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Circadian clock function by quantitative proteomic.. (Clockproteomics)
Circadian clock function by quantitative proteomics and phosphoproteomics
(Clockproteomics)
Date du début: 1 sept. 2009,
Date de fin: 9 juin 2014
PROJET
TERMINÉ
The circadian clock is an endogenous oscillator that drives daily rhythms in physiology and behavior. The mechanism underlying circadian timekeeping is cell-autonomous and oscillations are generated by interconnecting transcriptional and translational feedback loops. To date large-scale approaches to study molecular circadian rhythms have been limited to the analysis of the transcriptome (mRNA). However, the “true” mediators of cellular functions are proteins. Recent advances in mass spectrometric and bioinformatics technology now allow global quantitation of protein abundance and phosphorylation. The aim of this proposal is to apply the latest methodology in mass spectrometry and bioinformatics, in combination with stable isotope labeling by amino acids in cell culture (SILAC), or in mice, to the investigation of circadian clocks in mammals. I propose to perform a comprehensive quantitative analysis of protein and phosphoprotein circadian oscillations in mouse tissues. The results will be compiled in a database, linked to the European Bioinformatics Institute and available to the scientific community, containing the circadian profiles of all the identified proteins and phosphoproteins in different organs. In addition, I intend to use SILAC-based quantitative proteomics, to identify novel regulators of the circadian transcriptional activity by isolating and analyzing protein complexes bound to clock DNA consensus sequences, and to investigate the molecular interplay between the circadian clock and the DNA-repair machinery. In summary, the objectives of this proposal aim to pioneer the application of SILAC- and mass spectrometry-based proteomics in the quantification of circadian oscillations of proteins and phosphoproteins as well as in the identification of clock components and their interplay with proteins of the genotoxic response.
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