Chemical Biology of the E3 Ubiqutin Ligase Nedd4

泛素化是一种共价的翻译后修饰，影响着大量的正常 和疾病相关的细胞功能。神经前体细胞表达的发育下调4 酶(Nedd4)是调节蛋白平衡的Hect型E3泛素连接酶家族的创始成员 在各种情况下，包括癌症和神经退行性疾病。除了它在癌症中的重要性外， Nedd4最近也成为α-突触核蛋白蛋白毒性的重要调节因子，并可能成为靶标。 用于治疗以聚集性聚集性为特征的神经退行性疾病 A-突触核蛋白在脑内神经细胞和非神经细胞中的存在形式。NAB2是一个N- 从表型筛选中发现逆转α-突触核蛋白相关的芳基苯并咪唑小分子 A-突触核蛋白相关帕金森病细胞模型的毒性，包括内质网到高尔基体转运中断。最近我们 阐明了NAB2的作用方式，发现它与E3泛素连接酶Nedd4(Kdapp=42)紧密结合 Nm)，并扩展了Nedd4的底物专一性。我们确定NAB2与Nedd4结合会触发 新底物的泛素化，如运输相关蛋白，如TFG，TRAPP1亚单位， 和RER1。我们假设Nedd4信号会影响一条调控贩运过程的新途径 作为对以下问题的回应 A-突触核蛋白相关毒性。Nedd4对α-突触核蛋白的反应蛋白状态 NAB2的毒性和结合也是未知的。此外，NAB2结合位点在Nedd4内的位置也 是未知的。在这个提议中，我们希望测试NAB2单独作用于Ned4或作为分子作用的假设 以促进Nedd4和潜在底物之间的沟通。我们希望了解NAB2是如何 与NEDD4相互作用，以及这种相互作用如何使Ned4获得泛素修饰的功能活性 含PXY的底物，如TFG。如果成功，这些研究将更好地阐明Ned4在 蛋白毒性信号。此外，Nedd4活性可以被小分子调节的程度是 重要的是，鉴于Nedd4泛素化在癌症病理生物学和神经退行性变中发挥的重要作用 精神错乱。这些研究有望增加我们对E3连接酶在其中所起作用的基础知识 蛋白毒性信号，并可能激励未来的发现和发展的治疗可能解决 联体核病症的一些原因。

Ubiquitylation is a covalent posttranslational modification that influences a tremendous number of normal and disease-related cellular functions. The neuronal precursor cell-expressed developmentally downregulated 4 enzyme (Nedd4) is the founding member of a family of HECT-type E3 ubiquitin ligases that regulate proteostasis in various conditions including cancers and neurodegenerative disorders. In addition to its importance in cancer, Nedd4 has also recently emerged as a significant regulator of a-synuclein proteotoxicity, and may be a target for treating synucleinopathies, neurodegenerative disorders characterized by the accumulation of aggregated forms of the protein a-synuclein in both neuronal and non-neuronal cells in the brain. NAB2 is an N- arylbenzimidazole small molecule discovered from phenotypic screening that reverses a-synuclein associated toxicity in cellular models of a-synuclein-associated PD, including ER-to-Golgi trafficking disruption. Recently we elucidated the mode of action of NAB2, finding that it bound tightly to the E3 ubiquitin ligase Nedd4 (Kdapp = 42 nM) and expands the substrate specificity of Nedd4. We determined that NAB2 binding to Nedd4 triggers the ubiquitylation of new substrates, such as the trafficking-associated proteins such as TFG, TRAPP1 subunits, and RER1. We hypothesize that Nedd4 signaling influences a new pathway that regulates trafficking processes in response to a-synuclein-associated toxicity. The proteoform state of Nedd4 that responds to a-synuclein toxicity and binds NAB2 is also not known. In addition, the location of the NAB2 binding site within Nedd4 also is not known. In this proposal we wish to test the hypothesis that NAB2 acts on Nedd4 alone or as ‘molecular glue’ to facilitate communication between Nedd4 and potential substrates. We wish to understand how NAB2 interacts with NEDD4, and how this interaction affords Nedd4 a gain of function activity to ubiquitin modifiy non PxY-containing substrates like TFG. If successful, these studies will better illuminate the role Nedd4 plays in proteotoxicity signaling. In addition, the degree to which Nedd4 activity can be modulated by small molecules is important, given the important role Nedd4 ubiquitination plays in cancer pathobiology and neurodegenerative disorders. These studies will hopefully add to our fundamental knowledge of the role that E3 ligases play in proteotoxicity signaling, and may inspire the future discovery and development of therapeutics that may address some of the causes of synucleinopathies.