Probing ubiquitin topology

Lead Research Organisation: University of Nottingham
Department Name: Sch of Chemistry


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).


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Scott D (2016) Method for the Purification of Endogenous Unanchored Polyubiquitin Chains. in Methods in molecular biology (Clifton, N.J.)

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Shaw B (2014) Broad Utility of an Affinity-enrichment Strategy for Unanchored Polyubiquitin Chains in Journal of Proteomics & Bioinformatics

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 04/2011 
End 06/2013
Description PhD Studentship
Amount £56,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 10/2012 
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