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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:Inflammasome, activated by pathogen-derived and host-derived danger signals, constitutes a multimolecular signaling complex that serves as a platform for caspase-1 (CASP1) activation and interleukin-1beta (IL1B) maturation. The activation of NLRP3 inflammasome requires two-step signals. The first “priming” signal (Signal 1) enhances gene expression of inflammasome components. The second “activation” signal (Signal 2) promotes the assembly of inflammasome components. Deregulated activation of NLRP3 inflammasome contributes to the pathological processes of Alzheimer’s disease (AD) and multiple sclerosis (MS). However, at present, the precise mechanism regulating NLRP3 inflammasome activation and deactivation remains largely unknown. By genome-wide gene expression profiling, we studied the molecular network of NLRP3 inflammasome activation-responsive genes in a human monocyte cell line THP-1 sequentially given two-step signals. We identified the set of 83 NLRP3 inflammasome activation-responsive genes. Among them, we found the NR4A nuclear receptor family NR4A1, NR4A2, and NR4A3, the EGR family EGR1, EGR2, and EGR3, the IkappaB family NFKBIZ, NFKBID, and NFKBIA as a key group of the genes that possibly constitute a negative feedback loop for shutting down inflammation following NLRP3 inflammasome activation. By molecular network analysis, we identified a complex network of NLRP3 inflammasome activation-responsive genes involved in cellular development and death, and immune and inflammatory responses, where transcription factors AP-1, NR4A, and EGR serve as a hub. Thus, NLRP3 inflammasome activation-responsive genes constitute the molecular network composed of a set of negative feedback regulators for prompt resolution of inflammation. To load the Signal 1 (S1), THP-1 cells were incubated for 3 hours in the culture medium with or without inclusion of 0.2 microgram/ml lipopolysaccharide (LPS). To load the Signal 2 (S2), they were incubated further for 2 hours in the culture medium with inclusion of 10 microM nigericin sodium salt dissolved in ethanol or the equal v/v% concentration of ethanol (vehicle), followed by processing for microarray analysis on Human Gene 1.0 ST Array (Affymetrix).

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Dataset Information

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

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