Loss of Ubiquitin-Binding Associated With Paget's Disease of Bone p62 (SQSTM1) Mutations

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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. 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. 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. 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. 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.