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: 8776479
• 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/8776479
• 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。正常细胞控制蛋白质的丰度和活性， 细胞表面通过各种机制，调节蛋白质的血浆输送 膜（PM）和从PM中去除蛋白质。主要的清除机制之一包括 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。我感兴趣的是了解基板靶向机制，驱动 作为一名独立研究者，我的长期目标是确定 这些机制导致人类疾病。 本研究计划探讨Nedd4家族的底物靶向机制 泛素连接酶是泛素介导的内吞作用的关键酶，在许多人类细胞中起着重要作用。 疾病虽然人类Nedd4家族泛素连接酶仍然知之甚少，但对Nedd4家族泛素连接酶的了解更多。 酵母中Nedd4家族泛素连接酶Rsp5的功能。Rsp5作为一种调节剂的关键作用 泛素介导的内吞作用是公认的，部分原因是由于强大的实验工具， 酵母模型系统。这里提出的实验策略结合了详细的调查， Rsp5在酵母中的底物靶向机制及底物研究的平行策略 靶向WWP1，一种与人乳腺癌相关的Nedd4家族泛素连接酶。这将涉及 开发新的实验工具，以促进泛素连接酶底物选择的研究， 和体内。 本研究计划的目标1概述了确定磷酸化如何调节 Rsp5底物衔接蛋白在酵母中的功能。这项研究可能对以下方面产生重大影响： 人类Nedd4家族蛋白，因为磷酸化位点在哺乳动物抑制蛋白中是保守的 蛋白质，其已知作为Nedd4泛素连接酶的衔接子起作用。目标2描述了一种战略， 研究一类Rsp5相互作用蛋白的抑制功能。虽然负调节剂 Nedd4家族泛素连接酶尚未在人类中被描述，这项研究可能会导致 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 概述了一项计划，受酵母中Rsp5的机械见解的影响，以确定WWP1介导的 泛素化事件促进乳腺癌细胞生长。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。