title: Loss of ubiquitin-binding associated with Paget's disease of bone p62 (SQSTM1) mutations.
abstract: We have studied the effects of various PDB-causing mutations of SQSTM1 on the in vitro ubiquitin-binding properties of the p62 protein. All mutations caused loss of monoubiquitin-binding and impaired K48-linked polyubiquitin-binding, which was only evident at physiological temperature. This suggests that SQSTM1 mutations predispose to PDB through a common mechanism that depends on loss of ubiquitin-binding by p62.
  
  
  BACKGROUND
  Mutations in the SQSTM1 gene, which affect the ubiquitin-associated (UBA) domain of the p62 protein, are a common cause of Paget's disease of bone (PDB). We previously showed that the isolated UBA domain of p62 binds K48-linked polyubiquitin chains in vitro and that PDB-causing mutations in the UBA domain can be resolved in to those which retain (P392L and G411S) or lose (M404V and G425R) the ability to bind K48-linked polyubiquitin. To further clarify the mechanisms by which these mutations predispose to PDB, we have extended these analyses to study the ubiquitin-binding properties of the PDB-causing mutations in the context of the full-length p62 protein.
  
  
  METHODS
  We studied the effects of various PDB-causing mutations on the interaction between glutathione S-transferase (GST)-tagged p62 proteins and monoubiquitin, as well as K48-linked polyubiquitin chains, using in vitro ubiquitin-binding assays.
  
  
  RESULTS
  All of the PDB-causing mutations assessed (P392L, E396X, M404V, G411S, and G425R) caused loss of monoubiquitin binding and impaired K48-linked polyubiquitin-binding when introduced into the full-length p62 protein. However, these effects were only observed when the binding experiments were conducted at physiological temperature (37 degrees C); they were not seen at room temperature or at 4 degrees C.
  
  
  CONCLUSIONS
  Our in vitro findings suggest that PDB-causing mutations of SQSTM1 could predispose to disease through a common mechanism that is dependent on impaired binding of p62 to a ubiquitylated target and show that 5q35-linked PDB is the first example of a human disorder caused by loss of function mutations in a UBA domain.
authors.affiliation: School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: James R
authors.initials: JR
authors.lastname: Cavey
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Ralston
authors.forename: Lynne J
authors.initials: LJ
authors.lastname: Hocking
authors.forename: Paul W
authors.initials: PW
authors.lastname: Sheppard
authors.forename: Barbara
authors.initials: B
authors.lastname: Ciani
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D006801:Humans; D010001:Osteitis Deformans / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D000071456:Sequestosome-1 Protein; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D011506:Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D014452:Ubiquitins