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

揭示泛素化和 ESCRT 通路在 SV 蛋白降解分选中的作用。

基本信息

批准号：
10364729
负责人：
Clarissa Leigh Waites
金额：
$ 38.26万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-01-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10364729
关键词：
Action Potentials Alzheimer&apos s disease model American Animal Model Axon Axonal Transport Biochemical Biological Assay Biotinylation Caring Carrier Proteins Characteristics Communication Complex DOOR (deafness-onychodystrophy-osteodystrophy-retardation) syndrome Data Deubiquitinating Enzyme Disease Electron Microscopy Epilepsy Etiology Event Exhibits Fluorescence Genes Goals Health system Hippocampus (Brain)Image Impairment Intellectual functioning disability Kinesin Lead Lipids Lysosomes Mediating Membrane Membrane Proteins Molecular Monitor Monomeric GTP-Binding Proteins Morphology Movement Multivesicular Body Mutation Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neuroglia Neurons Neurotransmitters Organelles Parkinson Disease Parkinsonian Disorders Pathway interactions Phenotype Presynaptic Terminals Process Production Productivity Protein Sortings Proteins Recycling Resolution Role Series Site Sorting - Cell Movement Synapses Synaptic Cleft Synaptic Vesicles Techniques Testing UCHL1 gene Ubiquitin Ubiquitination Vesicle Work cell motility cost experimental study first responder fluorescence imaging hepatocyte growth factor-regulated tyrosine kinase substrate imaging approach insight loss of function nervous system disorder neural circuit neural network neurotransmission neurotransmitter release phosphatidylinositol 3-phosphate presynaptic prevent protein complex protein degradation protein transport proteostasis recruit response trafficking ubiquitin isopeptidase vesicle transport vesicular SNARE proteins

项目摘要

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.

项目概要突触小泡（SV）是高度专业化的细胞器，可以储存和释放神经递质。这旧的或受损的蛋白质在 SV 上的积累会损害神经传递，并可能导致功能失调的神经回路和网络。事实上，最近的研究表明，基因突变调节 SV 蛋白降解与神经系统和神经退行性疾病相关，证明 SV 蛋白更新对神经系统健康至关重要。然而分子导致 SV 更新和降解的机制仍知之甚少。本次活动的总体目标该项目旨在阐明这些机制，为困扰疾病的病因学提供重要见解数百万美国人。我们最近的工作表明，ESCRT 通路介导活性依赖性 SV 膜蛋白的降解。 ESCRT 途径包含一系列蛋白质复合物，依次招募泛素化货物并催化多泡体 (MVB) 的形成以进行递送这些货物到溶酶体。有趣的是，我们发现增加的神经元放电会刺激突触处的去/泛素化酶，以及携带初始蛋白的轴突转运囊泡的运动性 ESCRT 蛋白 Hrs 及其招募到 SV 池中的情况。我们假设这些事件是临界率- SV 膜蛋白活性依赖性周转的限制步骤。我们将用三个测试来检验这个假设目标。在目标 1 中，我们将评估去/泛素化在 SV 膜蛋白回收中的作用。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 蛋白海马神经元的回收与降解。我们还将研究去泛素化是否 UCHL1 酶对于维持 SV 蛋白在回收 SV 上、抵消其降解是必需的排序。在目标 2 中，我们将表征 Hrs 囊泡以及 Hrs 对下游 ESCRT 蛋白的影响招募到 SV 池。我们将使用超分辨率荧光/电子显微镜和接近技术生物素化来表征这些囊泡的形态和分子组成，以及小时增益和功能丧失结合实时成像来确定下游ESCRT是否招募蛋白质到 SV 池需要数小时。在目标 3 中，我们将研究活动依赖性 Hrs 的机制招募到 SV 池。我们将测试特定驱动蛋白在 Hrs 轴突运输中的作用，并测试其招募到 SV 池中是否需要脂质 PI(3)P、泛素化蛋白的存在和/或小 GTP 酶 Rab35。这些研究将共同揭示 SV 的基本机制神经元中的蛋白质稳态。