Characterization of Usher Syndrome Type 2-Associated Proteins in the Retina via Affinity Purification-Mass Spectrometry.

This research studied proteins involved in Usher syndrome type 2 (USH2), which is the most common form of inherited deaf-blindness. The scientists focused on understanding how three key proteins (usherin, ADGRV1, and whirlin) work together in the eye's light-sensing cells. They used laboratory techniques to identify what other proteins these USH2 proteins interact with, essentially mapping out their "partners" to better understand their normal jobs in healthy cells. The researchers discovered that these USH2 proteins act like a bridge system - they connect the outside structure of cells to the inside skeleton that gives cells their shape. They also found that these proteins are involved in several important cellular processes, including cell signaling, maintaining cell structure, and helping with the function of cilia (tiny hair-like structures on cells). Importantly, they identified connections to the Bardet-Biedl syndrome complex, which suggests these conditions may share some common biological pathways. This is early-stage laboratory research that helps scientists better understand what goes wrong in Usher syndrome. While it doesn't directly lead to treatments yet, this type of work is essential for eventually developing therapies. By understanding exactly how these proteins normally function and what other cellular components they work with, researchers can begin to design strategies to fix or work around the problems caused by mutations in USH2 genes.

Usher syndrome is the leading cause of inherited deaf-blindness, with type 2 (USH2) being the most common form. USH2A, ADGRV1, and WHRN are the three known USH2 causative genes, which are also linked to isolated retinal degeneration and hearing loss. These genes encode usherin, ADGRV1, and whirlin, respectively, collectively called USH2 proteins. These proteins form a multiprotein complex (USH2 complex) at the periciliary membrane in retinal photoreceptors and at the stereociliary ankle link in inner ear hair cells. The molecular function of the USH2 complex and its disease mechanisms are poorly understood. Currently, there is no cure for diseases caused by mutations in the three USH2 genes. In this study, we employed multiple affinity purification methods combined with mass spectrometry to systematically identify the interaction partners of USH2 proteins in the retina. The ADGRV1 intracellular bait pulled down proteins involved in actin-based cell projections, the chaperone-containing TCP-1 complex, and the Bardet-Biedl syndrome complex. The extracellular domains of ADGRV1 and usherin pulled down proteins related to peptidase regulation, collagen biosynthesis and modification, and elastic fiber formation. The EAR/EPTP repeats of ADGRV1 specifically pulled down TGFβ signaling proteins. Further immunoprecipitation experiments identified, with high confidence, Gαi and Gαq as ADGRV1-interacting proteins, and retinal degeneration and ciliary proteins as interaction partners of USH2 proteins. We also demonstrated that the usherin extracellular domains interact with each other and with ADGRV1. Overall, these findings suggest that the USH2 complex connects the extracellular matrix (ECM) to the intracellular actin network, signals through Gαi and Gαq, and participates in ECM remodeling, TGFβ signaling, cell adhesion, and ciliary function in photoreceptors.