Over 30 different mutations in p62 UBA have been identified in patients with Paget's disease of bone (PDB). The mechanisms which underlie PDB are poorly understood, although impaired ubiquitin binding has been identified as a mechanism in the onset of disease. However, the decrease in affinity is subtle for many PDB mutants. The p62 UBA is unique amongst UBAs since it exists as a highly stable dimer but binds to ubiquitin as a monomer. The dimerization interface partially occludes the ubiquitin binding interface resulting in competing equilibria. The factors which regulate the affinity of p62 UBA were examined in this thesis. In isolated p62 UBA the monomer-dimer equilibrium and the effects of phosphorylation were investigated. By mutating residues at the dimerization interface, weaker dimers which had a higher affinity for ubiquitin were produced. The weak dimers had an increased population of monomer at equilibrium. A phosphorylation site at Ser403 in p62 UBA was recently identified. Phosphomimetic mutants which showed subtle increases in affinity for ubiquitin were generated. The increase was attributed to the close proximity of Ser403 to the ubiquitin binding interface. Factors outside the UBA also have a role in regulating UBA affinity. Binding by p62 UBA was therefore probed in longer p62 constructs. A fragment of p62 encoding residues 300-440 was used to investigate p62 binding to multiple proteins. A ternary complex was formed, but an allosteric relationship was not observed by ubiquitin and MAP-Le3 in binding to the p62 fragment. A model of full length oligomeric p62 was generated to probe avid binding to polyubiquitin chains. The model showed higher affinity for linear diubiquitin than monoubiquitin suggesting avidity effects are influential in oligomeric p62 binding. Since the effects of PDB mutations and phosphorylation are subtle, they are likely to be amplified by avidity in vivo.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594862 |
| Date | January 2013 |
| Creators | Adlington, Jennifer |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/14519/ |
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