Structural role of a unique p62 UBA dimer in the regulation of signal transduction and autophagy

p62 is a multi-functional scaffold protein (also known as SQSTM1) which regulates a diverse range of cellular processes involving in vivo interaction partners relevant to bone cell (osteoclast), neuronal function and autophagetic clearance of protein aggregates from cells. Mutations in p62 are a common cause of Paget's disease of bone (PDB), however, the precise disease mechanism in this skeletal disorder is unclear. P62 is a ubiquitin-binding receptor and uses a UBA domain to achieve binding and recognition of ubiquitin. PDB mutations in p62 occur principally within the UBA recognition motif of p62, with the implication that these pathological mutations may compromise interactions of the p62 protein with osteoclast protein targets already tagged with ubiquitin. More recently, mutations in other parts of the p62 sequence have been identified in PDB patients which still result in loss or impairment of ubiquitin-binding function. We have recently demonstrated that the UBA domain of p62 is unique amongst this family of ubiquitin binding domains (UBDs) in forming a highly stable 'biologically inactive' dimer which appears to regulate the binding to poly-ubiquitin chains. The stability of the dimer also appears to be directly correlated with levels of NF-kB signalling activity in osteoclasts, linked to bone metabolism. Given the participation of p62 in multiple signalling complexes, the stability of the UBA dimer and the subsequent effects of PDB mutations will be investigated at the molecular and structural level to shed light on the physiological relevance of the formation of the p62 UBA dimer on p62's mode of action and the possible links to dysfunctional regulation of protein-protein interactions and human disease that result from mutations within the p62 receptor.

The p62/SQSTM1 protein acts as a scaffold protein in a number of signalling pathways that lead to NFB activation. The C-terminal region of the protein (residues 387-436) has been identified as a ubiquitin-associated (UBA) domain that occurs in enzymes of the ubiquitin conjugation/deconjugation pathway. Mutations in the p62 protein cause Paget's disease of bone (PDB), a common disorder of the elderly characterised by excessive bone resorption and formation. The majority of PDB mutations to date are found in the UBA domain with the implication that these pathological mutations must compromise interactions of the p62 protein with a ubiquitinated target. We have recently demonstrated that the UBA domain of p62 is unique amongst this family of ubiquitin binding domains (UBDs) in forming a highly stable 'biologically inactive' dimer which appears to regulate the binding to poly-ubiquitin chains. The stability of the dimer also appears to be directly correlated with levels of NF-kB signalling activity in osteoclasts. Given the participation of p62 in multiple signalling complexes, the stability of the UBA dimer and the subsequent effects of PDB mutations will be investigated at the molecular and structural level to shed light on the physiological relevance of the formation of the p62 UBA dimer on p62's mode of action and the possible links to dysfunctional regulation of protein-protein interactions and human disease. We plan to use naturally occurring PDB mutations in the UBA domain of p62 to probe the possible physiological effects of dimer stability on NF-kB signalling potential and ability to activate autophagy in a cellular context. We will investigate how the modest effects on protein binding function imparted by Paget's disease mutations are potentially amplified by the oligomeric properties of p62, resulting in effects on both UBA dimer formation at low protein concentrations, ability to bind to polyUb chains and effects on signal activation.

Relevance of the research The proposed research programme falls under the heading of expanding our fundamental science base in an area that is pivotal to our understanding of cellular physiology at the molecular level with regard to dysfunctional regulation of ubiquitin-mediated signalling processes linked to human disease. By understanding the fundamentals we are in a strong position to explore the design of molecules to regulate biological function. Thus, the beneficiaries will be (i) the academic research community of biochemists and cell biologists searching for a molecular basis for normal and malfunctioning cellular behaviour; (ii) medicinal chemistry and molecular modellers trying to predict the behaviour and function of important biomolecules, and (iii) the pharmaceutical companies engaged in programmes of drug discovery. In the short term, there will be no obvious and measurable economic benefit or directly associated wealth creation. No new drug candidate will be forthcoming nor will there be a spinout company formed around the outcomes of the project, or any new technology. However, such a fundamental understanding of molecular interactions in biology underpins much of the wealth creation from the biotech and pharmaceutical industry which will lead to new medicines to tackle 21st century 'grand challenges' in global healthcare. The highly skilled PDRAs and PhD students that we produce will most certainly lead ultimately to wealth creation through the applications of this transferable skills base. Communications and engagement The results of the research will be published in academic journals and presented by the PI, Co-I and PDRA at academic conferences, where they can also be accessed by non-academic beneficiaries. Further, materials will be used for purposes of public engagement both at BBSRC-sponsored events or at the University of Nottingham through Open Days, Science Fair events and through a wide range of other outreach activities within the School and across campus. Furthermore, structural data generated by the project will be made available as supporting information alongside publications, as well as online on the research group's website. If any non-academic beneficiaries are interested in further data or details on published research, the PI will provide these upon request. Collaboration The PI and Co-I have enjoyed research collaborations in related areas in the last 10 years with major players such as GSK, Roche-Discovery and AZ, reflecting the general relevance of fundamental studies of ligand-protein interactions to the pharmaceutical industry. Core facilities (particularly NMR and Mass Spec) are being established at Nottingham to provide a 'hub' for academic collaboration across the University lead by the PI. This is already leading to knowledge transfer opportunities and new projects of direct relevance to drug discovery (new Nottingham Priority Group). Exploitation and application The maximum impact of the research will be achieved through open access to the results, which will be achieved principally through rapid and open publication which will be accessible to the largest possible number of beneficiaries. In the event that there is something of potential commercial value, the School of Chemistry is well set up with a Business Partnership Unit and Manager for exploiting the impact of the research. Capability All members of the current research group are widely encourage to expand their transferable skills base and engage in presentation of their research and in School-led impact and outreach activities. At the School level, this is led by a full-time Outreach Officer with an extensive programme of activities with Schools across the East Midlands.