Dataset Information

Global identification ofphospho-dependent SCF ubiquitin ligase substrate reveal a role of ERK-FBXO22-BAG3 axis in tumorigenesis

ABSTRACT: 293T cells were cultured in SILAC medium. Heavy group was labeled with K6&R10. Light group was labeled with K0&R0. In light group, cells were treated with MLN4924. In heavy group, cells were treated with DMSO. Then cells were lysed, mixed and tryptic digested. Peptide mixture was subjected to phosphopeptide enrichment followed by LC-MS/MS analysis. MS raw data was processed using Maxquant(1.5.3.8) against a human proteome database from Uniprot. BAG3 is one of identified substrate. In order to find molecular machanism of BAG. TMT labeling based quantitative proteomics technique was used. Details were shown in manuscript.

ORGANISM(S): Homo Sapiens

SUBMITTER: Minjia Tan

PROVIDER: PXD027020 | iProX | Tue Jun 29 00:00:00 BST 2021

REPOSITORIES: iProX

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Publications

Global identification of phospho-dependent SCF substrates reveals a FBXO22 phosphodegron and an ERK-FBXO22-BAG3 axis in tumorigenesis.

Liu Ping P Cong Xiaoji X Liao Shengjie S Jia Xinglong X Wang Xiaomin X Dai Wei W Zhai Linhui L Zhao Lei L Ji Jing J Ni Duan D Liu Zhiwei Z Chen Yulu Y Pan Lulu L Liu Wei W Zhang Jian J Huang Min M Liu Bin B Tan Minjia M

Cell death and differentiation 20210702 1

SKP1-CUL1-F-box (SCF) ubiquitin ligases play fundamental roles in cellular functions. Typically, substrate phosphorylation is required for SCF recognition and subsequent degradation. However, phospho-dependent substrates remain largely unidentified. Here, using quantitative phoshoproteome approach, we performed a system-wide investigation of phospho-dependent SCF substrates. This strategy identified diverse phospho-dependent candidates. Biochemical verification revealed a mechanism by which SCF< ...[more]

PMID: 34215846

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Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.

Project description:Mycolactone is a diffusible macrolide produced by the skin pathogen Mycobacterium ulcerans that suppresses the development of protective immunity against Buruli ulcer disease. In this proteomics study we aimed to identify proteins of which the expression levels changed upon mycolactone treatment. Jurkat T cells were labeled light or heavy by stable isotope labeling by amino acids in cell culture (SILAC), then treated with mycolactone (light) or vehicle (heavy) for 1h prior to activation with PMA/IO for 6h. After mixing light- and heavy-labeled cultures, cells were lysed and extracted proteins were digested with trypsin. The resulting peptide mixture was analyzed by LC-MS/MS and proteins were identified by the MaxQuant software and quantified based on the intensity of the light and heavy signals in the MS spectra of their peptides. The analysis was repeated with reversed labeling, leading to a total of 4,636 proteins that were quantified in both analyses. Interestingly, 52 proteins were down-regulated in mycolactone-treated cells (mycolactone/control ratio < 1.4), while only two proteins were up-regulated (mycolactone/control ratio > 1.4). Gene ontology analysis further revealed that the down-regulated proteins were significantly enriched in proteins located in the plasma membrane and endoplasmic reticulum and we could show that this enrichment was not due to a bias in protein extraction, since the distribution of all identified proteins across different subcellular compartments was similar to those of all human proteins. Analysis of SP-PIR Family-Domains keywords in the UniProt database confirmed this observation and showed an additional enrichment in glycoproteins, proteins with an immunoglobulin domain and proteins involved in immune responses. Furthermore, we observed that the down-regulated proteins were significantly enriched in single-pass type I/II membrane proteins. In contrast, the fraction of multi-pass membrane proteins was comparable among the down-regulated and all identified proteins, suggesting that multi-pass membrane proteins may at least partially resist mycolactone-mediated down-regulation.

Dataset Information

Global identification ofphospho-dependent SCF ubiquitin ligase substrate reveal a role of ERK-FBXO22-BAG3 axis in tumorigenesis

Publications

Global identification of phospho-dependent SCF substrates reveals a FBXO22 phosphodegron and an ERK-FBXO22-BAG3 axis in tumorigenesis.

Similar Datasets

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