Regulation of cell function by protein modification by ubiquitin and ubiquitin like proteins

Lead Research Organisation: MRC Human Genetics Unit

Abstract

Proteins control most of the important reactions carried out within cells. It is widely recognised that the timely removal of proteins by protein destruction is easily as important as making proteins at the correct time. In humans when this process goes wrong the results are catastrophic for the individual causing a wide range of debilitating diseases such as cancer, Alzheimers and Parkinsons disease. Proteins are targeted for destruction by being marked by the addition of a ubiquitin chain. The chain acts as flag, which is recognised by the cell and the protein rapidly turned over by the 26S proteasome, which acts as a protein shredder. We study how the ubiquitin flag is recognised by the cell. Previously, we have identified a number of flag recognition proteins. We propose to carry out a number of experiments to investigate how these flag recognition proteins can interact with the flag adding machinery of the cell and then present the marked proteins for degradation by the 26S proteasome.||Recently, another protein flag called Nedd8 has been shown to be added to many different proteins. The function of Nedd8 addition to proteins is at present unclear but it is known that it does not target them for destruction. But as a substantial number of proteins seem to be modified in this way it seems that modification by this protein will have important implications for intracellular regulation. We propose to identify the different proteins modified by this protein flag and investigate how modification alters the properties of the protein.

Technical Summary

Postranslational modification by ubiquitin and ubiquitin like proteins is the most common form of modification in eukaryotic cells. This covalent modification is used to regulate many different intracellular processes such as endocytosis, vesicle sorting and virus budding. However most work on ubiquitin has been carried out on its role as a signal to target proteins for degradation by the 26S proteasome. Two projects will investigate different aspects of cell regulation by ubiquitin and ubiquitin like proteins.||In the first project, proteins which interact with E3 ubiquitin ligases, recognise and bind to ubiquitinated substrates and present the substrates to the 26S proteasome for degradation will be characterised in more detail. Both biochemical and genetic experiments will be carried out to determine how these presentation proteins carry out their function. In addition, genetic screens will be carried out to identify novel components of this presentation pathway.||The second project involves modification by the ubiquitin like protein Nedd8. Like ubiquitin Nedd8 is covalently added to lysine residues on substrate proteins by the action of a heterodimeric E1, and E2 enzymes. Mutation of the Nedd8 E1 or E2 genes has demonstrated that Nedd8 conjugation is essential in fission yeast. Also like ubiquitin the modification can be removed by the action of specific deneddylases. We propose to use biochemistry and genetics to study the Nedd8 modification pathway in fission yeast to work out what processes are regulated by this modification.

Publications

10 25 50
 
Description European Union
Amount £300,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start  
 
Title Specific antibodies to components of the ubiquitin pathway in fission yeast 
Description Specific antibodies 
Type Of Material Antibody 
Year Produced 2006 
Provided To Others? Yes  
Impact Many publications by numerous research groups 
 
Title Yeast strains 
Description Mutants that conditionally comprimise different aspects of the ubiquitin proteasome system in fission yeast 
Type Of Material Cell line 
Year Produced 2006 
Provided To Others? Yes  
Impact The strains have been disseminated to over 500 research groups. As a result many papers by many different research groups have been published using my mutants 
 
Description Recognition of the ubiquitin degradation signal 
Organisation University of Oxford
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified proteins involved in ubiquitin recognition for degradation by the proteasome
Collaborator Contribution The collaborator are experts in Biophysics and have characterised structurally protein complexes we have been studying in fission yeast.
Impact a PhD student has visited the collaborators lab to carry out some biophysical experiments. a student from the collaborators lab has visited my lab to carry out cell biology experiments. PMID:16138082
 
Description Regulation of the COP9/Signalosome 
Organisation Humboldt University of Berlin
Department Department of Surgery
Country Germany 
Sector Academic/University 
PI Contribution We charactersed a evolutionary conserved complex in fission yeast
Collaborator Contribution They chacterised a evolutionary complex in mammalian cells
Impact A PhD studented visited collaborators lab to carry out biochemical experiments. In addition PhD student from the collaborators lab visited my lab to carry out confocal miscropy.
Start Year 2007