Roles of multiple charged residues in membrane insertion of gasdermin-A3. Front Cell Dev Biol 10: 958957.

Lin, H.Y., P.H. Lin, S.H. Wu, and L.T. Yang. (2015). Inducible expression of gasdermin A3 in the epidermis causes epidermal hyperplasia and skin inflammation. Exp Dermatol 24: 897-899.

Loomis, W.P. and T. Bergsbaken. (2023). Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis. Methods Mol Biol 2641: 171-178.

Mari, S.A., K. Pluhackova, J. Pipercevic, M. Leipner, S. Hiller, A. Engel, and D.J. Müller. (2022). Gasdermin-A3 pore formation propagates along variable pathways. Nat Commun 13: 2609.

Masuda, Y., M. Futamura, H. Kamino, Y. Nakamura, N. Kitamura, S. Ohnishi, Y. Miyamoto, H. Ichikawa, T. Ohta, M. Ohki, T. Kiyono, H. Egami, H. Baba, and H. Arakawa. (2006). The potential role of DFNA5, a hearing impairment gene, in p53-mediated cellular response to DNA damage. J Hum Genet 51: 652-664.

Muendlein, H.I., D. Jetton, W.M. Connolly, K.P. Eidell, Z. Magri, I. Smirnova, and A. Poltorak. (2020). cFLIP protects macrophages from LPS-induced pyroptosis via inhibition of complex II formation. Science 367: 1379-1384.

Mulvihill, E., L. Sborgi, S.A. Mari, M. Pfreundschuh, S. Hiller, and D.J. Müller. (2018). Mechanism of membrane pore formation by human gasdermin-D. EMBO. J. [Epub: Ahead of Print]

Op de Beeck, K., G. Van Camp, S. Thys, N. Cools, I. Callebaut, K. Vrijens, L. Van Nassauw, V.F. Van Tendeloo, J.P. Timmermans, and L. Van Laer. (2011). The DFNA5 gene, responsible for hearing loss and involved in cancer, encodes a novel apoptosis-inducing protein. Eur J Hum Genet 19: 965-973.

Sborgi, L., S. Rühl, E. Mulvihill, J. Pipercevic, R. Heilig, H. Stahlberg, C.J. Farady, D.J. Müller, P. Broz, and S. Hiller. (2016). GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death. EMBO. J. 35: 1766-1778.

Shi, J., W. Gao, and F. Shao. (2016). Pyroptosis: Gasdermin-Mediated Programmed Necrotic Cell Death. Trends. Biochem. Sci. [Epub: Ahead of Print]

Shi, J., Y. Zhao, K. Wang, X. Shi, Y. Wang, H. Huang, Y. Zhuang, T. Cai, F. Wang, and F. Shao. (2015). Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526: 660-665.

Tang, S., C. Yang, S. Li, Y. Ding, D. Zhu, S. Ying, C. Sun, Y. Shi, J. Qiao, and H. Fang. (2022). Genetic and pharmacological targeting of GSDMD ameliorates systemic inflammation in macrophage activation syndrome. J Autoimmun 133: 102929.

Wang, Y., W. Gao, X. Shi, J. Ding, W. Liu, H. He, K. Wang, and F. Shao. (2017). Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547: 99-103.

Xia, S., J. Ruan, and H. Wu. (2019). Monitoring gasdermin pore formation in vitro. Methods Enzymol 625: 95-107.

Xia, S., Z. Zhang, V.G. Magupalli, J.L. Pablo, Y. Dong, S.M. Vora, L. Wang, T.M. Fu, M.P. Jacobson, A. Greka, J. Lieberman, J. Ruan, and H. Wu. (2021). Gasdermin D pore structure reveals preferential release of mature interleukin-1. Nature. [Epub: Ahead of Print]

Xie, W.J., S. Xia, A. Warshel, and H. Wu. (2022). Electrostatic influence on IL-1 transport through the GSDMD pore. Proc. Natl. Acad. Sci. USA 119:.

