Substrate Targeting Mechanisms of Nedd4 Family Ubiquitin Ligases

描述(申请人提供)：许多人类疾病是由细胞表面蛋白质的异常活性引起的，包括癌症、高血压和自身免疫性疾病。正常细胞通过各种机制控制细胞表面蛋白质的丰度和活性，这些机制既调节蛋白质向质膜的输送，又调节蛋白质从质膜中的去除主要的去除机制之一涉及与泛素的结合，泛素是一种针对PM蛋白的修饰，目的是将PM蛋白分类到囊泡中，然后通过内吞作用内化这些囊泡。我对了解推动泛素介导的内吞作用的底物靶向机制很感兴趣，作为一名独立研究员，我的长期目标是确定这些机制如何导致人类疾病。这项研究计划研究Nedd4家族泛素连接酶的底物靶向机制，这些连接酶在泛素介导的内吞作用中起关键作用，并与许多人类疾病有关。虽然人类Nedd4家族泛素连接酶仍然知之甚少，但人们对酵母中称为Rsp5的Nedd4家族泛素连接酶的功能有了更多的了解。Rsp5作为泛素介导的内吞作用的关键角色已经得到了充分的证实，在一定程度上 由于酵母模型系统提供了强大的实验工具。这里提出的实验策略结合了对酵母中Rsp5底物靶向机制的详细研究和对WWP1底物靶向的并行研究策略，WWP1是与人类乳腺癌相关的Nedd4家族泛素连接酶。这将涉及开发新的实验工具，以促进体外和体内泛素连接酶底物选择的研究。这项研究计划的目标1概述了一种确定磷酸化如何调节酵母中Rsp5底物适配器蛋白功能的策略。这项研究对人类Nedd4家族蛋白具有潜在的重要意义，因为这些磷酸化位点在哺乳动物arrestin蛋白中是保守的，这些蛋白被认为是Nedd4泛素连接酶的接头。目的研究一类Rsp5相互作用蛋白的抑制功能。尽管Nedd4家族泛素连接酶的负调控因子尚未在人类中被描述，但这项研究可能导致开发新的治疗策略来激活或失活Nedd4泛素连接酶功能。目标3概述了一个计划，受酵母中Rsp5的机械性见解的影响，以识别 WWP1介导的促进乳腺癌细胞生长的泛素化事件。这些在哺乳动物细胞上的实验将最终解决WWP1作为治疗乳腺癌的分子靶点的治疗潜力。 公共卫生相关性：各种人类癌症的标志之一是Nedd4家族泛素连接酶的放大，尽管它们促进癌细胞生长的机制尚不清楚。本文概述的研究计划将研究Nedd4家族泛素连接酶的底物靶向机制，并最终解决它们作为癌症治疗药物靶点的治疗潜力。

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. PUBLIC HEALTH RELEVANCE: One of the hallmarks of various human cancers is the amplification of Nedd4 family ubiquitin ligases, although the mechanism by which they facilitate cancer cell growth is unknown. The research plan outlined here will investigate the substrate targeting mechanisms of Nedd4 family ubiquitin ligases and ultimately address their therapeutic potential as drug targets for treatment of cancer.