Wednesday, April 24, 2024
4/24/2024
Abstract The canonical and noncanonical inflammasome signaling pathways can induce pyroptosis and secretion of inflammatory cytokines, which function as crucial immune defense mechanisms in microbial killing and clearance. Pyroptosis is a type of regulated lytic cell death that is mediated by members of the gasdermin family that assemble membrane pores upon cleavage by proteases. Gasdermin E (GSDME) can be cleaved by apoptotic caspases-3 and 7, which then triggers plasma membrane pore formation and pyroptosis instead of apoptosis. GSDME-mediated pyroptosis and cytokine release play crucial roles in host defense against viral or bacterial infections. On the other hand, GSDME in intestinal epithelial cells has been implicated in mucosal inflammation and pathogenesis of Crohn’s disease, and GSDME-mediated pyroptosis contributes to renal fibrosis and kidney injury. Despite recent progress in our understanding of the recruitment and recognition of gasdermin D (GSDMD) by inflammatory caspases, the molecular mechanisms of GSDME recognition and cleavage by apoptotic caspases have been poorly defined. This proposal aims to address the critical gaps in our knowledge using complementary structure-function approaches. We hypothesize that inactive GSDME is maintained in an autoinhibited conformation through intramolecular amino-terminal domain (NTD)-carboxy- terminal domain (CTD) interactions, which is recognized by apoptotic caspase-3 that cleaves at the NTD-CTD linker region to release the autoinhibition. This study will explore the structural mechanisms of GSDME autoinhibition and its recognition by caspase-3 to test the above hypothesis. The following specific aims are built on our past and ongoing studies on gasdermins and pyroptosis. Aim 1 will characterize the molecular mechanisms of GSDME autoinhibition. Our preliminary data show that GSDME protein can be crystallized and the crystals diffracted X-ray well. We will determine the structure of GSDME at its autoinhibitory state using crystallographic approaches, and validate the NTD-CTD interface through mutagenesis studies of the interface residues using LDH release, PI uptake, and liposome leakage assays. Aim 2 will define the mechanisms of GSDME recognition by caspase-3. We will probe the interaction between GSDME and caspase-3 through biochemical and cell biology techniques, and determine the structure of GSDME in complex with caspase-3 using X-ray crystallography. The success of this proposal will not only reveal important insights into GSDME autoinhibition and recognition by apoptotic caspases that move the field forward, but also facilitate the development of specific inhibitors for apoptotic caspase-3 based on the distinct GSDME-caspase-3 interface, which may be valuable reagents in the study of both pyroptosis and apoptosis signaling pathways.
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