NEDD4 IN T HELPER CELL DEVELOPMENT AND AUTOIMMUNITY

项目摘要 T辅助(Th)17细胞被认为在自身免疫的发展和发病机制中起关键作用 疾病，包括多发性硬化症(MS)及其小鼠模型，实验性自身免疫性脑脊髓炎 (EAE)、类风湿性关节炎、炎症性肠病和系统性红斑狼疮。最新进展 受rorγt控制，靶向rorγt的小分子可减弱th17的反应。 EAE和皮肤炎的严重程度。然而，Th17细胞中RoRγt活性的调节 TCR信号的分化在很大程度上是未知的。Nedd4(也称为Nedd4-1，神经元前体细胞- 表达的发育下调4)是Hect类型的E3泛素连接酶。通过序列分析， 我们发现了一个PPLYKEL基序，一个扩展的Nedd4 WW结构域结合基序，位于 RoRγt的配体结合区，提示RoRγt可能是Nedd4的结合伙伴。的确，Nedd4是 酪氨酸-在TcR/CD28刺激下被磷酸化和激活，并与经历K63-的RoRγt结合- 连接的多泛素化。这种泛素化在缺乏Nedd4的T细胞中被废除。进一步的分析显示， 尽管Nedd4缺乏不会损害Th1、Th2和可诱导的调节性T细胞分化，但Th17细胞 在没有Nedd4的情况下，分化会受到很大影响。为了支持这一观察结果，缺乏 Nedd4或T细胞中缺乏Nedd4，可以改善Th17反应受损的EAE。基于这些 初步数据，我们假设在T细胞抗原刺激下，NedD4被Src激酶(S)激活，并且 靶向K63连接的多泛素化的RoRγt，从而调节Th17反应和对EAE的易感性 老鼠，可能还有人类的多发性硬化症。在这个提案中，我们将研究1)Nedd4是如何增强Th17细胞的 2)Nedd4在体内是否促进Th17反应，调节EAE的易感性； (3)T细胞中Nedd4如何在TCR/CD28刺激下被激活。Nedd4基因的鉴定 调节Th17反应的关键分子和EAE的发病机制，可能还有MS，将具有潜在的作用 使用该分子作为治疗靶点治疗涉及Th17的自身免疫性疾病的意义。

Project Summary T helper (Th) 17 cells are thought to play a key role in the development and pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and its murine models, experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. The development of Th17 cells is controlled by RORγt, and small molecules that target RORγt attenuate Th17 response and the severity of EAE and cutaneous inflammation. However, the regulation of RORγt activity during Th17 cell differentiation by TCR signaling is largely unknown. Nedd4 (also known as Nedd4-1, neuronal precursor cell- expressed developmentally down-regulated 4) is a HECT-type E3 ubiquitin ligase. Through sequence analysis, we identified a PPLYKEL motif, an extended Nedd4 WW domain-binding motif, at the carboxyl terminus of the RORγt ligand-binding domain, suggesting that RORγt may be a binding partner of Nedd4. Indeed, Nedd4 is tyrosine-phosphorylated and activated upon TCR/CD28 stimulation and binds to RORγt which undergoes K63- linked poly-ubiquitination. This ubiquitination is abrogated in T cells lacking Nedd4. Further analysis showed that although Nedd4 deficiency does not impair Th1, Th2, and inducible regulatory T cell differentiation, Th17 cell differentiation is greatly compromised in the absence of Nedd4. In support of this observation, mice deficient for Nedd4, or deficient for Nedd4 in T cells, have ameliorated EAE with impaired Th17 responses. Based upon these preliminary data, we hypothesize that upon T cell antigenic stimulation Nedd4 is activated by Src kinase(s), and targets RORγt for K63-linked polyubiquitination, thus regulating Th17 responses and the susceptibility to EAE in mice and possibly MS in humans. In this proposal, we will investigate 1) How Nedd4 potentiates Th17 cell differentiation in vitro; 2) Whether Nedd4 promotes Th17 responses in vivo, regulating the susceptibility to EAE; and 3) How Nedd4 is activated in T cells in response to TCR/CD28 stimulation. The identification of Nedd4 as a key molecule regulating Th17 responses and the pathogenesis of EAE, and possibly MS, will have potential implications for using this molecule as a therapeutic target to treat autoimmune diseases involving Th17.