Probing ubiquitin topology

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.

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).