Substrate Targeting Mechanisms of Nedd4 Family Ubiquitin Ligases

描述（由申请人提供）：许多人类疾病是由细胞表面蛋白质的异常活动引起的，包括癌症、高血压和自身免疫性疾病。正常细胞通过多种机制控制细胞表面蛋白质的丰度和活性，这些机制调节蛋白质向质膜（PM）的传递和从质膜中去除蛋白质。其中一种主要的去除机制涉及到与泛素的结合，泛素是一种靶向PM蛋白的修饰，使其分选到囊泡中，然后通过内吞作用内化。我对了解驱动泛素介导的内吞作用的底物靶向机制很感兴趣，作为一名独立研究人员，我的长期目标是确定这些机制如何导致人类疾病。本研究计划拟研究Nedd4家族泛素连接酶的底物靶向机制，该酶对泛素介导的内吞作用至关重要，并与许多人类疾病有关。虽然人类对Nedd4家族泛素连接酶知之甚少，但对酵母中称为Rsp5的Nedd4家族泛素连接酶的功能了解更多。在某种程度上，Rsp5作为泛素介导的内吞作用的调节因子的关键作用是确定的

DESCRIPTION (provided by applicant): Many human diseases result from aberrant activity of proteins at the cell surface, including cancer, hypertension, and autoimmune disorders. Normal cells control the abundance and activity of proteins at the cell surface through a variety of mechanisms that regulate both the delivery of proteins to the plasma membrane (PM) and removal of proteins from the PM. One of the primary removal mechanisms involves conjugation to ubiquitin, a modification that targets PM proteins for sorting into vesicles which are then internalized by endocytosis. I am interested in understanding the substrate targeting mechanisms that drive ubiquitin-mediated endocytosis and my long-term goal as an independent researcher is to determine how these mechanisms contribute to human disease. This research plan proposes to investigate the substrate targeting mechanisms of Nedd4 family ubiquitin ligases, which are critical for ubiquitin-mediated endocytosis and are implicated in many human diseases. While human Nedd4 family ubiquitin ligases remain poorly understood, more is known about the function of a Nedd4 family ubiquitin ligase in yeast called Rsp5. The critical role of Rsp5 as a regulator of ubiquitin-mediated endocytosis is well-established, in part due to the powerful experimental tools available in the yeast model system. The experimental strategy proposed here combines a detailed investigation of substrate targeting mechanisms of Rsp5 in yeast with a parallel strategy for the investigation of substrate targeting of WWP1, a Nedd4 family ubiquitin ligase linked to human breast cancer. This will involve the development of new experimental tools to facilitate the study of ubiquitin ligase substrate selection both in vitro and in vivo. Aim 1 of this research plan outlines a strategy for determining how phosphorylation regulates the function of Rsp5 substrate adaptor proteins in yeast. This research has potentially significant implications for Nedd4 family proteins in humans because the phosphorylation sites are conserved in mammalian arrestin proteins, which are known to function as adaptors for Nedd4 ubiquitin ligases. Aim 2 describes a strategy to investigate the inhibitory function of a class of Rsp5-interacting proteins. Although negative regulators of Nedd4 family ubiquitin ligases have not been described in humans, this research could lead to the development of novel therapeutic strategies for activating or inactivating Nedd4 ubiquitin ligase function. Aim 3 outlines a plan, influenced by mechanistic insights from Rsp5 in yeast, to identify the WWP1-mediated ubiquitination event that facilitates breast cancer cell growth. These experiments in mammalian cells will ultimately address the therapeutic potential of WWP1 as a molecular target for treatment of breast cancer.