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Elucidating the regulation of Rsp5 a paradigm for the Nedd4-family of ubiquitin ligase proteins

Reference	BB/G004412/1
Principal Investigator / Supervisor	Dr James Sullivan
Co-Investigators / Co-Supervisors
Institution	Queen Mary University of London
Department	Sch of Biological and Chemical Sciences
Funding type	Research
Value (£)	362,710
Status	Completed
Type	Research Grant
Start date	10/10/2008
End date	09/04/2012
Duration	42 months

Abstract

The addition of ubiquitin to a protein can lead to degradation through targeting to a degradatory complex or compartment, regulated endocytosis or changes in the activity of the target protein. Central to the control of the ubiquitin pathway are a group of enzymes called ubiqutin ligases, the last step in a sequential cascade of reactions that leads to ubiquitination. It is ubiquitin ligases that control substrate specificity and to understand the ubiquitin pathway we must therefore understand how ubiquitin ligase activity is regulated. This proposal describes a series of experiments designed to investigate the yeast ligase Rsp5. Rsp5 is part of the important Nedd4-family of ubiquitin ligases, members of which are found in most eukaryotes and have implicated in cancers of the esophagus, prostate, breast and pancreas and diseases such as familial hypertension. Like other members of the Nedd4-family, Rsp5 has multiple targets in many different cellular locations and we propose to test the hypothesis that there are multiple ubiquitin ligase complexes in yeast of which Rsp5 is the core component. We will isolate Rsp5-containing complexes from yeast and purify them into distinct complexes. We will then identify the purified proteins using mass spectroscopy and confirm the activity and targets for these complexes using a combination of biochemical assays and in vivo genetic experiments. We also propose to use mass spectroscopy to identify the sites and type of any modifications on Rsp5 and its interacting proteins and to test the hypothesis that post-translational modification is used to regulate Rsp5 activity. By using Rsp5 as a paradigm for the Nedd4-family we can take advantage of yeast as a model organism with its rapid growth, excellent genetics and extensive range of bioinformatics and systems biology data, to perform experiments that would be impossible in other organisms and provide new and exciting information about the control of ubiquitin ligases.

Summary

All cells must have efficient mechanisms for identifying and removing unwanted or damaged proteins. The failure to remove a damaged protein can be catastrophic to an organism and many cancers and other diseases are often the result of an inability to degrade a particular protein. One method used by eukaryotic cells to remove unwanted proteins is to label them with a small protein called ubiquitin. The addition of ubiquitin acts as a signal that sends the tagged protein to a cellular 'dustbin' (either a degradatory complex or compartment) where the unwanted protein can be broken-down and recycled. Ubiquitin is a remarkably flexible signal since its addition, depending upon the site and the way in which it attached to another protein, can lead to outcomes other than destruction such as enzyme activation or causing the protein to move from one place in the cell to another. Understanding how ubiquitin is attached to other proteins and how this process is controlled is therefore vital to understanding many different biological processes. One group of enzymes that are essential for the addition of ubiquitin are ubiqutin ligases. Ubiquitin ligases are important because during the addition of ubiquitin they bind the target protein and control the specificity of the whole pathway. To understand the ubiquitin pathway you must therefore understand ubiquitin ligase enzymes. The aim of this proposal is to investigate a ubiqutin ligase called 'Rsp5' found in Baker's yeast (Saccharomyces cerevisiae). Rsp5 is an important enzyme involved in controlling many different cellular processes in multiple locations within the yeast cell. One obvious question relating to Rsp5 is how do you regulate an enzyme with multiple targets in different places? The experiments described in this proposal are designed to test the hypothesis that yeast cells contain multiple protein complexes of which Rsp5 is the core component and that this is how Rsp5 activity is controlled in different parts of thecell. Understanding Rsp5 is important because similar enzymes are found in other organisms, including humans where nine proteins similar in structure to Rsp5 have been identified. Like Rsp5, this family of enzymes (called the 'Nedd4'-family) regulates a huge number of different cellular processes, many of which are linked with cancers and other serious human diseases. By performing experiments on Rsp5 in yeast, which is simple and easy to grow, it is hoped that we may better understand all members of the Nedd4-family, which ultimately may lead to the development of new treatments for human diseases.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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