Nedd4-family ubiquitination networks that regulate T cell function

描述（由申请方提供）：当E3泛素连接酶将泛素转移至靶蛋白时，泛素介导的降解被激活。Nedd 4是HECT型E3泛素连接酶，特别与T细胞功能相关。我们已经发现Nedd 4通过降低Cbl-b的水平来促进T细胞活化。我们的假设是Nedd 4部分地通过泛素化并引起Cbl-b的随后降解来促进T细胞依赖性适应性免疫应答。本研究的实验重点是描绘T细胞中Nedd 4调节的途径，并表征其对免疫应答的影响。我们将通过以下方式实现这一目标。首先，我们将确定Nedd 4如何调节Cbl-b以促进T细胞活化。我们假设在T细胞活化后，Nedd 4被活化并启动Cbl-b的降解，从而延长T细胞活化的持续时间。为了测试这一点，我们将测量Cbl-b在静息和刺激的T细胞中的半衰期，并确定Cbl-b的降解是否通过溶酶体或蛋白酶体发生。然后，我们将确定Nedd 4和Cbl-b在细胞中的何处相互作用。最后，我们将确定Nedd 4 -1-/- T细胞中Cbl-b水平的增加是否是这些细胞中观察到的增殖和IL-2产生减少的原因。其次，我们将定义Nedd 4在免疫功能中的功能库。Nedd 4-/-FLCh小鼠不产生有效的免疫应答。这可能是因为T细胞的内在缺陷，或者部分是由于抗原呈递细胞的缺陷。因此，我们将测试这些小鼠中T细胞缺陷的程度。我们将确定观察到的缺陷是T细胞内在的还是由Nedd 4-/-抗原呈递细胞的缺陷加剧的。最后，我们将研究Nedd 4在适应性免疫反应中的作用。为了研究细胞介导的免疫应答是否需要Nedd 4，我们将使用淋巴细胞脉络丛脑膜炎病毒（LCMV）感染模型。为了测试Nedd 4是否是体液免疫应答所必需的，我们使用胶原诱导的关节炎模型诱导自身免疫性疾病。我们的长期目标是研究Nedd 4调控的泛素化途径如何调节T细胞活化和适应性免疫。我们相信Nedd 4依赖性通路可以在治疗上靶向治疗免疫介导的疾病，如自身免疫性疾病。公共卫生相关性：我们建议研究调节T细胞蛋白质去除的酶的功能。这些酶促进T细胞功能，从而影响许多免疫介导的事件。这些研究有可能确定调节免疫反应的新治疗靶点，并可能为临床管理自身免疫性和炎症性疾病提供新的治疗选择。

DESCRIPTION (provided by applicant): Ubiquitin-mediated degradation is activated when an E3 ubiquitin ligase transfers ubiquitin to a target protein. Nedd4 is a HECT-type E3 ubiquitin ligases, particularly relevant to T cell function. We have found that Nedd4 promotes T cell activation by decreasing levels of Cbl-b. Our hypothesis is that Nedd4 promotes T cell- dependent adaptive immune responses, in part, by ubiquitinating and causing the subsequent degradation of Cbl-b. The experimental focus of this research is to delineate pathways regulated by Nedd4 in the T cell and characterize their impact on the immune response. We will accomplish this in the following ways. First, we will determine how Nedd4 regulates Cbl-b to promote T cell activation. We hypothesize that following T cell activation, Nedd4 becomes activated and initiates degradation of Cbl-b, thus lengthening the duration of T cell activation. To test this, we will measure the half-life of Cbl-b in resting and stimulated T cells and determine whether degradation of Cbl-b occurs via the lysosome or the proteasome. We will then determine where in the cell Nedd4 and Cbl-b interact. Finally, we will determine whether the increased levels of Cbl-b in Nedd4-1-/- T cells are responsible for the decreased proliferation and IL-2 production observed in these cells. Second, we will define the functional repertoire of Nedd4 in immune function. Nedd4-/-FLCh mice do not mount an effective immune response. This could be because of an intrinsic defect in the T cell or it could be due, in part, to a defect in antigen presenting cells. Thus, we will test the extent of the T cell defect in these mice. We will determine whether the observed defects are T cell-intrinsic or exacerbated by defects in Nedd4-/- antigen presenting cells. Finally, we will study the role of Nedd4 in the adaptive immune response. To study whether Nedd4 is required for cell-mediated immune responses, we will use the Lymphocytic Choriomeningitis Virus (LCMV) infection model. The test whether Nedd4 is required for humoral immune responses, we induce autoimmune disease using the collagen induced arthritis model. Our long-term goal is to study how ubiquitination pathways regulated by Nedd4 regulate T cell activation and adaptive immunity. We believe that Nedd4-dependent pathways could be targeted therapeutically to treat immune-mediated diseases such as autoimmune disease. PUBLIC HEALTH RELEVANCE: We propose to study the function of enzymes that regulate protein removal from T cells. These enzymes promote T cell function and thus impact many immune mediated events. These studies have the potential to identify novel therapeutic targets for regulating the immune response, and might provide new treatments options to clinically manage autoimmune and inflammatory diseases.