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: 8279635
• 负责人: Jason A MacGurn
• 金额: $ 9万
• 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 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8279635
• 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 Delivery Systems; 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. 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.

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) 的输送以及蛋白质从 PM 的去除。主要去除机制之一涉及与​​泛素的缀合，这是一种针对 PM 蛋白的修饰，将其分选成囊泡，然后通过内吞作用将其内化。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。 该研究计划旨在研究 Nedd4 家族泛素连接酶的底物靶向机制，该机制对于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了解更多。 Rsp5 作为泛素介导的内吞作用调节剂的关键作用已得到充分证实，部分 由于酵母模型系统中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 底物靶向研究的平行策略，WWP1 是一种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。 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 底物接头蛋白功能的策略。这项研究对人类 Nedd4 家族蛋白具有潜在的重要意义，因为哺乳动物视紫红质抑制蛋白的磷酸化位点是保守的，已知这些蛋白可作为 Nedd4 泛素连接酶的接头。目标 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因子，但这项研究可能会导致开发用于激活或失活 Nedd4 泛素连接酶功能的新型治疗策略。目标 3 概述了一项计划，受酵母中 Rsp5 机制见解的影响，以识别 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进行的实验将最终解决 WWP1 作为乳腺癌治疗分子靶点的治疗潜力。 公共健康相关性：各种人类癌症的标志之一是 Nedd4 家族泛素连接酶的扩增，尽管它们促进癌细胞生长的机制尚不清楚。这里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。