title: The S349T mutation of SQSTM1 links Keap1/Nrf2 signalling to Paget's disease of bone.
abstract: Mutations affecting the Sequestosome 1 (SQSTM1) gene commonly occur in patients with the skeletal disorder Paget's disease of bone (PDB), a condition characterised by defective osteoclast differentiation and function. Whilst most mutations cluster within the ubiquitin-associated (UBA) domain of the SQSTM1 protein, and are associated with dysregulated NFκB signalling, several non-UBA domain mutations have also been identified. Keap1 is a SQSTM1-interacting protein that regulates the levels and activity of the Nrf2 transcription factor. This in turn controls the expression of numerous cytoprotective genes that contribute to the cell's capacity to defend itself against chemical and oxidative stress, through binding to the antioxidant response element (ARE). The PDB-associated S349T mutation maps to the Keap1-interacting region (KIR) of SQSTM1, however the effects of PDB mutant SQSTM1 on Keap1 function have not been investigated. Here we show that unlike other SQSTM1 mutations, the S349T mutation results in neither impaired ubiquitin-binding function in pull-down assays, nor dysregulated NFκB signalling in luciferase reporter assays. Keap1 is expressed in differentiating osteoclast-like cells and the S349T mutation selectively impairs the SQSTM1-Keap1 interaction in co-immunoprecipitations, which molecular modelling indicates results from effects on critical hydrogen bonds required to stabilise the KIR-Keap1 complex. Further, S349T mutant SQSTM1, but not other PDB-associated mutants, showed reduced ability to activate Nrf2 signalling as assessed by ARE-luciferase reporter assays. Thus, SQSTM1-mediated dysregulation of the Keap1-Nrf2 axis, which could potentially lead to aberrant production of oxidative response genes, may contribute to disease aetiology in a subset of PDB patients.
authors.affiliation: School of Biomedical Sciences, University of Nottingham, UK.
authors.forename: Tao
authors.initials: T
authors.lastname: Wright
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Rea
authors.forename: Alice
authors.initials: A
authors.lastname: Goode
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennett
authors.forename: Thomas
authors.initials: T
authors.lastname: Ratajczak
authors.forename: Jed E
authors.initials: JE
authors.lastname: Long
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Goldring
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
authors.forename: Ian M
authors.initials: IM
authors.lastname: Copple
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010001:Osteitis Deformans / Q000235:genetics*; D011485:Protein Binding; D016415:Sequence Alignment; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; C495635:NFE2L2 protein, human; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin