Poster Presentation The 6th Prato Conference on Pore Forming Proteins 2025

Gasdermins (GSDMs) are pore-forming proteins that execute pyroptosis for immune defense. Canonical GSDMs are two-domain proteins activated by proteolytic removal of the inhibitory domain. Here, we identified two novel cleavage-independent GSDM activation mechanisms in ancient eukaryotes. First, TrichoGSDM, a pore-forming domain-only protein from the basal metazoan Trichoplax adhaerens, is a disulfide-linked autoinhibited dimer activated by physiological reducing agents (e.g., glutathione or thioredoxin) via disulfide reduction. The cryo-electron microscopy (cryo-EM) structure illustrates the assembly mechanism for the 44-mer TrichoGSDM pore. Second, RCD-1-1 and RCD-1-2, encoded by the polymorphic regulator of cell death-1 (rcd-1) gene in filamentous fungus Neurospora crassa, are also pore-forming domain-only GSDMs. RCD-1-1 and RCD-1-2, when encountering each other, form pores and cause pyroptosis, underlying allorecognition in Neurospora. The cryo-EM structure reveals a pore of 11 RCD-1-1/RCD-1-2 heterodimers and a heterodimerization-triggered pore assembly mechanism. Thus, TrichoGSDM and RCD-1 represent two types of pore-forming-domain-only GSDMs, which come from simple and ancient eukaryotes and employ distinct cleavage-independent activation mechanisms. TrichoGSDM is a disulfide-linked autoinhibited dimer and activated by reduction of the disulfides, indicating a redox-responsive function. The pore-forming activity in RCD-1 is stimulated by hetero-recognition between RCD-1-1 and RCD-1-2 from genetically incompatible fungal strains, underlying allorecognition-caused cell death in N. crassa. These findings highlight mechanistic diversities in GSDM activation as well as versatile biological functions in the emerging GSDM family.