Inhibitors of the ubiquitin proteasome system rescue cellular levels and ion transport function of pathogenic pendrin (SLC26A4) protein variants

Pendrin (SLC26A4) is an anion exchanger highly expressed in the inner ear, kidney, and thyroid, where its dysfunction due to genetic mutations causes Pendred syndrome and non-syndromic deafness DFNB4. Pathogenic variants of pendrin typically show lower expression levels than the wild-type protein, though the mechanism behind this reduction remains unclear. In this study, we investigated whether enhanced protein degradation contributes to the decreased expression of these variants. To test this hypothesis, we assessed protein abundance and anion transport function of several pathogenic pendrin variants following treatment with inhibitors targeting the ubiquitin-proteasome system (UPS) and lysosomal/autophagosomal degradation pathways. Protein levels were quantified using western blotting and imaging, while anion transport was measured fluorometrically. Additionally, pendrin post-translational modifications were examined via immunoprecipitation and mass spectrometry. Results revealed that both the abundance and half-life of pathogenic pendrin variants were significantly reduced compared to wild-type, in both cellular models and a mouse model of Pendred syndrome/DFNB4, suggesting that accelerated degradation, rather than impaired production, accounts for the lower protein levels. Both wild-type and mutant pendrin proteins were ubiquitinated, but in a variant-specific manner. While C-terminal ubiquitination regulated wild-type pendrin stability, the p.R409H pathogenic variant showed preferential ubiquitination at lysine 77. Proteasome inhibition using either investigational (MG132) or clinical agents (bortezomib, delanzomib, or carfilzomib) restored protein expression, plasma membrane localization, and ion transport function of the pathogenic variants, whereas inhibition of the lysosomal/autophagosomal pathway had no effect. Notably, carfilzomib fully restored the ion transport capacity of the p.R409H variant to wild-type levels. These findings highlight the UPS as a key regulator of pendrin variant stability and suggest that targeting this pathway could represent a promising therapeutic strategy for Pendred syndrome/DFNB4.