Najat, Dereen
(2010)
SQSTM1 mutations and Paget's disease of bone.
PhD thesis, University of Nottingham.
Abstract
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.
In addition to interacting with ubiquitin, p62 also interacts with the LC3 (an autophagic marker) through its LC3 interacting region (LIR) to mediate the formation of autophagosomes. By co-transfecting p62 constructs with LC3 We found that some of the p62-positive cytoplasmic bodies were autophagosomes, and that the D335E mutation of p62 (which lies within the LIR) did not appear to affect the formation of autophagosomes. The effects of the wild type and PDB-mutant p62 proteins on NF-κB signalling were assessed in HEK293 cells co-transfected with an NF-κB luciferase reporter construct. A381V mutant p62 produced a level of activation of NF-κB signalling greater than wildtype and similar to that of UBA domain mutants, indicating that non-UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF-κB signalling. To further examine the function of p62 in the regulation of NF-κB signalling, we went on to determine possible effects of PDB-associated mutations on p62-CYLD (a DUB enzyme) interactions. Unexpectedly we found that CYLD expression appears to abrogate the formation of the p62 cytoplasmic bodies previously shown to be ubiquitin-positive. Finally, we went on to study the interaction of p62 (and its PDB mutants) with another important regulator of NF-κB signalling, IKKγ/NEMO. We concluded that wild type and PDB-mutant p62 proteins are capable of recruiting NEMO to cytoplasmic bodies which may represent autophagosomes, but do not appear to accelerate its degradation.
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