CLOCKPROTEOMICS

Circadian clock function by quantitative proteomics and phosphoproteomics

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 Obiettivo del progetto (Objective)

'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.'

Introduzione (Teaser)

The circadian or body clock is a 24-hour cycle that regulates various physiological processes. Understanding how circadian oscillations extend to metabolism was the subject of a European study.

Descrizione progetto (Article)

Circadian clocks rely on the master core transcription factors CLOCK and BMAL1 to drive rhythmic gene expression to ultimately regulate daily metabolism and physiology. To date, most studies have analysed oscillations of the transcriptome. However, since proteins are the true mediators of cellular functions, scientists need to re-evaluate their approach and extend their work at the proteomic level.

The EU-funded 'Circadian clock function by quantitative proteomics and phosphoproteomics' (CLOCKPROTEOMICS) project looked at the role of proteins in circadian clock function in mouse tissues. Scientists utilised state-of-the-art mass spectrometry (MS) in combination with quantitative proteomics to investigate circadian rhythms in mammalian protein expression.

Researchers discovered that approximately 6 % of the liver proteins cycled daily and their oscillations differed from those of their transcripts. This clearly indicated that post-transcriptional mechanisms critically shape the phase of rhythmic proteins and thus metabolic processes.

The circadian oscillations of liver proteins seem to be instrumental, not only for metabolism, but for other cellular processes. The next step in the project was to describe circadian oscillations in phosphorylated proteins, a modification associated with protein function. Results showed that a large set of proteins in mouse liver displayed rhythms in their phosphorylation levels.

Furthermore, scientists applayed MS to analyse protein complexes binding to clock DNA consensus sequences, and especially onto the promoter of the period circadian protein homolog 2 (per2) gene. They identified previously unknown interactors of BMAL and CLOCK that bind to consensus DNA sequences and appeared to have a role in chromatin modification.

The work by the CLOCKPROTEOMICS study has emphasised the importance of the circadian clocks- by mean of protein abundance- in the regulation of mammalian metabolism. The researchers have provided firm evidences on the mechanism by which CLOCK/BMAL1 complexes regulate gene transcription.