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: 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
• 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; 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

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异常活性（包括癌症，高血压和自身免疫性疾病）引起的。正常细胞通过多种机制来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（PM）的递送以及从PM中去除蛋白质。主要的去除机制之一涉及与​​泛素结合，该修饰的靶向PM蛋白用于分类囊泡，然后通过内吞作用将其内化。我有兴趣了解促进泛素介导的内吞作用的底物靶向机制以及作为独立研究人员的长期目标是确定这些机制如何促进人类疾病。 该研究计划建议研究NEDD4家族泛素连接酶的底物靶向机制，这对于泛素介导的内吞作用至关重要，并且与许多人类疾病有关。尽管人类NEDD4家族的泛素连接酶仍然鲜为人知，但对酵母中NEDD4家族泛素连接酶的功能众所周知，称为RSP5。 RSP5作为泛素介导的内吞作用的调节剂的关键作用是完善的 由于酵母模型系统中可用的功能强大的实验工具。此处提出的实验策略结合了对酵母中RSP5的底物靶向机制的详细研究，以及对WWP1的底物靶向研究的平行策略，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，以促进在体外和体内研究泛素连接酶底物选择的研究。 该研究计划的目标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。这项研究对人类的NEDD4家族蛋白具有潜在的显着影响，因为磷酸化位点在哺乳动物抑制蛋白中是保守的，该蛋白被称为NEDD4泛素连接酶的衔接子。 AIM 2描述了研究一类RSP5相互作用蛋白的抑制功能的策略。尽管尚未在人类中描述NEDD4家族泛素连接酶的负调节剂，但这项研究可能导致发展新型治疗策略，以激活或灭活NEDD4泛素连接酶功能。 AIM 3概述了一个计划，该计划受酵母中RSP5的机械见解的影响，以识别 WWP1介导的泛素化事件促进了乳腺癌细胞的生长。这些在哺乳动物细胞中的实验最终将解决WWP1作为治疗乳腺癌的分子靶标的治疗潜力。