Evolutionary and functional analysis of caspase-8 and ASC interactions to drive lytic cell death, PANoptosis

Abstract Caspases are evolutionarily conserved proteins essential for driving cell death in development and host defense. Caspase-8, a key member of the caspase family, is implicated in apoptosis, a non-lytic form of cell death, as well as lytic forms of cell death. Recently, caspase-8 has been identified as an integral component of PANoptosomes, multi-protein complexes formed in response to innate immune sensor activation. Several innate immune sensors can nucleate this caspase-8–containing PANoptosome complex to drive inflammatory lytic cell death, PANoptosis. However, how the evolutionarily conserved and diverse functions of caspase-8 drive PANoptosis remains unclear. To address this, we performed evolutionary, sequence, structural, and functional analyses to decode caspase-8’s complex-forming abilities and its interaction with the PANoptosome adaptor ASC. Our study distinguished distinct subgroups within the death domain superfamily based on their evolutionary and functional relationships, identified homotypic traits among sub-family members, and captured key events in caspase evolution. We also identified critical residues defining the heterotypic interaction between caspase-8’s death effector domain and ASC’s pyrin domain, validated through cross-species analyses, dynamic simulations, and in vitro experiments. Overall, our study elucidated recent evolutionary adaptations of caspase-8 that allowed it to interact with ASC, improving our understanding of critical molecular associations in PANoptosome complex formation and the underlying PANoptotic responses in host defense and inflammation. These findings have implications for understanding mammalian immune responses and developing new therapeutic strategies for inflammatory diseases.