Regulation of AP-3-dependent axonal targeting by HECT ubiquitin ligases

HECT 泛素连接酶对 AP-3 依赖性轴突靶向的调节

• 批准号: 8485412
• 负责人: Pearl Victoria Ryder
• 金额: $ 0.82万
• 依托单位: EMORY 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 2013-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8485412
• 关键词: 1-Phosphatidylinositol 4-Kinase; Adaptor Protein Complex 3; Binding; Biochemical; Biological; Brain; Cell Fractionation; Cell Line; Cell physiology; Cells; Clathrin-Coated Vesicles; Complex; Data; Disease; Docking; Endosomes; Family; Functional disorder; Genetic; Genetic Transcription; Goals; Health; Human; Immunofluorescence Microscopy; Laboratories; Lead; Ligase; Location; Maintenance; Mammalian Cell; Membrane Protein Traffic; Modeling; Mus; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neurons; Organelles; PC12 Cells; Parkinson Disease; Pathway interactions; Play; Post-Translational Protein Processing; Primary Cell Cultures; Process; Proteins; Recruitment Activity; Regulation; Resolution; Role; Signal Transduction; Sorting - Cell Movement; Specific qualifier value; Structure; Synapses; Synaptic Vesicles; Testing; Ubiquitin; Ubiquitination; Vesicle; Work; base; brain cell; functional outcomes; immunoaffinity chromatography; interest; late endosome; mammalian genome; member; new therapeutic target; novel; protein degradation; scaffold; stem; ubiquitin ligase

DESCRIPTION (provided by applicant): Numerous ubiquitin ligases and potential substrates are present at multiple locations within the cell, yet ubiquitination occurs only at specific organelles. While the subcellular location where a substrate is ubiquitinated has functional consequences for the fate of the substrate, the mechanisms that specify this location are incompletely understood. In neurons, ubiquitination regulates synapse assembly and function by a host of selectively targeted pre- and post-synaptic ubiquitin ligases. The mechanisms that target these ligases to their subcellular locations are poorly understood and constitute the focus of this proposal. Our interest in these mechanisms stems from the role of the ubiquitin machinery in sporadic and familial forms of neurodegenerative diseases, such as Parkinson's disease. Exciting new data in our laboratory demonstrate that a member of the HECT (homologous to E6-AP carboxy terminus) superfamily of ubiquitin ligases, Nedd4, interacts with the adaptor protein complex-3 (AP-3). While Nedd4 acts at the late endosome, the mechanisms for targeting it to this compartment are unknown. Since the AP-3 complex acts as a vesiculation scaffold that brings together multiple proteins at endocytic organelles, we postulate that Nedd4 is recruited to endocytic compartments by interaction with AP-3. Our overall hypothesis is that HECT family ubiquitin ligases recognize specific membranous organelles by binding to organelle-restricted "docking" factors, such as AP-3. We will test our hypothesis through the following specific aims: (1) to determine if the AP-3 complex plays a role in subcellular localization of the Nedd4 ubiquitin ligase and (2) to determine if AP-3 regulates Nedd-4-dependent substrate recognition and post-translational modification. We will use subcellular fractionation and quantitative high-resolution immunofluorescence microscopy of fixed primary cultured neurons from wild-type and Nedd4- deficient mice and neuronal model cell lines in order to test our hypothesis. Our long-term goal is to define subcellular compartmentalization as a novel biological regulatory principle of ubiquitin machineries. Given the importance of the ubiquitin machinery to the normal and pathological synapse, our work will contribute new mechanistic understanding of the processes involved in the establishment and maintenance of synapses.

描述(申请人提供)：许多泛素连接酶和潜在的底物存在于细胞内的多个位置，然而泛素化只发生在特定的细胞器。虽然底物泛素化的亚细胞位置对底物的命运有功能上的影响，但指定这一位置的机制尚不完全清楚。在神经元中，泛素化通过一系列选择性定向的突触前和突触后泛素连接酶来调节突触的组装和功能。将这些连接酶定位于它们的亚细胞位置的机制还不是很清楚，这是本提案的重点。我们对这些机制的兴趣源于泛素机制在散发性和家族性神经退行性疾病中的作用，如帕金森氏病。我们实验室的新数据表明，Hect(与E6-AP羧基末端同源)泛素连接酶超家族成员Nedd4与接头蛋白复合体-3(AP-3)相互作用。虽然Nedd4作用于晚期的内体，但其靶向该隔室的机制尚不清楚。由于AP-3复合体作为囊泡支架，将多种蛋白质聚集在内吞细胞器中，我们推测Ned4通过与AP-3的相互作用被招募到内吞体内。我们的总体假设是，Hect家族泛素连接酶通过与细胞器限制的“对接”因子(如AP-3)结合来识别特定的膜性细胞器。我们将通过以下具体目标来验证我们的假设：(1)确定AP-3复合体是否在Nedd4泛素连接酶的亚细胞定位中发挥作用；(2)确定AP-3是否调节Nedd-4依赖的底物识别和翻译后修饰。我们将使用来自野生型和Nedd4基因缺陷小鼠和神经元模型细胞系的固定原代培养神经元的亚细胞分离和定量高分辨率免疫荧光显微镜来验证我们的假设。我们的长期目标是将亚细胞区划定义为泛素机制的一种新的生物调节原理。鉴于泛素机制对正常和病理突触的重要性，我们的工作将有助于从新的机制上理解突触的建立和维持过程。