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， 神经回路和网络。事实上，最近的研究表明，调节SV蛋白的基因突变 降解与神经系统和神经退行性疾病有关，证明了关键的 SV蛋白周转对神经系统健康的重要性。然而导致SV的分子机制 对周转和退化的了解仍然很少。本项目的总体目标是阐明这些 机制，提供了重要的见解，疾病的病因折磨数百万美国人。我们最近 研究表明，ESCRT途径介导SV膜的活性依赖性降解 proteins. ESCRT途径包括一系列蛋白质复合物，其依次募集泛素化的 在一些实施方案中，所述载体可以被用于运载运载物并催化多泡体（MVB）的形成以将这些运载物递送至溶酶体。 有趣的是，我们发现增加的神经元放电刺激去/泛素化酶的激活， 突触，以及携带初始ESCRT蛋白Hrs的轴突运输囊泡的运动性，及其 招募到SV样本池。我们假设这些事件是活动依赖性的关键限速步骤。 SV膜蛋白的周转。我们将以三个目标来检验这一假设。在目标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，我们将使用生物化学和 荧光成像分析，以评估泛素化如何调节SV蛋白再循环与降解， 海马神经元我们还将研究去泛素化酶UCHL1是否是必需的， 维持SV蛋白在再循环SV上，抵消它们的降解分选。在目标2中，我们将描述 Hrs囊泡和Hrs对下游ESCRT蛋白募集至SV池的影响。我们将使用超级- 分辨率荧光/电子显微镜和邻近生物素化来表征形态， 这些囊泡的分子组成，以及Hrs功能的获得和丧失，结合实时成像， 确定下游ESCRT蛋白向SV池的募集是否需要Hrs。在目标3中，我们 研究活动依赖性Hrs募集到SV池的机制。我们将测试特定的 驱动蛋白在Hrs轴突运输中的作用，并测试其向SV池的募集是否需要脂质PI（3）P， 泛素化蛋白的存在，和/或小的GT3 Rab35。这些研究将共同揭示 神经元中SV蛋白稳定的基本机制。

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.