RESUMEN
The NLRP3 inflammasome is a key macromolecular complex of the innate immune system that activates the inflammatory signalling cascade in response to a wide range of stimuli. Structural studies have shown that the intracellular cytosolic receptor NLRP3 oligomerizes upon stimulation and serves as a scaffold to form the ASC filaments necessary for procaspase-1 activation. Despite the abundant structural evidences on NLRP3 inflammasome, the interactions of the NLRP3 Pyrin domain and its functional relevance are poorly understood. In this study, the split luciferase complementation assay is used as an alternative approach to investigate NLRP3PYD-NLRP3PYD interactions during inflammasome formation. Since the homotypic NLRP3 interaction is mainly based on electrostatic interactions, a phosphomimetic residue (S5) at the interface of the NLRP3PYDs interactions has been mutated to show a disruptive effect on luciferase activity. According to the results presented, the designed biosensor was able to monitor the NLRP3PYD-NLRP3PYD interaction in vitro. The current reporter assay not only provides a specific NLRP3PYD-NLRP3PYD assay to study the PYD-PYD interaction in vitro, but also provides a suitable system for screening chemicals and drugs to identify activators and inhibitors of NLRP3.