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

描述（由申请人提供）：许多泛素连接酶和潜在底物存在于细胞内的多个位置，但泛素化仅发生在特定的细胞器上。虽然底物泛素化的亚细胞位置对底物的命运具有功能后果，但指定该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靶向突触前和突触后泛素连接酶调节突触组装和功能。将这些连接酶靶向到亚细胞位置的机制尚不清楚，这是本研究的重点。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（如帕金森病）中的作用。我们实验室令人兴奋的新数据表明，泛素连接酶的HECT（与E6-AP羧基端同源）超家族成员Nedd4与接头蛋白复合物-3 （AP-3）相互作用。虽然Nedd4作用于内核体晚期，但其靶向内核体的机制尚不清楚。由于AP-3复合物作为一种囊泡支架，将内吞细胞器中的多种蛋白质聚集在一起，我们假设Nedd4通过与AP-3的相互作用被招募到内吞室中。我们的总体假设是HECT家族泛素连接酶通过与细胞器限制性“对接”因子（如AP-3）结合来识别特定的膜细胞器。我们将通过以下具体目的来验证我们的假设：(1)确定AP-3复合物是否在Nedd4泛素连接酶的亚细胞定位中起作用；(2)确定AP-3是否调节Nedd4依赖性底物识别和翻译后修饰。我们将使用亚细胞分离和定量高分辨率免疫荧光显微镜对野生型和Nedd4缺陷小鼠和神经元模型细胞系的固定原代培养神经元进行检测，以验证我们的假设。我们的长期目标是将亚细胞区隔化定义为泛素机制的一种新的生物调控原理。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对突触建立和维持过程的新的机制理解。

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.