Fungal gasdermin-like proteins are controlled by proteolytic cleavage

Area of Science:

• Molecular Biology
• Mycology
• Immunology

Background:

• Gasdermins are pore-forming proteins crucial for mammalian immune responses, activated by proteolytic cleavage.
• Fungal gasdermin-like proteins function in allorecognition, but their regulatory mechanisms are poorly understood.
• The het-Q genes in *Podospora anserina* are implicated in fungal allorecognition and cell death.

Purpose of the Study:

• To elucidate the molecular regulation of fungal gasdermin activity.
• To characterize the role of the *het-Q* genes in gasdermin-mediated cell death in *Podospora anserina*.
• To explore evolutionary conservation of gasdermin regulation between fungi and mammals.

Main Methods:

• Proteolytic cleavage assays of the HET-Q1 gasdermin.
• Site-directed mutagenesis of HET-Q1 and HET-Q2.
• Reconstitution of the cell death pathway in heterologous hosts (*Saccharomyces cerevisiae*, human 293T cells).
• Bioinformatic analysis of fungal gasdermin and protease gene clusters.

Main Results:

• The cytotoxic activity of the fungal HET-Q1 gasdermin is regulated by proteolysis, involving the loss of a C-terminal fragment.
• A subtilisin-like serine protease, HET-Q2, was identified as directly cleaving HET-Q1 to induce cell death.
• The fungal cell death pathway could be reconstituted in yeast and human cells, demonstrating conserved mechanisms.
• Genomic analysis revealed that fungal gasdermin genes are frequently clustered with protease-encoding genes, suggesting co-evolution.

Conclusions:

• Fungal gasdermin activity is regulated by proteolytic cleavage, analogous to mammalian gasdermins.
• The HET-Q2 protease directly activates HET-Q1, establishing a conserved molecular mechanism for gasdermin-dependent cell death.
• This study highlights evolutionary parallels in gasdermin regulation across kingdoms, from fungi to mammals.