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Substrate Targeting Mechanisms of Nedd4 Family Ubiquitin Ligases

Nedd4 家族泛素连接酶的底物靶向机制

基本信息

批准号：
8474635
负责人：
Jason A MacGurn
金额：
$ 1.5万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8474635
关键词：
Accounting Adaptor Signaling Protein Address Affect Apoptosis Arrestins Attenuated Autoimmune Diseases Autoimmunity Bacteria Biological Assay Breast Cancer Cell Breast Cancer Treatment C-terminal Cancer Cell Growth Cell membrane Cell surface Chemicals Development Disease Drug Targeting Endocytic Vesicle Endocytosis Epidermal Growth Factor Receptor Event Excision Family Future G-Protein-Coupled Receptors Goals Growth Health Human Human Genome Hypertension In Vitro Investigation Lead Ligands Link Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Mediating Membrane Proteins Modeling Molecular Molecular Target N-terminal Normal Cell Phosphorylation Phosphorylation Site Protein Family Proteins Proteomics Reaction Regulation Research Research Personnel Role Signal Transduction Site-Directed Mutagenesis Sorting - Cell Movement Stimulus System Therapeutic Ubiquitin Ubiquitination Vesicle Work Yeast Model System Yeasts cancer therapy computerized data processing endosome lumen high throughput screening human disease improved in vivo inhibitor/antagonist insight interest knock-down malignant breast neoplasm novel novel therapeutics receptor reconstitution research study technology development therapeutic target tool trafficking tumorigenesis ubiquitin ligase

项目摘要

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.

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）的递送和蛋白质从PM的去除。主要去除机制之一涉及与泛素缀合，这是一种靶向PM蛋白的修饰，用于分选成囊泡，然后通过内吞作用内化。我有兴趣了解驱动泛素介导的内吞作用的底物靶向机制，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。本研究计划旨在研究Nedd4家族泛素连接酶的底物靶向机制，这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Nedd4家族泛素连接酶仍然知之甚少，但对酵母中称为Rsp5的Nedd4家族泛素连接酶的功能了解更多。Rsp5作为泛素介导的内吞作用的调节剂的关键作用是公认的，部分地由于酵母模型系统中可用的强大实验工具。这里提出的实验策略结合了详细的调查底物靶向机制的Rsp5在酵母中与一个平行的策略调查底物靶向WWP1，一个Nedd4家族泛素连接酶连接到人类乳腺癌。这将涉及新的实验工具的发展，以促进泛素连接酶底物的选择在体外和体内的研究。本研究计划的目的1概述了确定磷酸化如何调节酵母中Rsp5底物衔接蛋白功能的策略。这项研究对人类Nedd4家族蛋白具有潜在的重要意义，因为磷酸化位点在哺乳动物arrestin蛋白中是保守的，已知其作为Nedd4泛素连接酶的衔接子发挥作用。目的2描述了一种研究一类Rsp5相互作用蛋白的抑制功能的策略。虽然Nedd4家族泛素连接酶的负调节因子尚未在人类中描述，但这项研究可能导致开发用于激活或灭活Nedd4泛素连接酶功能的新治疗策略。目标3概述了一个计划，受酵母中Rsp 5的机械见解的影响， WWP 1介导的促进乳腺癌细胞生长的遍在蛋白化事件。这些在哺乳动物细胞中的实验将最终解决WWP1作为治疗乳腺癌的分子靶点的治疗潜力。