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Characterization of GSDME in amphioxus provides insights into the functional evolution of GSDM-mediated pyroptosis [1]

['Xinli Wang', 'Guangdong Key Laboratory Of Pharmaceutical Functional Genes', 'Southern Marine Science', 'Engineering Guangdong Laboratory', 'Zhuhai', 'State Key Laboratory Of Biocontrol', 'School Of Life Sciences', 'Sun Yat-Sen University', 'Guangzhou', 'People S Republic Of China']

Date: 2023-05

Members of the gasdermin (GSDM) family are pore-forming effectors that cause membrane permeabilization and pyroptosis, a lytic proinflammatory type of cell death. To reveal the functional evolution of GSDM-mediated pyroptosis at the transition from invertebrates to vertebrates, we conducted functional characterization of amphioxus GSDME (BbGSDME) and found that it can be cleaved by distinct caspase homologs, yielding the N253 and N304 termini with distinct functions. The N253 fragment binds to cell membrane, triggers pyroptosis, and inhibits bacterial growth, while the N304 performs negative regulation of N253-mediated cell death. Moreover, BbGSDME is associated with bacteria-induced tissue necrosis and transcriptionally regulated by BbIRF1/8 in amphioxus. Interestingly, several amino acids that are evolutionarily conserved were found to be important for the function of both BbGSDME and HsGSDME, shedding new lights on the functional regulation of GSDM-mediated inflammation.

Funding: This work was supported by Ministry of Science and Technology of the People's Republic of China (2018YFD0900502 to SC.Y), National Natural Science Foundation of China (31770943 and 31970852 to SC.Y), the Guangdong Science and Technology Department (2020B1212060031 to SC.Y) and Innovation Group Project of Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai) (311021006 to SC.Y). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Although studies in evolutionary representatives have suggested the ancient origin and some functional conservations of the GSDM-mediated pyroptosis, the function of GSDM family at the transition from invertebrates to vertebrates is still unclear, remaining the gaps to fully understand the evolution of the GSDM family. Here, we conducted evolutionary analyses of the GSDM family and performed functional analyses of the GSDME homolog in amphioxus, the basal chordate harboring an extraordinarily complex innate immunity. We not only found the highly conserved roles of GSDME-mediated pyroptosis in antibacterial defense in amphioxus, but also shed new light on the feedback regulation of GSDME-mediated inflammatory responses via distinct N termini, alternative splicing and SNPs of GSDME.

After functional characterization of the GSDM family in mammals, GSDM-mediated pyroptosis has been revealed in teleost fish, cnidaria, and even fungi and bacteria. Danio rerio (Dr) has 3 GSDM members, the DrGSDMEa, DrGSDMEb, and DrPJVK [ 14 ]. Similar to mammalian GSDMD, DrGSDMEb is cleaved by inflammatory CASPs to release its N-terminus to mediate pyroptosis, which is responsible for lethal LPS-induced septic shock and NETosis for bacterial clearance in vivo [ 20 , 21 ]. Different to DrGSDMEs, turbot Scophthalmus maximus GSDMEa is cleaved by CASP3/6/7 to generate 2 N-fragments with different functions, while turbot GSDMEb can be cleaved by CASP8 and its NT fragment can not trigger pyroptosis in HEK293T cells [ 22 ]. GSDME homologs in coral or another teleost fish Cynoglossus semibreves (tongue sole) can be also cleaved by CASPs to exert pyroptotic and bactericidal activities through its N-terminal domain [ 23 , 24 ]. Notably, studies have shown that the regulator cell death (Rcd-1) in fungal and GSDM-like proteins in bacteria can trigger pyroptosis-like cell death [ 25 , 26 ].

GSDMD belongs to the pore-forming protein family [ 11 ]. Humans contain 6 GSDM members, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and Pejvakin (PJVK) [ 11 ]. In addition to inflammatory responses, important progress in GSDM-mediated cell death in septic shock and autoimmune diseases, especially in tumor growth, have been made [ 12 ]. GSDME, described as a genetic cause of hearing loss, has been considered as a tumor suppressor. The expression of GSDME is suppressed in many cancers, and such repression could enhance tumor growth but decrease survival [ 13 ]. When cleaved by CASP3, GSDME can switch noninflammatory apoptosis to pyroptosis in cancer cells [ 14 ]. GSDME can be also cleaved by granzyme B, therefore enhancing the phagocytosis of tumor cells by tumor-associated macrophages, as well as tumor-infiltrating natural killer and CD8 + T lymphocytes [ 15 ]. GSDMC, transcriptionally enhanced by nucleic PD-L1 in cancer cells, is a substrate of CASP8, leading to tumor necrosis and poor survival [ 16 ]. Similar to GSDME, GSDMB is cleaved by granzyme A to unleash its pore-forming activity, resulting in the killing of GSDMB-positive cancer cells through pyroptosis [ 17 ]. In addition to targeting the membrane of cancer cells, a new study showed that GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on gram-negative bacterial membranes, placing GSDMB as a center executioner of intracellular bacterial killing [ 18 ]. Recently, GSDMA was reported to trigger pyroptosis in keratinocytes after being cleaved by streptococcal pyrogenic exotoxin B (SpeB) in the defense against skin microbial pathogen [ 19 ].

Cytokines of the interleukin-1 (IL-1) family are essential determinants of inflammation. Due to the absence of an amino-terminal secretion signal, the mechanisms underlying their protein secretion from phagocytes have attracted intense attention for more than 30 years [ 1 ]. In 2015, 3 independent research groups using genetic screening or quantitative mass spectrometry-based analysis identified the pore-forming protein gasdermin D (GSDMD) as the conduit for IL-1 secretion [ 2 – 4 ]. GSDMD is a substrate of activated caspase-1 (CASP1) or CASP4/5/11, allowing it to release the N-terminus and to form inner diameters of 10 to 14 nm oligomeric pores in the cell membranes [ 5 – 8 ]. In addition to inflammasome-mediated pore formation in macrophages, GSDMD can mediate neutrophil pyroptosis, which is essential in NETosis and antimicrobial NET extrusion, revealing fundamental differences in GSDMD trafficking between neutrophils and macrophages [ 9 , 10 ].

Results

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002062

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