Apoptotic and proinflammatory caspases orchestrate necrotic cell death by cleaving three gasdermin orthologs in the highly adaptable killifish

The Atlantic killifish Fundulus heteroclitus represents a remarkable estuarine model for studying physiological adaptation to environmental stress and even industrial pollution. Assessing cell death, including apoptosis and necrosis, is essential to understanding its phenotypic plasticity and adaptability. However, previous studies predominantly focus on apoptosis, while the regulation of necrosis remains largely unexplored. Pyroptosis is a type of regulated necrotic cell death executed by the pore-forming protein, gasdermin. Herein, we revealed the presence of three phylogenetically segregated gasdermin E (GSDME) homologs, designated GSDMEa/b/c, in killifish. All three GSDME orthologs harbor the pore-forming N-terminal domain, which is capable of perforating the cell membrane and inducing pyroptosis. Mechanistically, GSDMEa is activated by the cleavage of proinflammatory caspase-1 and apoptotic caspase-3/7, GSDMEb is proteolytically activated by proinflammatory caspase-1 and apoptotic caspase-3/7/8, while GSDMEc specifically relies on activation by apoptotic caspase-7. Following cleavage, the pyroptosis-inducing NT fragment is liberated from the auto-inhibitory C-terminal domain, and executes pyroptosis with massive release of intracellular contents. Site-directed mutagenesis reveals that GSDMEa and GSDMEb possess evolutionarily conserved functional residues required for pore-forming activity, whereas the functional residues involved in the C-terminal auto-inhibition are highly diverged among these three orthologs. Our findings collectively unveil a sophisticated and intertwined signaling pathway in regulating killifish pyroptosis, improving our understanding of cell death in killifish phenotypic adaptability and plasticity. • Killifish harbor three phylogenetically distinct pyroptosis-inducing gasdermins. •Apoptotic and proinflammatory caspases cleave gasdermins with different patterns. •Functional residues are conserved in the N-terminal domain whereas diverged in the C-terminal domain.