Dr Colin Gordon: Chromosomes and Gene Expression
Regulation of Cell Function through Protein Modification by Ubiquitin and Ubiquitin like Proteins
To identify the mechanisms by which post-translational covalent modification of proteins by ubiquitin and ubiquitin like proteins regulate intracellular processes.
2. The identification and function of modification by the ubiquitin like protein Nedd8
The ubiquitin like protein Nedd8 is a 78amino acid protein that is 50% identical to ubiquitin. Like ubiquitin the Nedd8 protein is first activated by its own E1 and E2 enzymes. Again like ubiquitin Nedd8 attaches via an isopeptide bond to a lysine residue in the substrate protein. Until recently only members of the cullin family had been identified as substrates for neddylation. Cullins are subunits of the multi-subunit SCF E3 ligases and their neddylation is thought to activate the E3 ligase activity. We have recently identified two enzymes which act as nedd8 proteases to remove Nedd8 from its substrate.
In strains deleted for these proteases neddylated substrates are stabilised. In
addition to cullins a large number of additional proteins appear to be Nedd8 substrates.
Using a quantitative mass spectrometry approach we are carrying out experiments to
identify what proteins, in addition, to cullins, are neddylated and therefore what
pathways are potentially regulated by this ubiquitin like protein.
- O'Donoghue, J. and Gordon, C.. Proteasome interacting proteins. In:The ubiquitin/proteasome system Volume II Ciechanover, A.; Mayer, R.J. and Rechsteiner, M.(eds.) 157-182, 2006
- Trempe, J.F.; Brown, N.R.; Lowe, E.D.; Gordon, C., Campbell, I.D.;
Noble, M.E.M. and Endicott.J.A. Mechanism of Lys48-linked polyubiquitin chain recognition
by the Mud1 UBA domain. EMBO Journal 24:3178-3189, 2005
- Welchman,R.L.; Gordon, C. and Mayer.R.J. Ubiquitin and
ubiquitin-like proteins as multifunctional signals. Nature Mol Cell Biol Rev
- Hartmann-Petersen, R.; Wallace, M.; K. Hofmann, K.; Koch, G.; Johnsen, A.H.; Hendil,
K.B. and Gordon, C. The Ubx2 and Ubx3 co-factors direct Cdc48 activity
to proteolytic and non-proteolytic ubiquitin-dependent processes. Current
Biology 14:824-828, 2004
- Hartmann-Petersen, R. and Gordon, C. Integral UBL domain proteins: a
family of proteasome interacting proteins. Semin Cell Dev Biol 15:247-259,
- Seeger, M.; Hartmann-Petersen, R.; Wilkinson, C.R.M.; Wallace, M.; Samejima, I.;
Taylor, M. and C.Gordon. Interaction of the APC/Cyclosome and Proteasome
protein complexes with multiubiquitin chain binding proteins. J Biol Chem
- Hartmann-Petersen, R.; Hendil K.B. and Gordon, C. Ubiquitin binding proteins protect ubiquitin conjugates from disassembly. FEBS Letters 535:77-81, 2003 PubMed Abstract
- Yen, H-Y. S.; Gordon, C. and Chang. E. Schizosaccharomyces pombe
Int6 and Ras homologs regulate cell division and mitotic fidelity via the proteasome.
Cell 112:207-217, 2003
- Hartmann-Petersen, R.; Seeger, M. and Gordon, C.. Transferring
substrates to the 26S proteasome Trends Biochem Sci 28:26-31, 2003
- Wilkinson, C.R.M., Seeger, M., Hartmann-Petersen, R., Stone, M., Wallace, M., Semple,
C. and Gordon, C. Proteins containing the UBA domain are able to bind to
Nature Cell Biol 3:939-943, 2001
- Professor Stuart Ralston Edinburgh University, UK.
- Professor Jane Endicott Oxford University, UK.
- Dr Daniel Mulvihill Kent University, Canterbury, UK.
