Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function

Abstract

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.

Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

“…GST pull-down analysis indicated that G425R and A427D mutants failed to bind ARIP4, while P392L and G411S mutants had significantly increased binding activity ( Fig.4C , upper panel). These data demonstrate that the binding characteristics between ARIP4 and the UBA domain of p62 are very similar to those reported for ubiquitin 35 37 .…”

“…We further examined several mutations of the p62 UBA domain that were previously identified in human patients suffering from Paget’s disease of bone (PDB) 35 37 . GST pull-down analysis indicated that G425R and A427D mutants failed to bind ARIP4, while P392L and G411S mutants had significantly increased binding activity ( Fig.4C , upper panel).…”

p62/SQSTM1 – steering the cell through health and disease

“…Mutations in SQSTM1 are strongly associated with PDB (Laurin et al, 2002), a chronic and progressive skeletal disorder characterized by focal areas of increased and disorganized bone turnover (Numan et al, 2015). These mutations mainly localize to the UBA domain of SQSTM1 and are causative in ∼50% of the cases of familial PDB (Goode et al, 2014;Rea et al, 2014). Although additional research is required to clarify the exact molecular mechanism, SQSTM1 can regulate osteoclast activity through the recruitment of the deubiquitinating enzyme CYLD to the RANK-TRAF6 complex (Jin et al, 2008).…”

Paget's disease of bone—genetic and environmental factors

“…These mutations differentially stimulate nuclear factorκB (NFκB) in vitro; furthermore, enhanced acti vation of NFκB is associated with an increased number of bone lesions in affected patients. 24 Seldom have two mutations been found in the same allele and rarely has a patient been found to be homozygous for the Pro392Leu mutation. 18 Disease manifestations have been reported to be more severe in patients who have an SQSTM1 muta tion (especially a truncation mutation) than in those who do not.…”