Caspase-1 Activation by the Inflammasomes

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Inflammasomes are multi-protein complexes that assemble to activate caspase-1 in response to tissue damage and infection by microbial or viral pathogens. Once activated, caspase-1 processes the inactive proforms of interleukin-1β (IL-1β) and interleukin-18 (IL-18) to produce the active pro-inflammatory cytokines IL-1β and IL- 18, respectively. In addition, caspase-1 processes the gasdermin protein GSDMD to induce pyroptosis or inflammatory cell death. The NLRP3 inflammasome is unique among the different inflammasomes in that it is activated by diverse pathogen-associated and danger-associated molecular patterns (PAMPs and DAMPs) derived from infection with microbial pathogens, or tissue damage. As a result, uncontrolled NLRP3 activation can lead to a number of human inflammatory diseases, including gout, arthritis, atherosclerosis, and type 2 diabetes. The mechanism of NLRP3 activation by these seemingly unrelated stimuli is poorly understood but is currently believed to require two distinct signals; a priming signal or “signal 1” produced by Toll-like receptors (TLRs) and an activation signal or “signal 2” that induces fragmentation of trans Golgi network (TGN) and binding to NEK7. Studies in the applicant's laboratory demonstrated that signal 1 induces post-translational modification (PTM) of NLRP3 at critical sites via the MyD88 and TRIF signaling pathways and partial oligomerization of NLRP3. In this application, studies are proposed to elucidate how TLR-induced PTM contributes to posttranslational priming of the NLRP3 inflammasome by employing mass spectrometry to identify and characterize all critical changes in the phosphorylation and other PTM profile of NLRP3 induced by signal 1, and investigating how these changes contribute to activation of NLRP3. Additional aims will investigate the effect of signal 1 and signal 1-induced PTM on NLRP3 association with dispersed TGN and NEK7, and identify the TGN-associated kinases required for final assembly and activation of the inflammasome. Finally, preliminary evidence suggest that kinases involved in the regulation of intracellular ion homeostasis exert negative control on activation of NLRP3 by signal 2. Thus, additional experiments will investigate how signaling from these kinases in macrophages impacts NEK7 phosphorylation and interaction with NLRP3, and how genetic deficiency in these kinases impacts NLRP3-mediated pro-inflammatory responses to PAMPs and DAMPs in vivo. Results from this research will provide fundamental new insights into the pathways that regulate the assembly and activation of the NLRP3 inflammasome, and the cellular mechanisms that control its activation. Successful completion of this study should have a high impact on the field by providing a unifying paradigm for how NLRP3 can be regulated by an exceptionally diverse group of activating stimuli. Understanding these mechanisms is of great scientific and health significance as this should better our understanding of the molecular basis of NLRP3-related diseases and should in the long term help in the development of therapeutics to alleviate these inflammatory diseases.