- Dr Rasmus Hartmann-Petersen Copenhagen University, Denmark.
- Dr Kay Hofmann Memorec Biotech GmbH, Cologne, Germany.
- Professor Mattius Mann Max Planck Institute for
Biochemistry, Martinsfried, Germany.
Current lab members involved in this work are:
Bethan Medina - PhD student
Mairi Wallace - Research support
Morag Robertson - Research support
Dr Konstantinos Paraskevopoulos
These fall into two parts:
The role of the multi-chain recognition proteins Dph1, Pus1 and Rhp23 in ubiquitin dependent proteolysis by the 26S proteasome
The identification and function of modification by the ubiquitin like protein Nedd8
Postranslational modification by ubiquitin and ubiquitin like proteins is the most
common form of modification in eukaryotic cells. The 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. In humans when the
modifications are misregulated the results can be catastrophic for the individual
resulting in a wide range of diseases such as cancer and neurodegenerative disease such
as Alzheimer’s and Parkinson’s diseases. Therefore,
knowledge of how these basic intracellular pathways work should have direct implications
for novel therapies for a wide range of different human diseases.
Approach, Progress and Future Work
We study regulation of cell function by ubiquitin and ubiquitin like proteins in the fission yeast Schizosaccharomyces pombe. Fission yeast is an attractive model organism for the study of cell biology. It is amenable to genetic manipulation and also provides suitable material for biochemical experiments allowing a combined approach to solve biological problems. Protein modification by ubiquitin and ubiquitin like proteins shows striking degree of conservation in all eukaryotes, from yeast to man. Therefore the results from studies in fission yeast can be easily extrapolated to higher organisms, including humans, to understand how misregulation of the pathway can lead to clinical disease.
Two main projects are currently being studied in the laboratory
1. The role of the multi-chain recognition proteins Dph1, Pus1 and Rhp23 in ubiquitin dependent proteolysis by the 26S proteasome
The ubiquitin pathway is found in all eukaryotes. In this pathway target proteins are covalently modified by the addition of ubiquitin a 76 amino acid protein to specific lysine residues. Ubiquitin molecules are added to target proteins by an enzyme cascade (See Figure1). First the ubiquitin activating enzyme or E1 enzyme, activates the ubiquitin molecule by forming a thioester bond between a conserved cysteine residue in the enzymes active site and the glycine at position 76 in the carboxy-terminal end of the ubiquitin molecule. This reaction requires ATP. The ubiquitin is then passed on to the ubiquitin conjugating enzyme or E2 enzyme, again with the formation of a thioester bond between the ubiquitin molecule and the E2 enzyme. The E2 enzyme transfers the activated ubiquitin to a lysine residue of the substrate protein, either directly or in co-operation of specific ubiquitin ligases or E3 enzymes. Most species have only one E1 enzyme and multiple E2 and E3 enzymes. The multi-ubiquitin signal is then recognised by receptor proteins.
The E1, E2 and E3 enzyme cascade attaches a multi-ubiquitin chain to the target substrate protein at the correct time. A key question is how is the multi-ubiquitin recognised as a signal within the cell. We have demonstrated that a previously identified domain, the UBA domain, acts as a multi-ubiquitin receptor. Searching the fission yeast database has identified at least 13 different fission yeast proteins that contain a UBA domain in their structure. Two of these proteins, Dph1 and Rhp23, are able to interact with the 26S proteasome. In addition to the UBA domain both proteins contain another conserved domain called a UBL domain. We have shown that the UBL domain is responsible for the interaction of the proteins with the 26S proteasome. In Schizosaccharomyces pombe we carried out biochemical and genetical experiments which implicated the Dph1 and Rhp23 proteins along with the fission yeast Pus1/Rpn10 proteasome subunit in the recognition and turnover of proteins by the 26S proteasome. Our current model is that these proteins act as shuttle proteins to transport ubiquitinated proteins to the 26S proteasome for destruction. Further research will be to understand how these proteins carry out their role in ubiquitin dependant proteolysis.