Probing ubiquitin topology
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Chemistry
Abstract
Ubiquitin is a small protein found in organisms as diverse as yeast and man. It is responsible for the regulation of a host of processes within the cell, including targeting proteins for destruction, gene transcription, cell signalling, and controlling traffic through the cell membrane. Ubiquitin is attached to one or more sites on a target protein by the action of a set of enzymes in a process known as ubiquitination. Ubiquitin is coupled to the amino acid lysine within the target protein structure and, because ubiquitin itself contains lysine residues, it is possible for polymeric ubiquitin chains to build up during ubiquitination / through linkage at any of seven lysines. Thus, poly-ubiquitin can form defined shapes, or topologies, depending upon its linkage pattern. It is believed that different topologies and chain lengths maybe responsible for different functions within the cell, but probing the complete topology of a poly-ubiquitin chain has not previously been possible. Here we propose methods to map poly-ubiquitin linkage, and determine the binding of different topologies with ubiquitin-interacting proteins to allow us to tackle the key question 'does poly-ubiquitin shape and size matter?' in the context of some important biological processes.
Technical Summary
Protein ubiquitination is a key regulatory mechanism in eukaryotic cells. Post-translation modification of proteins with ubiquitin (Ub) results in covalent attachment of the C-terminus of Ub to lysine residues on the target protein. Given that Ub contains lysine residues itself, poly-Ub chains can build-up through multiple isopeptide bonds. It has been demonstrated that all 7 Lys on Ub are able to form linkages, resulting in varied topologies (and lengths) of poly-Ub. We have a limited understanding of the roles of different poly-Ub topologies within the cell. K48-linked poly-Ub is associated with proteasomal protein degradation, whilst K63-linked poly-Ub modification has been implicated in the cell cycle, transcription and DNA repair, but we know little about the roles of other linkages, or of mixed linkages or forked poly-Ub chains. Major limitations are placed on our understanding of this fundamental regulatory pathway by the lack of sensitive and high-throughput methodologies to probe (a) non-covalent interactions between protein Ub-binding domains and Ub/poly-Ub, which often precede covalent Ub attachment, or regulate the fate of ubiquitinated proteins, and (b) the topology of intact poly-Ub chains (the current state-of-the-art is able to identify the types of linkage present, but not their position or relationship in a poly-Ub chain). We will develop methods to address both of these key areas and apply them to study the biological role of Ub and ubiquitination in a number of key systems. A sensitive ESI-MS based assay for detecting and quantifying non-covalent UBD-polyUb interactions will used to study the effects of chain length and linkage on poly-Ub binding in the p62, hHR23A, Vps9, Rabex-5 and Hrs UBDs. We will employ ESI-FTICR-MS to map the topology of intact poly-Ub chains, including those attached to CNBr-cleaved target proteins / such as MuRF1 (autoubiquitinated by UbcH5 and other E2s) and luciferase ubiquitinated with CHIP and UbcH5 (and other E2s).
Publications
Sokratous K
(2012)
Cyclisation of Lys48-linked diubiquitin in vitro and in vivo.
in FEBS letters
Sokratous K
(2012)
The effects of cation adduction upon the conformation of three-helix bundle protein domains
in International Journal for Ion Mobility Spectrometry
Strachan J
(2012)
Insights into the molecular composition of endogenous unanchored polyubiquitin chains.
in Journal of proteome research
Shaw B
(2014)
Broad Utility of an Affinity-enrichment Strategy for Unanchored Polyubiquitin Chains
in Journal of Proteomics & Bioinformatics
Sokratous K
(2012)
Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry.
in Journal of the American Chemical Society
Scott D
(2016)
Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.
in Methods in molecular biology (Clifton, N.J.)
Manzi L
(2016)
Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions.
in Nature communications
Scott D
(2015)
Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry.
in Protein science : a publication of the Protein Society
Scott D
(2015)
Ion mobility-mass spectrometry reveals conformational flexibility in the deubiquitinating enzyme USP5.
in Proteomics
Description | a)Development of a sensitive and quantitative mass spectrometry (MS)-based method for probing the selectivity of ubiquitin (Ub) and polyubiquitin (Ubn) interactions with ubiquitin-binding domains (UBDs). The method was validated against UBDs of known affinity and found to be in excellent agreement. Application to previously unstudied Ub interactions provided new insights into this important signalling pathway. b)Identification of linkage topology profiles for unanchored Ubn in rat skeletal muscle using analysis of intact Ubn chains by MS. K-48 and K-11 isopeptide-linked topologies were found to be the most abundant forms of Ub2 in vivo. Cyclic K-48 linked Ub2 was identified as a biologically relevant species for the first time. c)Development of an ion mobility-MS method to distinguish between K-11, K-27, K-48 and K-63 Ub2 using collisional activation of low charge state ions. Different linkages of Ub2 were found to exhibit different unfolding profiles in the gas-phase, allowing their discrimination. |
Exploitation Route | Ubiquitination, a post-translational modification (PTM) resulting in the covalent attachment of ubiquitin (Ub) to a target protein, is a fundamental regulatory mechanism in many eukaryotic cellular processes. Protein degradation, endocytosis, vesicular trafficking, cell-cycle control, DNA repair, and various signalling pathways are all regulated by this important PTM. A better understanding of the topology of poly-Ub and its interactions with proteins possessing Ub-binding domains have potential for significant application for drug development by the pharmaceutical industry. As one example we are currently exploring the inhibition of interactions between isopeptidase-T and unanchored poly-Ub chains, which leads to stabilisation of the cell cycle-controlling protein p53, and is a potentially powerful anti-cancer strategy. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | Knowledge of the existence of cyclic di-ubiquitin, which was identified as a result of research conducted under this award (see publication) has been used by the company Boston Biochem in improving the quality of their commercial di-ubiquitin product. |
First Year Of Impact | 2012 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | Molecular and Functional Characterisation of Unanchored Polyubiquitin Chains |
Amount | £396,753 (GBP) |
Funding ID | BB/I006052/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2011 |
End | 06/2013 |
Description | PhD Studentship |
Amount | £56,000 (GBP) |
Organisation | University of Nottingham |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2012 |
End | 04/2016 |
Title | MS Ub binding assay |
Description | As a direct result of work funded by this award we have developed a sensitive and rapid method to detect binding between ubiquitin and ubiquitin binding proteins. |
Type Of Material | Technology assay or reagent |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | The methodology has been adopted by a number of research groups, including Heck's group in the Netherlands. |
Description | Met4 UIM |
Organisation | University of California, Irvine |
Country | United States |
Sector | Academic/University |
PI Contribution | Using mass spectrometry methodology developed under the grant to determine the binding of phosphorylated and unphosphorylated Met4 protein to ubiquitn. |
Collaborator Contribution | Supply of Met4 peptides to assess binding. |
Impact | This work has informed the role of phosphorylation in Met4 regulation. The protein is important in cancer. The work is as yet unpublished. |
Start Year | 2013 |