Dataset Information

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

ABSTRACT: The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

SUBMITTER: Pouly D

PROVIDER: S-EPMC4710524 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

Pouly Daniel D Chenaux Sébastien S Martin Virginie V Babis Maja M Koch Rafael R Nagoshi Emi E Katanaev Vladimir L VL Gachon Frédéric F Staub Olivier O

PloS one 20160112 1

The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output ...[more]

PMID: 26756164

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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.

Dataset Information

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

Publications

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

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