Mapping the reactive cysteine landscape of the NOD-like receptor pyrin domain-containing 3 protein

Abstract

NOD-like receptor pyring-containing protein 3 (NLRP3) is a cytosolic pattern recognition receptor that drives assembly of an oligomeric inflammasome complex to stimulate pro-inflammatory cytokine production and pyroptotic cell death. Extensive research has shown that NLRP3 activation is dependent on intracellular reactive oxygen species (ROS). Despite this, the precise targets of ROS in this process remain undefined. In this study, a combination of chemoproteomics, in vitro biochemical assays and molecular dynamics simulations were used to characterize reactive Cys and how they may impact NLRP3 conformation and activity status. Affinity-captured NLRP3 was subjected to reverse-thiol trapping to reveal a pool of reactive Cys residues that were identified in subsequent chemoproteomic analyses. C150, C196, and C884 were consistently detected with both in vitro and in cellulo experiments. Several NLRP3 mutants were generated; C150A and C196A (NACHT domain) as well as C933A and C941A (LRR domain) reduced IL-1b output in a reconstituted inflammasome assay. Molecular simulation of Cys sulfenylation in the LRR suggests structural alterations that reposition the trLRR loop to destabilize the inactive NLRP3 structure. Structural modelling provided insights for how Cys oxidation of the LRR may impact on NLRP3 structure. Collectively, the results establish a mechanistic understanding for how ROS may promote NLRP3 inflammasome activation.