iProXProteomicsJian WangMus Musculushttp://www.iprox.org/page/project.html?id=IPX0015078000wangj.bio@foxmail.comNational Center for Protein Sciences (Beijing)iProXfalseIntegrated Optoproteomics and AP-MS Reveals interactome Remodeling Upon NLRP3 Inflammasome ActivationNOD-like receptor family pyrin domain-containing 3 (NLRP3) is a critical intracellular danger sensor that forms inflammasome complexes to drive inflammatory caspase activation; however, the components and dynamic organization of the NLRP3 inflammasome remain incompletely defined. Here, we systematically characterized stimulus-dependent perturbations of the NLRP3 inflammasome interaction landscape by generating antibody-based endogenous immunoprecipitation-mass spectrometry (IP-MS) datasets for both NLRP3 and the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) in bone marrow-derived macrophages (BMDMs). Across these conditions, we identified 1,541 inflammasome-associated protein interactions (1,048 non-redundant), enabling quantitative comparison of interaction dynamics across first- and second-signal activation states. Differential interaction scoring revealed networks selectively enriched during inflammasome priming and activation. Integration of these interaction datasets with microscopy-guided photo-labelling (optoproteomics) of ASC specks identified inner membrane mitochondrial protein (IMMT) as a previously unrecognized component of the NLRP3 inflammasome that associates with both NLRP3 and ASC upon activation. Functional analyses demonstrated that Immt knockdown enhanced caspase-1 and gasdermin D cleavage, IL-1β secretion, lactate dehydrogenase (LDH) release, and ASC oligomerization in response to NLRP3 agonists, establishing IMMT as a negative regulator of inflammasome activation. Together, this study provides a comprehensive and stimulus-resolved proteomic map of NLRP3 and ASC interaction networks in macrophages and reveals IMMT as a regulatory factor in NLRP3 inflammasome assembly and signaling.Thu Jun 11 00:00:00 GMT+01:00 2026PXD07955510090