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Title: Effects of Paget's disease of bone-associated P62 mutations on protein structure and function
Author: Goode, Alice
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2013
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Mutations within the SQSTMl gene occur in 25-30% of patients with familial Paget's disease of bone (POB). Over 30 mutations have been described which commonly affect the C-terminal ubiquitin-associated (UBA) domain of the p62 protein, which SQSTMl encodes. In order to gain an understanding into how these mutations exert their effects I investigated the impact of several on p62 protein function and structure. Notably, over-expression of truncating mutants of p62, which genotypephenotype analyses suggest to be associated with more severe POB than common missense mutants, was previously suggested to be associated with greater activation of NF-KB activity than missense mutants in reporter assays. These observations led to the simple hypothesis that changes in p62'S ability to regulate (RANK-mediated) NF-KB signalling are directly related to disease severity in POB patients. In further support of this notion, I found that, A4270 a rare missense mutant of p62 associated with very severe POB produced a level of activation of NF-KB activity above that seen for any other mutant (including truncating mutants). All the POB-associated p62 mutants tested did not impact on p62'S interactions with aPKC and CYLO in co-immunoprecipitation studies. Conversely, unlike all the other POB-associated mutants tested, the non-UBA S349T mutant was not associated with activation of basal NF-KB signalling, but directly affected the interaction of p62 with Keapl; selectively inhibiting Nrf2 signalling. These observations highlight the complexities of disease aetiology in POB and suggest that in addition to dysregulated NF-KB signalling, aberrant production of oxidative response genes may be related to disease mechanism in some POB patients. As an example of a relatively common POB-associated p62 mutation, the 1424S missense mutant was further structurally characterised in the UBA model. Biophysical analysis of the I424S-UBA revealed that the mutation caused major structural rearrangement of the entire UBA domain; a 11 r significantly weakened UBA dimer; and a destabilised UBA-monomer altogether resulting in weakening of the binding affinity for ubiquitin. The interplay between the PDB-associated mutations and phosphorylation of the p62-UBA was also investigated as recently it was reported that phosphorylation of the UBA domain at S403 enhances ubiquitin-binding function. In mouse macrophage samples homozygous for the PDB-associated P394L-p62 mutation (equivalent to the most common P392L mutation in humans) increased phosphorylation of p62 at S403 was detected compared to heterozygotes and wild-type samples. Using UBA-phosphomimetic mutants I confirmed an enhanced ability to bind ubiquitin through weakening the UBA dimer. However the effects of the phosphomimetic mutation were found to further exaggerate the difference in binding affinity between wild-type and PDB-mutant (1424S) UBA domains, presumably due to the PDB mutation disrupting the UBA's structural integrity and weakening the binding interface. Although phosphorylation causes only small changes in binding affinity, this effect is likely to be amplified by avidity in vivo along with the larger effects of the PDB mutations. III
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available