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Potential interactors of NLRP3 in NLRP3-inducible THP-1 cells treated with LPS

ABSTRACT: The NLRP3-inducible THP-1-cell line was generated by introducing Flag-WT NLRP3 into NLRP3 KO THP-1 cells through a tetracycline (Tet) responsive element (TRE) promoter (Tet-On) lentiviral infection system. Cells were treated with doxycycline (Dox, 200 ng/mL, Sigma-Aldrich, cat# D9891) for 18 h to induce the expression of Flag-NLRP3 in THP-1-derived macrophages, followed by treatment with lipopolysaccharide (LPS, 200 ng/ml, Sigma-Aldrich, cat# L4524) for 3 h. We used the cells without doxycycline treatment as a negative control and mixed the cells from three individual flasks for mass spectrometry. The cells were collected in low-salt lysis buffer (50 mM Tris-HCl (GCRF, China), pH 8.0, 150 mM NaCl (GCRF, China), 1 mM EDTA (Vetec), 1 mM DTT (Sigma-Aldrich) and 0.5% IGEPAL CA630 (Sigma-Aldrich) containing phosphatase inhibitor (Roche) and EDTA-free protease inhibitor (Biosharp)) and incubated on a rocker with ice for 30 min and centrifuged at 12000 g/4 °C for 15 min to obtain cell lysates. The cell lysates were immunoprecipitated with an anti-Flag antibody. The beads were washed three times with low-salt lysis buffer, separated by SDS-PAGE, and then stained with Coomassie Blue. The entire lane was excised and digested with trypsin at 37 °C for 20 h. The enzymatic hydrolysis product was desalted, lyophilized and redissolved in 0.1% FA solution for chromatography-tandem mass spectrometry (LC-MS/MS) identification of peptide mixtures at Applied Protein Technology (aptbiotech, Inc. Shanghai, China) with a Q Exactive mass spectrometer (Thermo Fisher, California, USA). RAW files generated from the spectrometer were subjected to Proteome Discoverer 1.4 software for protein identification.

ORGANISM(S): Homo Sapiens

SUBMITTER: Jun Cui

PROVIDER: PXD038215 | iProX | Fri Nov 18 00:00:00 GMT 2022

REPOSITORIES: iProX

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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).

Project description:Human keratinocytes, HaCaT cells were transfected with the dsRNA-analogue polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich) using Lipofectamine 2000 reagent (Invitrogen) for intracellular delivery. The cells were transfected with 7 ug/ml poly I:C for 1 h or left untreated. The cells were collected in PBS with a cell scraper and washed once with PBS before they were lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The cell lysates were centrifuged 2,264 x g for 15 min at 4°C, divided into smaller fractions and centrifuged again 11,686 x g for 15 min at 4°C . The supernatant was collected and the protein content was measured with Bio-Rad DC protein assay (Bio-Rad). For the samples, 8 mg of protein was used. The proteins were precipitated with 10 % TCA/acetone at -20°C overnight. Samples were centrifuged 10,733 x g for 20 min at 4°C and the supernatant was removed. The precipitation was washed once with acetone and dissolved in urea buffer (8 M urea, 400 mM NH4CO3, 20 mM DL-dithiotheitol, 1 mM EDTA, pH 8.5). The proteins were reduced, alkylated, and enzymatically digested in-solution with lysyl endopeptidase (Wako Chemicals) and trypsin (Promega). The peptides were fractionated by strong cation exchange chromatography. Phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich). The enriched phosphopeptides were analyzed by nanoLC-MS/MS using an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization.23,24 The LC-MS/MS data were submitted through the ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) to an in-house Mascot database search engine version 4.0 (Matrix Science), and to the ProteinPilot algorithm Paragon. The data were searched against the human canonical sequences in the Swiss-Prot database (decoy version 08132012 for the Mascot searches and version 04012013 with 539,829 sequences for the Paragon searches). The Mascot search criteria were: fixed modifications: carbamidomethyl of cysteine, variable modifications: oxidation (M), phospho (ST), phospho (Y), acetylation (K), 1 missed cleavage allowed, enzyme: trypsin. The Paragon search criteria were: modifications: cystein alkylation with iodoacetamide, ID focus: biological modifications, phosphorylation emphasis, identification threshold: 95 % confidence (Unused ProtScore (Conf) > 1.3), Thorough search (ID), enzyme: trypsin.

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Potential interactors of NLRP3 in NLRP3-inducible THP-1 cells treated with LPS

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