Department of Chemistry, Duke University, Durham, NC 27708, USA.
Proteomics and Metabolomics Core Facility, Duke University Medical Center, Durham, NC 27710, USA.
Cell Chem Biol. 2021 Jan 21;28(1):14-25.e9. doi: 10.1016/j.chembiol.2020.10.008. Epub 2020 Nov 10.
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 = 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 通过依赖 Nedd4 的方式挽救与早发性帕金森病相关的α-突触核蛋白相关的运输缺陷。尽管已鉴定出 E3 泛素连接酶 Nedd4 是 NAB2 的一个潜在靶点，但它的作用机制仍未阐明。因此，通过生化、生物物理和蛋白质组学分析研究了 NAB2 对 Nedd4 活性和特异性的影响。结果发现 NAB2 与 Nedd4 结合（Kd=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 与运输细胞器共定位，而 NAB2 的暴露并未改变其共定位。泛素富集结合蛋白质组学表明，NAB2 刺激与运输相关的蛋白质的泛素化，很可能是通过调节 Nedd4 的底物特异性，提供了一个可能参与 NAB2 机制的蛋白质网络，并揭示了运输支架蛋白 TFG 是 Nedd4 的底物。
