Characterization of a Non-UBA Domain Missense Mutation of Sequestosome 1 (SQSTM1) in Paget's Disease of Bone

Mutations affecting the ubiquitin-associated (UBA) domain of sequestosome 1 (SQSTM1/p62) are commonly found in Paget's disease of bone (PDB) and impair SQSTM1's ability to bind ubiquitin, resulting in dysregulated NF-kappa B signaling. In contrast, non-UBA domain mutations are rarer, and little is known about how they manifest their effects. We present the first characterization at the molecular, cellular, and functional level of a non-UBA domain missense mutation (A381V) of SQSTM1. Direct sequencing of exon 7 of the SQSTM1 gene in all Italian PDB patient detected a heterozygous C to T transversion at position 1182, resulting in all alanine to valine substitution at codon 381. Pull-down assays showed the non-UBA region of SQSTM1. that contains A381 is important in mediating ubiquitin-binding affinity and that the A381V mutation exerts weak negative effects on ubiquitin binding. Structural and binding analyses of longer UBA constructs containing A381, using NMR spectroscopy and circular dichroism, showed this region of the protein to be largely unstructured and confirmed its contribution to increased ubiquitin-binding, affinity. Co-transfections of U20S cells showed that the A381V mutant SQSTM1 co-localized with ubiquitin with a cellular phenotype indistinguishable from wildtype. Finally, effects of the wildtype and mutant SQSTM1 on NF-kappa B signaling were assessed in HEK293 cells co-transfected with an NF-kappa B luciferase reporter construct. A381V mutant SQSTM1 produced a level of activation of NF-kappa B signaling greater than wildtype and similar to that of UBA domain mutants, indicating, that non-UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF-kappa B signaling.