Chengliang Wang, et al.
Citation: Wang C, Ruan J (2023) An ancient defense mechanism: Conservation of gasdermin-mediated pyroptosis. PLoS Biol 21(5):
Published: May 4, 2023
Copyright: © 2023 Wang, Ruan. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Pyroptosis is a highly pro-inflammatory form of lytic cell death that serves as the first line of host defense by eliminating intracellular replication niches and enhancing the host’s defensive responses. The gasdermins, a family of pore-forming proteins, are executioners of pyroptosis. Gasdermins have a well-conserved domain architecture that contains an N-terminal pore-forming domain, a C-terminal autoinhibitory domain, and a highly divergent interdomain linker, and share a unified mechanism of autoinhibition and activation [1,2]. In response to microbial infections and other danger signals, activated caspases, granzymes, and bacterial proteases can recognize and cleave the interdomain linker in gasdermins, unleashing the pyroptotic N-terminal fragments that form pores in cell membranes and trigger pyroptosis [3–7]. Gasdermins have been widely studied in recent years, and emerging roles in autoimmune and inflammatory diseases, deafness, and cancers have highlighted their strong potential as therapeutic targets.
GSDM (the gene encoding gasdermin) and GSDM-like genes are evolutionarily ancient and have been identified across a wide range of animal and nonanimal species, including fungi and bacteria. In humans, the GSDM gene family consists of 6 paralogs, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME (also known as DFNA5), and DFNB59 (also known as PJVK, encoding pejvakin). Previous studies on gasdermins mostly focused on their functional roles in the immune response, and their origin and evolution have not been well studied. In this issue of PLOS Biology, Wang and colleagues investigated the evolutionary history of GSDM genes across metazoan species using bioinformatic methods . Consistent with another recent study , Wang and colleagues found that GSDME is the most ancient GSDM gene and that the evolution of gasdermins can be divided into 2 clades: the gasdermin A/B/C/D clade, which is specific to jawed vertebrates, and the gasdermin E/pejvakin clade, which is widely distributed from cnidaria to mammals . Within the gasdermin E/pejvakin clade, PJVK was a duplication from GSDME but lost the last 3 exons that encode the C-terminal autoinhibitory domain in early vertebrates. In the gasdermin A/B/C/D clade, GSDMA and GSDMB arose about 400 million years ago as a duplication of GSDME in cartilaginous fish. GSDMD emerged by duplication from GSDMB and then duplicated to form GSDMC in early mammals. Moreover, analysis of the intron phases of GSDM genes indicated that the evolution of the gasdermin family is dynamic and influenced by the coevolution of host and pathogen.
Gasdermin-mediated pyroptosis can be activated through a variety of signaling pathways. Canonical and noncanonical inflammasome activation induces gasdermin D–mediated pyroptosis, promoting protective immune responses [3,4]. Alternatively, granzyme A, delivered by cytotoxic lymphocytes, cleaves gasdermin B, triggering pyroptosis in target cells and promoting tumor clearance in mice , and gasdermin A is cleaved by group A Streptococcus cysteine protease SpeB in response to infection . Gasdermin E was first known to have a role in antitumor immunity by inducing pyroptosis in certain cancer cells through cleavage by caspase-3 . A later study in teleosts demonstrated that gasdermin E–mediated pyroptosis is also important for immune defense against bacterial infection . However, pyroptosis in invertebrates, which only have ancestral gasdermin E, is currently unclear.
In their study, Wang and colleagues focused on the invertebrate amphioxus and found that amphioxus gasdermin E (BbGSDME) induced significant pyroptosis in HeLa cells stimulated with TNFα and cycloheximide for the activation of caspase-3, and identified 3 caspase homologs (BbCASP1/2/3-like) as the upstream enzymes of BbGSDME . Interestingly, BbGSDME contains 2 caspase cleavage sites (at D253 and D304) responsible for generating a p30 or a p40 N-terminal fragment of BbGSDME, respectively, upon cleavage by caspases. However, only the p30 fragment could induce pyroptosis in HeLa cells, whereas the p40 fragment exhibited no pyroptotic activity unless it was further processed by caspases to become p30. Using AlphaFold, the authors predicted an autoinhibited conformation of p40 mediated by the region spanning residues 254–304 in the C-terminus. The region of 254–304 binds to the N-terminal β1-β2 loop in p40, thereby preventing its attachment to the membrane but not affecting its self-oligomerization. Surprisingly, p40 functioned as a negative regulator of BbGSDME-mediated pyroptosis by directly interacting with the p30 fragment, indicating a novel feedback mechanism fine-tuning gasdermin-mediated pyroptosis (Fig 1). Notably, expression of BbGSDME is also alternative-splicing regulated, which may increase the complexity of regulating pyroptosis in amphioxus.
