Integrated IP-MS and Optoproteomics Reveal Dynamic Remodeling of Protein Interaction Networks During NLRP3 Inflammasome Activation

Abstract NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a key intracellular sensor that assembles inflammasome complexes to activate inflammatory caspases, yet the molecular composition and stimulus-dependent organization of the NLRP3 inflammasome remain incompletely defined. Here, we systematically characterized dynamic remodeling of the NLRP3 inflammasome interactome using endogenous immunoprecipitation coupled with mass spectrometry (IP-MS) targeting both NLRP3 and its adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) in bone marrow-derived macrophages. Quantitative interactome profiling across NLRP 3 inflammasome priming and activation states identified 1,496 stimulus-associated protein interactions (1,032 non-redundant), revealing extensive and stage-specific reorganization of NLRP3- and ASC-centered interaction networks. Integration of these datasets with microscopy-guided in situ protein photo-labeling (optoproteomics) of ASC specks provided spatial context to inflammasome assembly and identified inner membrane mitochondrial protein (IMMT) as an activation-associated binding partner of both NLRP3 and ASC. Functional analyses demonstrated that Immt depletion enhanced caspase-1 and gasdermin D cleavage, interleukin-1β secretion, lactate dehydrogenase release, and ASC oligomerization in response to NLRP3 agonist, indicating a negative regulatory role for IMMT in inflammasome activation. Together, this study establishes a stimulus-resolved proteomic framework for NLRP3 inflammasome interaction networks and reveals mitochondrial IMMT as a regulator of inflammasome assembly and inflammatory signaling.