Caspase-4/-5/-11 non-canonical inflammasomes have been known to play pivotal roles in various inflammatory and infectious diseases, such as sepsis. Current studies demonstrate that caspase-4/-5/-11 are activated by directly sensing intracellular microbial infections, such as lipopolysaccharide (LPS, also known as endotoxin), or endogenous products, such as oxidized phospholipids (e.g., OxPAPC), through their N-terminal caspase activation and recruitment domains (CARDs) and C-terminal catalytic domains, respectively. However, the molecular mechanisms of how caspase-4/-5/-11 recognize their ligands and how caspase-4/-5/-11 are activated upon binding ligands remain unknown. In this proposal, we aim to elucidate the structural basis of non-canonical inflammasome signaling by characterizing the interactions between caspase-4/-5/-11 and ligands - LPS and OxPAPC, and determining the high-resolution structures of caspase/-4/-5/-11 in complex with ligands. Our structural findings will provide new therapeutic strategies for sepsis and other non-canonical inflammasome- associated diseases. We propose three specific aims to achieve our goal: 1) Biochemical characterization of the interactions between caspase-4/-5/-11 and LPS-including the identification of the essential structural elements in the LPS molecule and key residues on caspase-4/-5/-11 CARDs that are required for caspase-4/-5/-11 activation; 2) Determine high-resolution structures of caspase-4/-5/-11 CARDs both in their inactive form and in complex with LPS; 3) Characterize the interactions between caspase-4/-11 and OxPAPC, and determine the high resolution structures of caspase-4/-11 catalytic domains in complex with OxPAPC. We will pursue these aims using cutting-edge experimental approaches including biochemical and biophysical characterization, X-ray crystallography, mass spectrometry, electron microscopy, and cellular experiments. The proposed studies will significantly expand our current knowledge on the mechanisms of non-canonical inflammasome signaling, and provide rationale and a structural basis for designing novel strategies to control the activation of the non- canonical inflammasome for better treatment of related diseases.
The proposed research seeks to understand the molecular mechanisms of caspase-4/-5/-11 noncanonical inflammasomes activation. Non-canonical inflammasomes are large multiprotein complexes that play key roles in the innate immune surveillance of infections and danger, and have been shown to play pivotal roles in various inflammatory and infectious diseases, including sepsis. Completion of the research will produce critical insights into the non-canonical inflammasome signaling, and open a new avenue for developing therapeutic strategies against sepsis and other noncanonical inflammasome-related diseases.