The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson's disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (K_D^app = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its co-localization. Ubiquitin enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking-associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism and revealing trafficking scaffold protein TFG as a Nedd4 substrate.

苯并二咪唑 NAB2 以 Nedd4 依赖性方式挽救与早发帕金森病相关的 α-突触核蛋白相关运输缺陷。尽管将 E3 泛素连接酶 Nedd4 鉴定为 NAB2 的推定靶标，但其分子作用机制尚未阐明。因此，通过生化、生物物理学和蛋白质组学分析研究了 NAB2 对 Nedd4 活性和特异性的影响。发现 NAB2 与 Nedd4 结合（K _D ^app  = 42 nM），但这种结合是侧链介导的，不会改变其构象或体外泛素化动力学. Nedd4 与贩运细胞器共定位，NAB2 暴露并未改变其共定位。泛素富集偶联蛋白质组学揭示 NAB2 刺激贩运相关蛋白的泛素化，很可能是通过调节 Nedd4 的底物特异性，提供参与 NAB2 机制的推定蛋白质网络，并揭示贩运支架蛋白 TFG 作为 Nedd4 底物。