Dataset Information

Integrated Optoproteomics and AP-MS Reveals interactome Remodeling Upon NLRP3 Inflammasome Activation

ABSTRACT: NOD-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.

ORGANISM(S): Mus Musculus

SUBMITTER: Jian Wang

PROVIDER: PXD079555 | iProX | Thu Jun 11 00:00:00 BST 2026

REPOSITORIES: iProX

ACCESS DATA

Similar Datasets

Project description:The cellular stress response plays a vital role in regulating homeostasis by modulating cell survival and death. Stress granules (referred to as SGs) are cytoplasmic compartments that allow cells to survive various stressors. Defects in SG assembly and disassembly have been found to play important roles in neurodegenerative diseases, antiviral responses and cancer1–5. Inflammasomes are multi-protein heteromeric complexes that sense intracellular pathogen- and damage-associated molecular patterns and assemble into cytosolic compartments called ASC specks to facilitate caspase-1 (CASP1) activation. This activation of inflammasomes induces secretion of the leaderless proinflammatory cytokines IL-1β and IL-18 and also drives cell fate towards pyroptosis, a form of programmed inflammatory cell death that plays major role in health and disease6–12. Cellular stress sensing can trigger both SGs and inflammasomes; however, they drive contrasting cell fate decisions during stress conditions. Crosstalk between SGs and inflammasomes to decide cell fate has not been well studied. Here we show that the induction of SGs specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The SG protein DDX3X interacts with NLRP3 to drive inflammasome activation in the absence of SGs. Assembly of SGs leads to the sequestration of DDX3X to inhibit NLRP3 inflammasome function. SGs and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell fate decisions under stress conditions. Induction of SGs or loss of DDX3X in the myeloid compartment leads to decreased production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages utilize DDX3X availability to interpret stress signals and choose between pro-survival SGs and pyroptotic ASC specks. Together, our data show the role of DDX3X in driving NLRP3 inflammasome and SG assembly, informing a new rheostat-like mechanistic paradigm for regulating live or die cell fate decisions under stress conditions.

Dataset Information

Integrated Optoproteomics and AP-MS Reveals interactome Remodeling Upon NLRP3 Inflammasome Activation

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets