Dataset Information

Identification of Drosophila USP2 partners

ABSTRACT: We used Drosophila flies model system for the identification of new enzymes of the ubiquitin specific protease family regulating evolutionary conserved immune signals in both Drosophila and Humans. These enzymes specifically control the stability or the activated status of target proteins by hydrolysing ubiquitin. We report here results of USP2 partners pull-down.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Yohann Couté

LAB HEAD: Christophe Bruley

PROVIDER: PXD000881 | Pride | 2014-07-18

REPOSITORIES: pride

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Publications

Identifying USPs regulating immune signals in Drosophila: USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome.

Engel Elodie E Viargues Perrine P Mortier Magda M Taillebourg Emmanuel E Couté Yohann Y Thevenon Dominique D Fauvarque Marie-Odile MO

Cell communication and signaling : CCS 20140716

<h4>Background</h4>Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NF-κB dependent signals which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, we created an ...[more]

PMID: 25027767

Similar Datasets

Project description:In order to identify molecular targets of USP2-45 in the small intestine, we carried out a quantitative proteomics iTraq (Isobaric tag for relative and absolute quantitation) screen to compare proteins from WT and knockout mice. With this analysis we identified, among others, the scaffolding protein Na(+)/H(+) Exchange Regulatory cofactor NHERF4 to be up-regulated (4.81-fold) in the duodenum of Usp2-KO mice. Methods : Usp2-KO and WT littermates males were entrained to 12h L:12h D for at least two weeks in light-light housing boxes. Mice were sacrificed by cervical disolcation at ZT13 under red dim light and duodenal mucosa was dissected by scraping. The tissue samples were quickly frozen in liquid nitrogen before total protein extraction. Proteins were extracted in a lysis buffer containing 8M Urea, 20mM Tris-HCl, pH 7.5, 14 µg/ml Aprotinin, 0.7 µg /ml PepstatinA 0.7 µg /ml Leupeptin, 1 mM NaVO4, 10 mM Na-Pyrophosphate, 50 µM MG132 and 1mM PMSF. Protein concentration was measured by Bradford quantitation. Relative quantitation of proteins in 4 pools of 3 KO and 4 pools of 3 WT tissue samples was carried out by iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). After reduction and alkylation of cysteines, proteins were precipitated, redissolved in 8M Urea buffer and digested with trypsin (1:50 w:w) overnight. 80ug of digested material for each sample was labeled by reaction with one vial of iTRAQ 8-plex reagent (ABSciex). Samples were pooled , desalted and fractionated by off-gel electrofocusing. The 24 fractions obtained were analysed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) on a hybrid linear trap LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) on a two-hour gradient. Full MS survey scans were performed at 60’000 resolution and the ten most intense multiply charged precursor ions detected in the full MS survey scan were selected for HCD (Higher energy Collision Dissociation) fragmentation and Orbitrap analysis (7500 resolution). Each spectrum was acquired at two relative collision energies (35% and 45%) and the spectra were summed. Data files were analysed with MaxQuant 1.3.0.5 incorporating the Andromeda search engine. Further data analysis was performed using the R statistical programming language version 2.15.2 (R core team 2012). Intensities for the reporter ions 113 to 121 were normalized using the Variance Stabilizing method (R package version 3.26.0). Proteins with non-zero intensities in all channels were evaluated for differential expression using the Local-Pooled-Error method (R package version 1.32.0) followed by correction for multiple testing according to Benjamini and Hochberg.

			Action	DRS
	GST-USP2.raw	Raw
	GST.raw	Raw
	USP2partners.pride.mgf.gz	Mgf
	USP2partners.pride.mztab.gz	Mztab
	USP2partners.xml.gz	Xml