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HEK293T cells were transfected with Flag-tagged NLRP3 treated with or without nigericin (10 μM, 1 h).

ABSTRACT: In order to identificate NLRP3 acetylation, we utilized an acetylproteomic approach to identify the acetylation sites within NLRP3. We first reconstituted the NLRP3 inflammasome in HEK293T cells by co-transfected with plasmids encoding NLRP3, ASC, Caspase-1 and Pro-IL-1β, then stimulated the genetically modified cells with nigericin.Flag-tagged NLRP3 was immunoprecipitated and analyzed by liquid chromatography-mass spectrometry.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Kai Zhao

LAB HEAD: Kai Zhao

PROVIDER: PXD041763 | Pride | 2023-11-25

REPOSITORIES: Pride

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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

HEK293T cells were transfected with Flag-tagged NLRP3 treated with or without nigericin (10 μM, 1 h).

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