The SANS-USH2a-whirlin ternary complex of Usher syndrome type 1 and type 2 proteins is disrupted by pathogenic mutations

Abstract

Purpose: Usher syndrome (USH) is the most common form of combined deaf-blindness. Among three clinical types (USH1-3), USH1 is the most severe and USH2 the most common type. Previous in vitro studies have indicated that USH genes encode for proteins of diverse families which interact within an interactome. Furthermore, we have described a USH1-USH2 protein network in the periciliary compartment of rodent photoreceptor cells. Here we aim to further characterize USH1-USH2 protein networks in photoreceptor cells and to get insights into the molecular pathomechanisms underlying USH by testing the affects of diverse USH causing mutation on these networks.

Methods: We analyzed direct binding in vitro (GST-pull downs), and in cell culture (membrane targeting assays). Ternary complexes assembly was accessed by co-precipitation experiments. USH mutations were introduced by site-specific mutagenesis. PTC124 and gentamicine were applied as translational read-through drugs (TRIDs) targeting nonsense mutations. The subcellular localization of complex partners was analyzed by immunofluorescence and electron microscopy in mouse and human retinas.

Results: We demonstrate for the first time the mutual direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein USH2A. Furthermore, we identified a ternary USH1-USH2 protein complex of SANS, USH2A and the PDZ-domain protein whirlin (USH2C) mediated by mutual direct interactions and dimerization. Co-localization pattern emphasize that the SANS-USH2A-whirlin complex assembles in the periciliary region of human and rodent photoreceptor cells and in calyceal processes of human photoreceptor cells. USH causing mutations in USH1G severely affected complex formation. TRIDs treatment targeting the USH1G_C728A nonsense mutation restored SANS scaffold and complex function.

Conclusions: Present data revealed that the USH1 and USH2 proteins are interwoven in protein complexes. In addition we show that pathogenic mutations in complex partners disrupt these complexes which can be targeted by gene-based treatments. Our results further suggest that the ternary SANS-USH2A-whirlin protein complex participate in functional modules of photoreceptor cilia and support the structure of calyceal processes in human photoreceptor cells.