Nedd4-family ubiquitination networks that regulate T cell function

描述(由申请人提供)：当E3泛素连接酶将泛素转移到目标蛋白时，泛素介导的降解被激活。Nedd4是一种Hect型E3泛素连接酶，与T细胞功能密切相关。我们发现Nedd4通过降低Cbl-b水平促进T细胞活化。我们的假设是，Nedd4促进T细胞依赖的适应性免疫反应，部分是通过泛素化并导致Cbl-b随后的降解。这项研究的实验重点是描绘T细胞中Nedd4调节的通路，并表征它们对免疫反应的影响。我们将通过以下方式实现这一目标。首先，我们将确定Nedd4是如何调节Cbl-b来促进T细胞激活的。我们推测，在T细胞激活后，Nedd4被激活，并启动Cbl-b的降解，从而延长T细胞激活的持续时间。为了验证这一点，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的降解是通过溶酶体还是通过蛋白酶体发生的。然后我们将确定Nedd4和Cb1-b在细胞内的哪里相互作用。最后，我们将确定Nedd4-1-/-T细胞中Cbl-b水平的增加是否与这些细胞中观察到的增殖和IL-2产生减少有关。其次，我们将确定Nedd4在免疫功能中的功能谱系。Nedd4-/-Flch小鼠不会产生有效的免疫反应。这可能是因为T细胞的固有缺陷，也可能部分是由于抗原提呈细胞的缺陷。因此，我们将测试这些小鼠T细胞缺陷的程度。我们将确定观察到的缺陷是T细胞固有的，还是由于Nedd4-/-抗原提呈细胞的缺陷而加剧。最后，我们将研究Nedd4在获得性免疫反应中的作用。为了研究细胞免疫反应是否需要Nedd4，我们将使用淋巴细胞性脉络膜脑膜炎病毒(LCMV)感染模型。在体液免疫反应是否需要Nedd4的测试中，我们使用胶原诱导的关节炎模型来诱导自身免疫性疾病。我们的长期目标是研究Nedd4调节的泛素化途径如何调节T细胞激活和获得性免疫。我们认为，Nedd4依赖的通路可以作为治疗的靶点，用于治疗免疫介导性疾病，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，从而影响许多免疫调节事件。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.