SQSTM1 mutations and Paget's disease of bone
Najat, Dereen (2010) SQSTM1 mutations and Paget's disease of bone. PhD thesis, University of Nottingham.
Mutations affecting the p62 signalling adapter protein are commonly found in patients with the skeletal disorder Paget‟s disease of bone (PDB). We have extended previous in vitro functional analyses of PDB-mutant p62 proteins (Cavey et al., 2006) to study the effects of several uncharacterised PDB-associated mutations on the ubiquitin-binding properties of p62. These include mutations which affect regions of p62 outside of the ubiquitin-binding UBA domain (A381V, D335E and a mutant equivalent to a predicted product of the G1205C splice-site mutation which lacks amino acids 351-388), as well as a double mutation involving the P392L and S399P changes on the same allele. In accordance with previous findings, both of the non-UBA domain mutations (A381V, ∆351-388) showed deleterious effects on ubiquitin-binding by p62 in pull-down assays, further emphasising the important role of non-UBA domain sequences in mediating ubiquitin-recognition, as well as in PDB aetiology. The D335E mutant retained its ubiquitin-binding function in vitro. The P392L/S399P double mutant showed a more severe effect on ubiquitin-binding than either of the single P392L or S399P missense mutations alone; as this double mutation is associated with a particularly severe phenotype, our findings are supportive of the proposal that disease severity in PDB with p62 mutations may be directly related to the effects of the mutations on the ubiquitin-binding function of the p62 protein. Since the in vitro pull-down assays are semi-quantitative at best, we sought to investigate if a more quantitative biophysical approach, two dimensional Heteronuclear Single Quantum Coherence (2D-HSQC) protein NMR, might be applied to investigate the effects of PDB-associated mutations on protein (ubiquitin-binding) function. Our results showed that protein NMR was not optimal to quantitatively assess the effects of the mutations on the interaction between p62 and ubiquitin in vitro. Using confocal microscopy, co-transfection of U20S cells showed that the selected PDB-associated p62 mutants (A381V, P392L, G425R) co-localised with ubiquitin with a cellular phenotype indistinguishable from wild type, as each PDB mutant formed cytoplasmic bodies with an area ranging from the detection limit of the microscope to 40μm2 or higher; in contrast the E396X truncating mutant did not form cytoplasmic bodies nor co-localise with ubiquitin.
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