Substrate Targeting Mechanisms of Nedd4 Family Ubiquitin Ligases

Nedd4 家族泛素连接酶的底物靶向机制

• 批准号: 8786568
• 负责人: Jason A MacGurn
• 金额: $ 24.9万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786568
• 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; 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; signal processing; technology development; therapeutic target; tool; trafficking; tumorigenesis; ubiquitin ligase

Project Summary 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.

项目摘要 许多人类疾病是由蛋白质在细胞表面的异常活性所引起的，包括癌症， 高血压和自身免疫性疾病。正常细胞控制蛋白质在 细胞表面通过多种调节蛋白质递送到血浆的机制 膜（PM）和从PM中去除蛋白质。主要去除机制之一涉及 与泛素结合，一种靶向PM蛋白的修饰，用于将其分类为囊泡 通过内吞作用内在。我有兴趣了解驱动的底物靶向机制 泛素介导的内吞作用和我作为独立研究人员的长期目标是确定如何 这些机制有助于人类疾病。 该研究计划建议研究NEDD4家族的底物靶向机制 泛素连接酶，对泛素介导的内吞作用至关重要，与许多人有关 疾病。尽管人类NEDD4家族的泛素连接酶仍然很了解，但对此有更多了解 NEDD4家族泛素连接酶在酵母中称为RSP5的功能。 RSP5作为调节器的关键作用 泛素介导的内吞作用是良好的，部分是由于可用的强大实验工具 酵母模型系统。这里提出的实验策略结合了对 酵母中RSP5的底物靶向机制，具有研究底物的平行策略 WWP1的靶向是NEDD4家族泛素连接酶与人类乳腺癌有关。这将涉及 开发新的实验工具，以促进泛素连接酶底物选择的研究均在体外选择 和体内。 本研究计划的目标1概述了确定磷酸化如何调节的策略 酵母中RSP5底物适配器蛋白的功能。这项研究对 NEDD4家族蛋白在人类中，因为磷酸化位点在哺乳动物中保守 蛋白质，被称为NEDD4泛素连接酶的衔接子。 AIM 2描述了一种策略 研究一类RSP5相互作用蛋白的抑制功能。虽然负面监管因子 NEDD4家族泛素连接酶尚未在人类中描述，这项研究可能导致 开发新的治疗策略，用于激活或灭活NEDD4泛素连接酶功能。目标3 概述了一个计划，该计划受酵母RSP5的机械见解的影响，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中的实验将 最终将WWP1作为治疗乳腺癌的分子靶标的治疗潜力。