Fig 1. Regulation and activation of gasdermin E in amphioxus.
In amphioxus, transcription factors IRF1 and IRF8 promote the transcription of GSDME by binding to the IRF1/RelA binding sites in the promoter region. Full-length amphioxus gasdermin E (BbGSDME) can be cleaved by BbCASP1/2/3-like at 2 different sites, D253 and D304, generating 2 distinct N-terminal fragments, p30 and p40. p40 can be further processed by BbCASP1/2/3-like to form the pyroptotic p30. p30 translocates to the plasma membrane and forms pores, leading to pyroptosis. p40 adopts an autoinhibited conformation and can interact with p30 to inhibit BbGSDME pore formation, providing a negative feedback mechanism for BbGSDME-mediated pyroptosis. The figure was created in BioRender.
Wang and colleagues then identified several interferon regulatory factor 1 (IRF1) or RelA binding sites in the promoter region that control the expression of BbGSDME (Fig 1). Transcription factors BbIRF1 and BbIRF8 are responsible for recognizing these regulatory elements and triggering the expression of BbGSDME. It is noteworthy that IRFs primarily regulate immune response genes in response to pathogen invasion and are considered crucial mediators of pro-inflammatory responses. Previous studies have also identified IRF1/IRF2 binding sites in the promoter regions of mammalian GSDMB and GSDMD , further confirming the immune relevance of BbGSDME in amphioxus and suggesting a conserved mechanism regulating the transcription of GSDM genes.
Wang and colleagues then generated an atomic model of BbGSDME in the pore conformation based on the structure of human gasdermin D . The simulated model, combined with their biochemical studies, revealed highly conserved lipid-binding and oligomerization interfaces in BbGSDME. Interestingly, many single nucleotide polymorphisms (SNPs) of GSDME were identified that altered the pyroptotic function of gasdermin E, including the mutations K120Q or P212L. K120 is predicted to be a potential ubiquitination site, suggesting a possible mechanism of posttranslational modification to regulate human gasdermin E activity. Other GSDM genes, such as GSDMB and GSDMD, also exhibit SNPs, highlighting the importance of identifying functionally relevant SNPs in GSDM genes to better understand the regulation of gasdermin-mediated pyroptosis and its relevance to various diseases.
Gasdermins are associated with many diseases, including inflammatory bowel disease, asthma, Alzheimer’s disease, and cancers. In this study, Wang and colleagues found that BbGSDME was involved in muscle necrosis in amphioxus upon bacterial infection. Specifically, infection by the bacteria Edwardsiella tarda caused significant tissue damage in the pharyngeal gill slits, skin, and intestines of amphioxus, which all express BbGSDME. Treatment with Ac-VHTD-CHO, a BbGSDME-D253 cleavage-specific inhibitor, significantly alleviated necrosis in these tissues.
Overall, the new study provides insights into the origin and evolution of the gasdermin family across metazoans . The functional analysis of BbGSDME in amphioxus highlighted its ancient role in innate immune response and revealed a novel negative feedback regulation of gasdermin-mediated pyroptosis. However, the regulation of the 2 cleavages of BbGSDME in amphioxus and any potential preference of BbCASPs for these cleavage sites remain unclear. Additionally, while human (and other mammalian) gasdermin E lacks the extra caspase-3 cleavage site, it is unknown whether this feedback regulation is unnecessary in these organisms or if alternative mechanisms exist. Given the significance of gasdermins in immune responses and disease, further research may uncover other regulatory mechanisms for their activation and inhibition.