Fungal gasdermin-like proteins are controlled by proteolytic cleavage

Abstract

Gasdermins are a family of pore-forming proteins controlling an inflammatory cell death reaction in the mammalian immune system. The pore-forming ability of the gasdermin proteins is released by proteolytic cleavage with the removal of their inhibitory C-terminal domain. Recently, gasdermin-like proteins have been discovered in fungi and characterized as cell death–inducing toxins in the context of conspecific non-self–discrimination (allorecognition). Although functional analogies have been established between mammalian and fungal gasdermins, the molecular pathways regulating gasdermin activity in fungi remain largely unknown. Here, we characterize a gasdermin-based cell death reaction controlled by the het-Q allorecognition genes in the filamentous fungus Podospora anserina. We show that the cytotoxic activity of the HET-Q1 gasdermin is controlled by proteolysis. HET-Q1 loses a ∼5-kDa C-terminal fragment during the cell death reaction in the presence of a subtilisin-like serine protease termed HET-Q2. Mutational analyses and successful reconstitution of the cell death reaction in heterologous hosts (Saccharomyces cerevisiae and human 293T cells) suggest that HET-Q2 directly cleaves HET-Q1 to induce cell death. By analyzing the genomic landscape of het-Q1 homologs in fungi, we uncovered that the vast majority of the gasdermin genes are clustered with protease-encoding genes. These HET-Q2–like proteins carry either subtilisin-like or caspase-related proteases, which, in some cases, correspond to the N-terminal effector domain of nucleotide-binding and oligomerization-like receptor proteins. This study thus reveals the proteolytic regulation of gasdermins in fungi and establishes evolutionary parallels between fungal and mammalian gasdermin-dependent cell death pathways.

Bacterial NLR-related proteins protect against phage

“…NLRC4 requires the pore-forming protein Gasdermin D to execute cell death (pyroptosis) (Broz and Dixit, 2016; Shi et al, 2015). Gasdermin D homologs can also be found in fungi, where they mediate heterokaryon incompatibility, and in bacteria, where they mediate antiphage signaling (Clavé et al, 2022; Daskalov et al, 2020; Johnson et al, 2022). However, bacterial NLR-related proteins do not require Gasdermin D homologs for signaling, unlike the mammalian NLRs which activate cell death via Gasdermin D. Heterokaryon incompatibility loci are highly polymorphic across fungi, and there are many more than het-d/e (NLRs) and rcd-1 (Gasdermin D) (Dyrka et al, 2014; Van der Nest et al, 2014).…”

RNA-triggered protein cleavage and cell death by the RNA-guided type III-E CRISPR-Cas nuclease-protease complex

“…Moreover, given unexpected structural similarity between the Csx30 CTD and pore-forming proteins in type IV secretion systems, it is possible that Csx29 proteolysis might liberate the Csx30 NTD from being near the cell membrane into the cytoplasm to exert its cellular effects. This type of programmed cell death might be analogous to that caused by the bacterial membrane pore-forming toxins gasdermins that are activated via proteolytic cleavage and release of auto-inhibitory peptides by associated proteases activated during phage infection( 24 , 25 ). Moreover, given the high diversity of Csx30 CTDs (fig.…”

Homing in on gasdermins: How fungi regulate cell death

“…Taken together, the findings by Clavé et al. ( 1 ) indicate that GSDM-mediated cell death is an ancient defense mechanism and that its regulation by proteolytic cleavage is likely equally old. In this respect it will be interesting to uncover the molecular activation mechanism of RCD-1, a GSDM from N. crassa that has previously been characterized by the authors and does not seem to be activated by proteases: RCD-1 is also part of an allorecognition system in which the two alleles rcd1-1 and rcd1-2 are exclusive and induce lytic cell death when coexpressed even in human HEK-293T cells ( 17 ).…”

“…In PNAS, Clavé et al. ( 1 ) now demonstrate that proteolytic cleavage, the mechanism governing the activation of vertebrate GSDMs, is also conserved in fungi. Intriguingly, the proteases that putatively cleave fungal GSDMs are related to caspases, the proteases mediating GSDM activation in vertebrate cells.…”

“…Working in P. anserina , Clavé et al. ( 1 ) find that the polymorphic het-Q locus, an allorecognition determinant, contains one of two alleles, het-Q1 or het-Q2. Genetic complementation and deletion experiments confirm that cells expressing both alleles display reduced viability.…”