Dataset Information

EGF-dependent ubiquitinome, phosphorylation and interactome analysis in control and CYLD-deficient cells

ABSTRACT: The goal of the present project was to investigate the EGF-dependent ubiquitination changes occurring in HeLa cells upon a CYLD deficiency. We applied SILAC-based quantitative mass spectrometry proteomics together with genetic engineering and biochemical approaches, in order to enrich the ubiquitinated proteins of the control and silenced cells in response to the growth factor. In order to get a deeper insight of the obtained results, we subsequently combined the enrichment of CYLD protein through immunoprecipitation with SILAC-based proteomics. This allowed us to decipher a specific tyrosine phosphorylation of CYLD that responds to the treatment with EGF and, therefore, we next aimed to analyse the ability of this modification to recruit potential interaction partners. We thus performed a peptide pull-down assay that uncovered a set of proteins specifically associated to the phosphorylated tyrosine and helped to uncover an unexpected, previously unreported role for this deubiquitinase.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: VIRGINIA Sanchez-Quiles

LAB HEAD: BLAGOY BLAGOEV

PROVIDER: PXD003423 | Pride | 2017-06-05

REPOSITORIES: Pride

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Publications

Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor.

Sanchez-Quiles Virginia V Akimov Vyacheslav V Osinalde Nerea N Francavilla Chiara C Puglia Michele M Barrio-Hernandez Inigo I Kratchmarova Irina I Olsen Jesper V JV Blagoev Blagoy B

Molecular & cellular proteomics : MCP 20170601 8

Cylindromatosis tumor suppressor protein (CYLD) is a deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitin ...[more]

PMID: 28572092

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Project description:miRNA-mediated gene expression silencing has previously been shown to be important for a variety of physiological and pathological processes. Here, we have explored the role of one bona fide human-specific miRNA (miR-941) in evolution of the human-specific expression and function. Using combination of high-throughput sequencing (GSE26545), miRNA transfection and large-scale PCR of various human populations, we have shown that emergence and rapid expansion of miR-941 might take place on the human evolutionary linage between six and one million years ago. Functionally, miR-941 could be associated with hedgehog and insulin signaling pathways, and thus might potentially play a role in evolution of human longevity. Human-specific effects of miR-941 regulation are detectable in human brain and affect genes involved in neurotransmitter signaling. Furthermore, emergence of miR-941 on the human evolutionary linage was accompanied by the accelerated loss of its binding sites. Taken together, these results strongly implicate the contribution of miR-941 in evolution of the human-specific phenotype. Ago2 Immunoprecipitation (Ago2-IP) experiments after miR-941 overexpression were conducted in 293T cell line. Briefly, All transfections were performed using human 293T cells cultured in 6-well tissue culture plates. Lipofectamine 2000 (Invitrogen) was used for a Synthetic miR-941 or a scrambled oligo transfection, at 30 nmol/l each (final concentration) per 1x106 cells/well of a 6-well plate using DharmaFECT (GE Healthcare). Total 5x106 cells were collected and subjected to Ago2 immunoprecipitation (Ago2-IP) using the RNA isolation kit Mouse Ago2 (Wako Chemicals) according to the manufacturer's instructions. For a negative control, immunoprecipitation was performed using nonimmune IgG beads prepared with the antibody immobilization bead kit (Wako Chemicals). The IP pull down RNA was used as template for an “in vitro” transcription reaction generating biotin-labeled antisense cRNA. The cRNA was analyzed on affymetrix Human Genome U133 Plus 2.0 arrays following the manufacturer’s instructions. R RMA package was used to quantify gene expression levels.

Dataset Information

EGF-dependent ubiquitinome, phosphorylation and interactome analysis in control and CYLD-deficient cells

Publications

Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor.

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