Zhang, Z., Y. Zhang, S. Xia, Q. Kong, S. Li, X. Liu, C. Junqueira, K.F. Meza-Sosa, T.M.Y. Mok, J. Ansara, S. Sengupta, Y. Yao, H. Wu, and J. Lieberman. (2020). Gasdermin E suppresses tumour growth by activating anti-tumour immunity. Nature 579: 415-420.

Zhou, Z., H. He, K. Wang, X. Shi, Y. Wang, Y. Su, Y. Wang, D. Li, W. Liu, Y. Zhang, L. Shen, W. Han, L. Shen, J. Ding, and F. Shao. (2020). Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science 368:.

Zihlif, M., N.M. Obeidat, N. Zihlif, T. Mahafza, T. Froukh, M.T. Ghanim, H. Beano, F.M. Al-Akhras, and R. Naffa. (2016). Association Between Gasdermin A and Gasdermin B Polymorphisms and Susceptibility to Adult and Childhood Asthma Among Jordanians. Genet Test Mol Biomarkers 20: 143-148.

Examples:

TC#NameOrganismal TypeExample
1.C.123.1.1

Pore-forming Gasdermin D (Gasdermin-A3, GSDMD, DFNA5L, GSDMDC1, FKSG10) of 484 aas (Ding et al. 2016). GSDMD is activated by inflammasome-activated caspases-1/-4/-5/-11 as well as a caspase-8-mediated pathway during Yersinia infection. These caspases cleave GSDMD to release its functional N-terminal fragment (GSDMD-NT) from its auto-inhibitory C-terminal fragment (GSDMD-CT). GSDMD-NTs bind to acid lipids in mammalian cell membranes and bacterial membranes, oligomerize, and insert into the membranes to form large transmembrane pores. Consequently, cellular contents including inflammatory cytokines are released (e.g., IL-1β), and cells can undergo pyroptosis, a highly inflammatory form of cell death (Xia et al. 2019; Muendlein et al. 2020). As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D. Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1beta and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis. Cryo-EM structures of the pore and the prepore reveal the different conformations of the two states, as well as membrane-binding elements including a hydrophobic anchor and three positively charged patches. The pore conduit is predominantly negatively charged, but IL-1 precursors have an acidic domain that is proteolytically removed by caspase-1. When permeabilized, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favor the passage of IL-1beta and IL-18 over that of their precursors (Xia et al. 2021). Gasdermin-A3 oligomers assemble on the membrane surface where they remain attached and mobile. Once inserted into the membrane it grows variable oligomeric stoichiometries and shapes, each able to open transmembrane pores. Molecular dynamics simulations resolved how the membrane-inserted amphiphilic beta-hairpins and the structurally adapting hydrophilic head domains stabilize variable oligomeric conformations and open the pore. Without a vertical collapse, gasdermin pore formation propagates along a set of multiple parallel but connected reaction pathways to ensure a robust cellular response (Mari et al. 2022). Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Du et al. 2023 reported that GSDMD cleavage is not sufficient for its pore formation; GSDMD must be lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation. Thus, GSDMD palmitoylation is induced by ROS and required for pore formation (Du et al. 2023).

Gasdermin D or A3 of Homo sapiens

 
1.C.123.1.10

Pajvakin (Gasdermin homologue) of 247 aas with 8 or 9 short peaks of hydrophobicity.

Pejvakin (Gasdermin homolog) of Exaiptasia diaphana

 
1.C.123.1.11

Uncharacterized protein of 472 aas

UP of Nematostella vectensis (starlet sea anemone)

 
1.C.123.1.2

Gasdermin A of 445 aas.  Gasdermins A and B may be involved in asthma (Zihlif et al. 2016). Induction in the epidermis leads to skin inflammation (Lin et al. 2015). Roles of multiple charged residues in membrane insertion of gasdermin-A3 have been identified (Korn and Pluhackova 2022).

