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Linking the proteome and transcriptome - assessment of the relationship between proteome ubiquitination and turnover

Reference	BB/C507510/1
Principal Investigator / Supervisor	Professor Robert Beynon
Co-Investigators / Co-Supervisors	Professor Simon James Gaskell
Institution	University of Liverpool
Department	Veterinary Preclinical Science
Funding type	Research
Value (£)	263,226
Status	Completed
Type	Research Grant
Start date	01/10/2004
End date	30/09/2007
Duration	36 months

Abstract

A full description of any proteome must include the definition of the processes whereby is synthesised and degraded, as these two opposing processes lead to changes in protein concentration and the ultimate steady state level that is attained. In particular, the key regulatory step is the commitment process, which for many eukaryotic proteins is the conjugation of a polyubiquitin sequence. In this programme, we aim to determine the sites of ubiquitin conjugation for proteins in the yeast and chicken skeletal muscle proteome, and correlate the site of ubiquitination with the physicochemical properties of the target protein, and its rate of turnover. This is the first attempt to bring together an integrated and proteome-wide view of the dynamics of ubiquitin medicated protein targeting for degradation. Proteins from yeast or chicken skeletal muscle will be labelled with stable isotope labelled amino acids in vivo, using previously established protocols and then recovered. The labelled proteins will be resolved on 2-D gel electrophoresis and ubiquitin conjugates identified by western blotting; alternatively the ubiquitinated proteins will be recovered by an affinity pull-down experiment using antibodies to ubiquitin, or a GT-S5a fusion protein, which selectively binds polyubiquitin chains. The sites of ubiquitination will be determined by a combination of high resolution peptides mass fingerprinting, by selective product ion analysis and novel precursor/neutral loss strategies, based on knowledge of peptide ion fragmentation chemistry and selective derivatisations. At the same time, the rates of turnover of individual proteins will be assessed as the relative incorporation of stable isotope labelled amino acids, determined either by established approaches based on simple peptide mass fingerprinting or by new approaches based on scanning for heavy and light precursor ions. The outcome of this work is that we shall derive two data sets that define the interaction betweenthe commitment step for protein breakdown and the subsequent rate of turnover. These data sets will be used to compare rates of protein ubiquitination and breakdown in fast and slow growing pectoralis muscle in the chicken, and in the definition of ubiquitination sites for proteins in yeast, where the scope for genetic intervention is greater. In both instances we will be able to include turnover rates in an integrated model that links transcriptome data with steady state protein levels. We also expect to be able to explore predictive approaches to the identification of ubiquitination sites in proteins.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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