The transcription factor Nrf2 and its repressor protein Keap1 play key roles in the regulation of antioxidant stress responses and both Keap1-Nrf2 signalling and oxidative stress have been implicated in the pathogenesis of the ALS-FTLD spectrum of neurodegenerative disorders. The Keap1-binding partner and autophagy receptor SQSTM1/p62 has also recently been linked genetically to ALS-FTLD, with some missense mutations identified in patients mapping within or close to its Keap1-interacting region (KIR, residues 347–352). Here we report the effects on protein function of four different disease associated mutations of SQSTM1/p62 which affect the KIR region. Only mutations mapping precisely to the KIR (P348L and G351A) were associated with a loss of Keap1 binding in co-immunoprecipitations comparable to wild-type SQSTM1/p62. These selective effects on Keap1 recognition were entirely rational based on protein structural models. Consistent with impaired Keap1 binding, the P348L and G351A KIR mutants showed reduced ability to activate Nrf2 signalling compared to wild-type SQSTM1/p62 in antioxidant response element (ARE)-luciferase reporter assays. The results suggest that SQSTM1 mutations within the KIR of SQSTM1/p62 contribute to aetiology of some cases of ALS-FTLD through a mechanism involving aberrant expression or regulation of oxidative response genes.

SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface.

Paget's disease of bone (PDB) has a strong genetic component. Variants in SQSTM1 are found in up to 40% of patients with a family history of the disease, where a pattern of autosomal dominance with incomplete penetrance is apparent. By contrast, SQSTM1 variants are only found in up to 10% of patients with sporadic disease. It has been hypothesised that the remaining genetic susceptibility to PDB, particularly in familial cases, could be explained by rare genetic variants in loci previously identified by Genome Wide Association Studies. It is likely that polygenic factors are involved in many individuals. In this study we utilised whole exome sequencing to investigate predisposing genetic factors in an unsolved PDB kindred and identified a c.1189C > T p.L397F variant in DC-STAMP , also known as TM7SF4 , that co-segregated with disease. DCSTAMP was identified as a gene of interest in PDB following Genome Wide Association Studies and has been previously shown to play critical roles in osteoclast fusion. The variant we identified has also been reported in association with PDB in a French-Canadian cohort however the significance of this variant was inconclusive. Targeted screening of DCSTAMP in our familial cohort of PDB patients revealed an additional 8 variants; however we did not find a significant association between any of these, including p.L397F, with PDB. Osteoclastogenesis assays from the affected proband and his unaffected brother demonstrated an increase in osteoclast number and nucleation, consistent with the pagetic phenotype. In converse to other established Paget's associated genetic variations such as SQSTM1 , TNFRSF11A and OPTN , expression of the mutant DC-STAMP protein attenuated the activation of transcription factors NFκB and AP-1 when exogenously expressed. We found that the p.L397F variant did not influence the subcellular localization of the protein. Based on these findings we conclude that genetic variation in DCSTAMP is not a significant predisposing factor in our specific cohort of PDB patients and the p.L397F variant is unlikely to be a contributing factor in PDB pathogenesis.