Uncovering the roles of ubiquitination and the ESCRT pathway in degradative sorting of SV proteins.

项目摘要 突触囊泡（SV）是高度特化的细胞器，储存和释放神经递质。的 SV上旧的或受损的蛋白质的积累会损害神经传递， 功能失调的神经回路和网络。事实上，最近的研究表明， 调节SV蛋白降解与神经和神经变性疾病有关， 证明了SV蛋白质周转对神经系统健康的关键重要性。然而， 对SV周转和降解的机制仍知之甚少。这个项目的总体目标是 该项目的目的是阐明这些机制，提供关键的见解，疾病的病因，折磨 数百万美国人。我们最近的工作表明，ESCRT途径介导了活性依赖性的 SV膜蛋白的降解。ESCRT途径包括一系列蛋白质复合物， 依次募集泛素化的货物并催化多泡体（MVB）的形成以用于递送 这些货物的溶酶体。有趣的是，我们发现增加的神经元放电刺激了 在突触的去/泛素化酶，以及轴突运输囊泡携带初始的运动性， ESCRT蛋白Hrs，及其向SV库的募集。我们假设这些事件是临界速率- SV膜蛋白活性依赖性周转的限制步骤。我们将用三个例子来检验这个假设。 目标。在目的1中，我们将评估去/泛素化在SV膜蛋白再循环中的作用。在这里， 我们将使用生化和荧光成像分析来评估泛素化如何调节SV蛋白 海马神经元的再循环与降解。我们还将研究去泛素化是否 酶UCHL 1是维持SV蛋白在回收SV上所必需的， 分类在目标2中，我们将表征Hrs囊泡和Hrs对下游ESCRT蛋白的影响 招募到SV样本池。我们将使用超分辨率荧光/电子显微镜和近距离 生物素化来表征这些囊泡的形态和分子组成， 功能丧失与实时成像相结合，以确定下游ESCRT的募集是否 蛋白质到SV池需要小时。在目标3中，我们将研究活动依赖性Hrs的机制 招募到SV样本池。我们将测试特定驱动蛋白在Hrs轴突运输中的作用，并测试 是否需要脂质PI（3）P，泛素化蛋白的存在，和/或 小GTSTUDIER Rab 35.总之，这些研究将揭示SV的基本机制 神经元中的蛋白质稳态。

PROJECT SUMMARY Synaptic vesicles (SVs) are highly specialized organelles that store and release neurotransmitters. The accumulation of old or damaged proteins on SVs compromises neurotransmission and can lead to dysfunctional neural circuits and networks. Indeed, recent studies have shown that mutations in genes that regulate SV protein degradation are associated with neurological and neurodegenerative disorders, demonstrating the critical importance of SV protein turnover for nervous system health. Yet the molecular mechanisms responsible for SV turnover and degradation remain poorly understood. The overall goal of this project is to elucidate these mechanisms, providing critical insights into the etiology of diseases that afflict millions of Americans. Our recent work has shown that the ESCRT pathway mediates the activity-dependent degradation of SV membrane proteins. The ESCRT pathway comprises a series of protein complexes that sequentially recruit ubiquitinated cargo and catalyze the formation of multivesicular bodies (MVBs) for delivery of these cargo to lysosomes. Intriguingly, we find that increased neuronal firing stimulates the activation of de/ubiquitinating enzymes at the synapse, as well as the motility of axonal transport vesicles carrying initial ESCRT protein Hrs, and their recruitment to SV pools. We hypothesize that these events are critical rate- limiting steps for activity-dependent turnover of SV membrane proteins. We will test this hypothesis with three aims. In Aim 1, we will evaluate the role of de/ubiquitination in the recycling of SV membrane proteins. Here, we will use biochemical and fluorescence imaging assays to evaluate how ubiquitination regulates SV protein recycling vs. degradation in hippocampal neurons. We will also investigate whether the deubiquitinating enzyme UCHL1 is necessary for maintaining SV proteins on recycling SVs, counteracting their degradative sorting. In Aim 2, we will characterize Hrs vesicles and the impact of Hrs on downstream ESCRT protein recruitment to SV pools. We will use super-resolution fluorescence/electron microscopy and proximity biotinylation to characterize the morphology and molecular composition of these vesicles, and Hrs gain- and loss-of-function combined with live imaging to determine whether the recruitment of downstream ESCRT proteins to SV pools requires Hrs. In Aim 3, we will investigate the mechanisms of activity-dependent Hrs recruitment to SV pools. We will test the roles of specific kinesins in the axonal transport of Hrs, and test whether its recruitment to SV pools requires the lipid PI(3)P, the presence of ubiquitinated proteins, and/or the small GTPase Rab35. Together, these studies will uncover fundamental mechanisms underlying SV proteostasis in neurons.