Inflammasomes are multiprotein complexes that assemble in response to infection or tissue damage. Among them, the NLRP3 inflammasome stands out as it responds to an exceptionally wide range of stimuli, including microbes, amyloids, asbestos fibers, and ionic imbalance. Upon sensing such signals, NLRP3 oligomerizes and recruits the adaptor ASC and pro-caspase-1, which together form the inflammasome and enable the activation of pro-caspase-1. Active caspase-1 cleaves pro-inflammatory cytokines IL-1β and IL-18, as well as gasdermin D (GSDMD), whose N-terminal fragment (NtGSDMD) inserts into the plasma membrane and induces lytic cell death known as pyroptosis. Uncontrolled NLRP3 inflammasome activity contributes to common diseases such as diabetes, cardiovascular disorders, neurodegenerative diseases and cancer. Despite intensive research, many aspects of the upstream and downstream signaling pathways that govern inflammasome activation remain poorly understood. In the first part of this doctoral thesis, we addressed how the same stimulus can trigger NLRP3 activation at distinct subcellular locations. Using a synthetic biology-based approach we found that NLRP3 can be activated at various membranes and even at protein scaffolds. We showed that NLRP3 forms clusters at these scaffolds, which promote inflammasome assembly. We propose that diverse NLRP3 activators induce disturbances in cellular homeostasis and ultrastructural changes that lead to scaffold formation, where NLRP3 can bind to, leading to active NLRP3 oligomer formation and inflammasome assembly. Inflammasome-driven inflammation is also shaped by pyroptosis, an immunogenic form of cell death. We showed that an oxidative environment induced by various stressors enhanced NtGSDMD activity, and that redox sensing occurs through cysteine 192. Using synthetic biology approach, we designed synthetic inflammasomes combining the proinflammatory cytokine release and cell death through oligomerization and activation of key components. By fusing signaling motifs of innate immune adaptors MAVS, TRIF, and MyD88 to an oligomerizing scaffold protein, we achieved the activation of specific signaling pathways. Coupling these modules with effectors of lytic cell death, GSDMD or MLKL, enabled construction of synthetic inflammasomes that induced cytokine production and cell death. We tested these synthetic inflammasomes in cancer cells, where they triggered cytokine expression, indication potential application in cancer immunotherapy for modulating immune responses within the tumor microenvironment. This doctoral work provides new mechanistic insights into NLRP3 activation and regulation of pyroptosis, while also establishing a synthetic biology-based strategy for building minimal inflammatory modules.
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