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

项目总结 突触小泡是储存和释放神经递质的高度专门化细胞器。这个 SVS上陈旧或受损的蛋白质堆积会损害神经传递，并可能导致功能障碍 神经电路和网络。事实上，最近的研究表明，调控SV蛋白的基因突变 退化与神经和神经退行性疾病有关，显示出关键的 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 人们对周转和退化仍然知之甚少。本项目的总体目标是阐明这些 机制，为困扰数百万美国人的疾病的病因学提供了关键的见解。我们最近 研究表明，ESCRT途径介导了SV膜的活性依赖的降解 蛋白质。ESCRT途径由一系列蛋白质复合体组成，这些复合体依次招募泛素化 并催化形成多囊泡体(MVB)，以便将这些货物运送到溶酶体。 有趣的是，我们发现神经元放电的增加刺激了去泛素化酶在 突触，以及携带初始ESCRT蛋白HRS的轴突运输小泡的运动性，以及它们的 招募到SV人才库。我们假设这些事件是依赖于活动的关键速率限制步骤 SV膜蛋白的周转。我们将通过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膜蛋白循环中的作用在这里，我们将使用生化和 荧光成像分析评估泛素化如何调节SV蛋白循环与降解 海马神经元。我们还将调查去泛素化酶UCHL1是否对 在回收SVS时保持SV蛋白，抵消其降解排序。在目标2中，我们将描述 HRS囊泡和HRS对下游ESCRT蛋白重新募集到SV池的影响。我们将使用超级- 分辨荧光/电子显微镜和邻近生物素化来表征形态和 这些囊泡的分子组成，以及HRS功能的获得和丧失结合活体成像 确定下游ESCRT蛋白募集到SV池是否需要HRs。在《目标3》中，我们将 研究依赖活动的人力资源重新招募到SV池的机制。我们将测试特定的角色 Kinesins在HRS轴突运输中的作用，并测试其重新聚集到SV池是否需要脂质PI(3)P， 泛素化蛋白和/或小GTP酶Rab35的存在。总之，这些研究将揭示 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.