Gasdermin A of Homo sapiens

 
1.C.123.1.3

Gasdermin B of 411 aas.  Promotes invasioin and metastasis in breast cancer (Hergueta-Redondo et al. 2014).

Gaseremin B of Homo sapiens

 
1.C.123.1.4

Gasdermin C of 508 aas.  The N-terminal moiety promotes pyroptosis. It may be acting by homooligomerizing within the membrane and forming pores (Ding et al. 2016). Pyroptosis and its role in central nervous system diseases have been reviewed (Hu et al. 2021).

 

Gasdermin C of Homo sapiens

 
1.C.123.1.5

Non-syndromic hearing impairment protein 5, DFNA5, (Gasdermin E precursor; GSDME, ICERE1) of 496 aas.  After cleavage by CASP3, it moves to the plasma membrane, homooligomerizes within the membrane and forms pores of 10-15 nanometers (nm) of inner diameter, triggering pyroptosis (Wang et al. 2017, Zhang et al. 2020). It plays a role in hearing loss and the TP53-regulated cellular response to DNA damage, probably by cooperating with TP53 (Masuda et al. 2006; Kim et al. 2008; Op de Beeck et al. 2011). The N-terminal moiety promotes pyroptosis (inflamatory cell death) and exhibits bactericidal activity (Ding et al. 2016).

DFNA5 of Homo sapiens

 
1.C.123.1.6

DNFB59 protein of 361 aas.

DNFB59 protein of Danio rerio (Zebrafish) (Brachydanio rerio)

 
1.C.123.1.7

Pejvakin (DFNB59; PJVK) of 357 aas. It is a constituent of the afferent auditory pathway, causing DFNB59 auditory neuropathy (Delmaghani et al. 2006), autosomal recessive nosyndromic hearing impairment (Collin et al. 2007). It is also called the diaphanous homologue 3 (DIAPH3).

Pejvakin of Homo sapiens

 
1.C.123.1.8

Gasdermin family protein of 252 aas and 1 or 2 central TMSs. The 3-D structure is known (7N52_A-D). Bacterial gasdermins are activated by caspase-like proteases, oligomerize into large membrane pores, and defend against pathogenic bacteriophage (Johnson et al. 2022). They mediate an ancient mechanism of prokaryotic cell death (Johnson et al. 2022).

Gasdermin protein of Salmonella enterica subsp. enterica serovar Typhi (Salmonella typhi)

 
1.C.123.1.9

Gasdermin Eb of 472 aas and 1 or 2 TMSs.

Gasdermin Eb of Danio rerio (zebrafish)

 
Examples:

TC#NameOrganismal TypeExample
1.C.123.2.1

Uncharacterized protein of 285 aas with one TMS between residues 70 and 90.

UP of Fusarium solani-melongenae

 
1.C.123.2.2

Uncharacterized protein of 336 aas and 1 TMS between residues 70 and 90.

UP of Lasiodiplodia theobromae

 
1.C.123.2.3

Uncharacterized protein of 267 aas and probably 0 TMSs.

UP of Trichoderma atroviride

 
1.C.123.2.4

Uncharacterized protein of 261 aas and possibly 4 TMSs, one N-terminal, one at residue 70, one at residue 130, and one at residue 170.

UP of Acephala macrosclerotiorum

 
Examples:

TC#NameOrganismal TypeExample
1.C.123.3.1

Uncharacterized protein of 323 aas and 4 regions of hydrophobicity that might be TMSs.

UP of Ceratodon purpureus

 
1.C.123.3.2

Uncharacterized protein of 319 aas and 1 N-terminal TMS.

UP of Sphagnum fallax

 
1.C.123.3.3

Uncharacterized protein of 314 aas and an N-terminal TMS plus several possible TMSs.

UP of Ceratodon purpureus