title: 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.
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, UK; Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Goode
authors.affiliation: Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, UK.
authors.forename: Jed E
authors.initials: JE
authors.lastname: Long
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, UK.
authors.forename: Barry
authors.initials: B
authors.lastname: Shaw
authors.affiliation: Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK.
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Ralston
authors.affiliation: Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, UK.
authors.forename: Micaela Rios
authors.initials: MR
authors.lastname: Visconti
authors.affiliation: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", Italian National Research Council, Naples, Italy.
authors.forename: Fernando
authors.initials: F
authors.lastname: Gianfrancesco
authors.affiliation: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", Italian National Research Council, Naples, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Esposito
authors.affiliation: Department of Medicine, Surgery and Neurosciences, University of Siena, Siena, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Gennari
authors.affiliation: Department of Medicine, Surgery and Neurosciences, University of Siena, Siena, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Merlotti
authors.affiliation: Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Rendina
authors.affiliation: Harry Perkins Institute of Medical Research, University of Western Australia, Australia; Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Rea
authors.affiliation: Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia.
authors.forename: Melanie
authors.initials: M
authors.lastname: Sultana
authors.affiliation: Centre for Biomolecular Sciences, School of Chemistry, University of Nottingham, Nottingham, UK.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, UK. Electronic address: robert.layfield@nottingham.ac.uk.
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010001:Osteitis Deformans / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D016328:NF-kappa B; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin
keywords: NF-κB; Paget disease of bone; SQSTM1; Sequestosome 1; Ubiquitin; p62