Kagan, J. C. | Search bioRxiv

bioRxiv2020

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-1{beta} (IL-1{beta}). No caspase is known to contain both activities, yet distinct caspases with complementary activities bookend an LPS-induced pathway to IL-1{beta} cleavage. Using unique caspases present in carnivorans as a guide, we identified molecular determinants of IL-1{beta} 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-1{beta} 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.

immunology↗

bioRxiv2020

Downstream of gasdermin D cleavage, a Ragulator-Rag-mTORC1 pathway promotes pore formation and pyroptosis

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins are recognized to promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that was designed to identify regulators of pyroptosis that act downstream of GSDMD cleavage. This screen identified several components of the Ragulator-Rag complex, which is known for its metabolic control of mTOR. Mechanistic studies revealed that Ragulator-Rag is not necessary for GSDMD localization to the plasma membrane, but is necessary for pore formation and mitochondrial inactivation. Downstream of Ragulator-Rag is mTORC1, which we found to promote pyroptosis in response to diverse natural stimuli, including infection. GSDMD therefore requires a Ragulator-Rag-mTORC1 pathway in order to form pores and execute pyroptosis.

immunology↗