Evolution-inspired dissection of caspase activities enables the redesign of caspase-4 into an LPS sensing interleukin-1 converting enzyme

Innate immune signaling pathways comprise multiple proteins that promote inflammation. This multistep means of information transfer suggests that complexity is a prerequisite for pathway design. Herein, we examined this possibility by studying caspases that regulate inflammasome-dependent inflammation. Several caspases differ in their ability to recognize bacterial LPS and cleave interleukin-1β (IL-1β). No caspase is known to contain both activities, yet distinct caspases with complementary activities bookend an LPS-induced pathway to IL-1β cleavage. Using unique caspases present in carnivorans as a guide, we identified molecular determinants of IL-1β cleavage specificity within caspase-1. This knowledge enabled the redesign of human caspase-4 to operate as a one-protein signaling pathway, which intrinsically links LPS detection to IL-1β cleavage and release, independent of inflammasomes. Strikingly, cat caspase-4 displays the activities of redesigned human caspase-4. These findings illustrate natural signaling pathway diversity and highlight how multistep innate immune pathways can be condensed into a single protein. ### Competing Interest Statement J.C.K. holds equity and consults for IFM Therapeutics, Quench Bio and Corner Therapeutics. None of these relationships influenced the work performed in this study.