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Protein-ligand interactions: specificity in ubiquitin recognition by the p62 UBA domain

Reference	BB/C503754/1
Principal Investigator / Supervisor	Professor Mark Searle
Co-Investigators / Co-Supervisors	Professor Robert Layfield
Institution	University of Nottingham
Department	Sch of Biomedical Sciences
Funding type	Research
Value (£)	194,270
Status	Completed
Type	Research Grant
Start date	04/04/2005
End date	03/04/2008
Duration	36 months

Abstract

We plan to investigate using a range of biophysical and structural techniques (NMR in particular) the origin of binding specificity between the p62 UBA domain and ubiquitin chains. The p62/SQSTM1 protein acts as a scaffold protein in a number of signalling pathways that lead to NFkB 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, as well as in proteins that are downstream regulators of ubiquitin-dependent proteolysis. 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. All of the mutations identified to date cause amino acid substitutions in the ubiquitin-associated (UBA) domain. We propose that like other proteins containing the UBA domain, p62 directly regulates ubiquitin-dependent degradation of specific osteoclast proteins, and that PDB mutations modify ubiquitylated substrate-binding resulting in dysfunctional protein degradation via the ubiquitin-proteasome pathway and defective bone proteolysis. Specifically, we plan to investigate the structural basis for di-ubiquitin recognition by the p62 UBA domain and rationalise the consequences of PDB mutations in the UBA domain of p62 in the context of the inhibition of normal cellular recognition process. Using multi-nuclear NMR spectroscopy of 15N/13C-labelled proteins, we plan to map the interaction surface and determine a high resolution structure of the UBA-Ub2 complex to understand the origin of binding specificity. The effects of the P392L, M404V, G411S and G425R PDB mutations on the structure and stability of the UBA domain of P62 will be examined by NMR and quantitative thermal and chemical denaturation experiments using CD, fluorescence and calorimetry. The structural data will enable us to rationalise the effects of PDB mutations on the binding of ubiquitylated substrates by p62 to provide the first molecular insights into effects on protein-ligand interactions that ultimately lead to dysfunctional protein degradation.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Ageing, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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