In innate immunity, the inflammasomes are large molecular machineries that are responsible for combating microbial infections. A canonical inflammasome complex contains the sensor protein, the adaptor protein called apoptosis-associated speck-like protein (ASC), and the cysteine protease caspase-1. The inflammasome machinery promotes the maturation of caspase-1, which can further recognize and cleave its specific substrates pro-interleukin-1β (pro-IL1β), pro-interleukin-18, and gasdermin D (GSDMD). After cleavage, the N-terminal domain of GSDMD can induce pyroptosis by forming plasma membrane pores. The mature IL1β and IL-18 are released via the GSDMD pores and induce inflammation. Dysfunction of inflammasome and pyroptosis contributes to the development of many human diseases. Despite the physiological and pathological importance, the molecular mechanism of inflammasome activation and pyroptosis is not well defined. We investigated the structure, function and regulation of the inflammatory caspases. Inflammatory caspases such as caspase-1 and caspase-11 are able to directly bind to GSDMD. The cleavage peptide Phe-Leu-Thr-Asp (FLTD) from human GSDMD participates in interaction with inflammatory caspases. Based on the binding data, we designed a GSDMD-derived peptide inhibitor Ac-FLTD-CMK for inflammatory caspases. Biophysical and biochemical studies demonstrated that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases, but not for apoptotic caspases. Crystal structure of Ac-FLTD-CMK in complex with caspase-1 not only revealed the inhibition mechanism, but also shed light on how GSDMD is recognized by inflammatory caspases.
In a non-canonical inflammasome signaling pathway, after intracellular bacterial infection, the caspase activation and recruitment domains (CARDs) of caspases-4, -5, -11 can directly bind to bacterial cell wall component lipopolysaccharide (LPS), and thus be activated through ligand-mediated oligomerization. The activated caspase-4, -5, -11 can recognize and cleave GSDMD and induce pyroptosis. We determined the crystal structures of the human caspase-4 and murine caspase-11 catalytic domains. Structural analysis of caspases-4, -11 suggested an enzymatic activation mechanism that is similar to that for caspase-1.
VX-765 is an inhibitor for inflammatory caspases and it is in clinical trial for treating epilepsy and psoriasis. In order to better understand the mechanism by which VX-765 inhibits inflammatory caspases, we determined the crystal structure of caspase-1 in complex with VRT-043198, which is the active metabolite of VX-765. Both hydrophobic and hydrophilic interactions contribute to the binding of VRT-043198 and its specific inhibition of inflammatory caspases. Together with other biophysical, biochemical, and animal studies, we characterized the molecular mechanism by which XV-765 inhibits inflammatory caspases. This study will provide a basis for future drug development based on VX-765.
Together, the studies reported in this thesis contribute to our understanding of inflammasome activation and pyroptotic cell death in innate immunity.
