Investigation of the E306K NLRP3 mutation in inflammasome complex formation by split-luciferase complementation assay

NOD-like receptor family pyrin domain-containing 3) NLRP3(is a key inflammasome component that oligomerizes upon activation. We employed a split-luciferase complementation assay to investigate the quantitative monitoring of early-stage NLRP3-NLRP3 interactions and assess E306K as a disease-associated NLRP3 mutation. NLRP3-NLRP3 proximity increased within 1 min of nigericin stimulation, whereas E306K did not show a detectable increase. MCC950, an NLRP3 inhibitor, reduced early NLRP3-NLRP3 luciferase complementation more in the wild-type model. Apoptosis-associated speck-like protein containing a CARD (ASC) may affect luciferase reconstitution while promoting ASC-NLRP3 interactions. Collectively, this assay is useful for monitoring early-stage NLRP3-NLRP3 interactions, with caution for NLRP3 mutants.