Dataset Information

Molecular characterization of a fungal gasdermin-like protein.

ABSTRACT: Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

SUBMITTER: Daskalov A

PROVIDER: S-EPMC7414189 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Molecular characterization of a fungal gasdermin-like protein.

Daskalov Asen A Mitchell Patrick S PS Sandstrom Andrew A Vance Russell E RE Glass N Louise NL

Proceedings of the National Academy of Sciences of the United States of America 20200723 31

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassarcd-1< ...[more]

PMID: 32703806

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Project description:Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28-34 of pGSDMD (LQTSDRF), 19H3 recognizes 257-260aa (PPQF), 23H10 and 27A10 recognize 78-82aa (GPFYF), and 25E2 recognizes 429-435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10-9, 3.66 × 10-9, 9.04 × 10-9, 1.83 × 10-9, and 8.00 × 10-8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis. KEY POINTS: • A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus. • The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized. • All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.

Dataset Information

Molecular characterization of a fungal gasdermin-like protein.

Publications

Molecular characterization of a fungal gasdermin-like protein.

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