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

  • Mol Cell Proteomics. 2026 May 26;25(7):101595. doi: 10.1016/j.mcpro.2026.101595.
Yuehui Zhang  1 ,  Kairong Zhang  2 ,  Chaozhi Jin  1 ,  Limin Shang  1 ,  Shanshan Wu  1 ,  Mingyang Zhang  1 ,  Yanzhi Yuan  1 ,  Yuchen Liu  3 ,  Jian Wang  4
Affiliations
  • 1. State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Beijing, China.
  • 2. State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Beijing, China; College of Life Sciences, Hebei University, Baoding, China.
  • 3. State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Beijing, China. Electronic address: [email protected].
  • 4. State Key Laboratory of Medical Proteomics, National Center for Protein Sciences (Beijing), Beijing, China. Electronic address: [email protected].
PMID: 42202946 DOI: 10.1016/j.mcpro.2026.101595
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 1496 stimulus-associated protein interactions (1032 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.

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