Molecular Targets of Neurosteroid Anti-depressant Action

神经类固醇抗抑郁作用的分子靶点

基本信息

批准号：
10662406
负责人：
ALEX S. EVERS
金额：
$ 55.77万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10662406
关键词：
Affinity Agonist Alkynes Allopregnanolone Amino Acids Anesthetics Anti-Anxiety Agents Anti-Inflammatory Agents Antidepressive Agents Antiepileptic Agents Automobile Driving Autophagocytosis Behavior Behavior assessment Behavioral Binding Binding Proteins Binding Sites Biological Biological Assay Biology Brain Carrier Proteins Cell membrane Cellular Stress Chemicals Chemistry Cholesterol Clustered Regularly Interspaced Short Palindromic Repeats Coupled Data Development Docking Electrophysiology (science)Goals Golgi Apparatus Hippocampus Inflammatory Intracellular Membranes Ion Channel Ketamine Knock-out Label Link Lipids Maps Mass Spectrum Analysis Mediating Membrane Mental Depression Methods Molecular Molecular Target Mutation N-Methyl-D-Aspartate Receptors NMDA receptor A1 Neurons Nitrous Oxide Organelles Output Pathway interactions Phospholipids Photoaffinity Labels Physiological Physiological Processes Physiology Protein Chemistry Protein Isoforms Proteins Proteome Proteomics Psychiatric therapeutic procedure Reagent Receptor Inhibition Research Research Support Role Signal Transduction Site Sterols Techniques Therapeutic Treatment Efficacy Work analog antagonist antidepressant effect beta Tubulin biological adaptation to stress enantiomer in vivo insight lipid transfer protein lipid transport liquid chromatography mass spectrometry molecular modeling network dysfunction neuroinflammation neuronal excitability neurosteroids novel novel therapeutics oxysterol binding protein positive allosteric modulator protein expression receptor response sedative steroid analog trans-Golgi Network

项目摘要

Project description Neuroactive steroids (NAS) are important modulators of neuronal excitability with demonstrated therapeutic efficacy as anesthetics and rapidly acting anti-depressants. While the anesthetic actions of NAS are mediated by GABAA receptors, the mechanism(s) of their anti-depressant effect remains unknown. NAS functionally modulate the activity of NMDA receptors and specifically bind several intracellular proteins that may contribute to their anti-depressant actions. Notably, NAS also localize to trans-Golgi membranes, suggesting that they are either selectively transported to Golgi or have an abundant selective binding partner in Golgi. The goals of this project are to characterize NAS binding sites on putative anti-depressant protein targets and identify novel intracellular NAS binding proteins that may contribute to the anti-depressant effects of NAS. To achieve these goals, we will utilize NAS analogue photo-affinity labeling in concert with state-of-the-art protein chemistry and expression techniques and cutting-edge mass spectrometry (MS) methods. The Evers lab has developed these methods and successfully applied them to delineating the molecular details of NAS binding to VDAC, β- tubulin and isoform-specific sites on GABAA receptors. Novel photo-affinity labeling reagents for this project will be synthesized in the Chemistry Core. The output of our labeling studies will be identification of novel NAS binding proteins and mutations in NAS binding sites that enable characterization of the role of these target proteins in the anti-depressant physiological and behavioral effects of NAS (Projects 2 and 3). The Project has two specific aims: In Aim 1, we will identify and characterize the binding sites for NAS that are negative- and positive- allosteric modulators (NAMs and PAMs) of NMDA receptors. GluN1/GluN2B NMDA receptors will be labeled with NAM and PAM NAS photo-affinity analogues and the number of labeling sites on each subunit will be determined using intact protein MS. The specific amino acids modified by the photolabeling reagents will be identified using middle-down MS. Molecular modeling will be used, in conjunction with the photolabeling data, to predict critical residues that may be necessary for NAS binding or effect. In Aim 2A we will perform an unbiased search to identify the “proteome” of NAS binding proteins in intact hippocampal neurons using photo- affinity labeling reagents with a “”click chemistry” tag to enrich binding proteins, followed by LC-MS/MS-based protein identification. In Aim 2B, we will perform a focused proteomic search to identify cytosolic neuronal proteins that selectively bind/transport NAS and may alter the lipid composition of organelle membranes and modulate cellular stress responses. The physiological and behavioral effects of identified NAS-binding proteins involved in cellular stress response pathways (Aim 2A) and NAS/lipid transport (Aim 2B) will be evaluated using CRISPR knockout methods (Projects 2 and 3).

项目描述神经活性类固醇 (NAS) 是神经元兴奋性的重要调节剂，具有已被证明的治疗作用 NAS 的麻醉作用是介导的，具有麻醉药和快速作用的抗抑郁药的功效。通过 GABAA 受体，其抗抑郁作用的机制在功能上仍不清楚。调节 NMDA 受体的活性并特异性结合几种可能有助于值得注意的是，NAS 也定位于跨高尔基体膜，这表明它们是要么选择性地转运到高尔基体，要么在高尔基体中具有丰富的选择性结合伙伴。该项目旨在表征推定的抗抑郁蛋白靶点上的 NAS 结合位点并鉴定新的细胞内 NAS 结合蛋白可能有助于 NAS 的抗抑郁作用，以实现这些目标。为了实现这一目标，我们将利用 NAS 模拟光亲和标记与最先进的蛋白质化学相结合， Evers 实验室开发了表达技术和尖端质谱 (MS) 方法。这些方法并成功应用于描绘 NAS 与 VDAC、β- 结合的分子细节该项目的新型光亲和标记试剂将检测微管蛋白和 GABAA 受体上的异构体特异性位点。我们的标记研究的输出将是新型 NAS 的鉴定。结合蛋白和 NAS 结合位点的突变能够表征这些靶标的作用 NAS 的抗抑郁生理和行为效应中的蛋白质（项目 2 和 3）。两个具体目标：在目标 1 中，我们将识别并表征 NAS 的正负结合位点 NMDA 受体的变构调节剂（NAM 和 PAM）将被标记。与 NAM 和 PAM NAS 光亲和类似物，每个亚基上的标记位点数量为使用完整蛋白质 MS 确定被光标记试剂修饰的特定氨基酸。将使用中下质谱鉴定，并结合光标记数据，为了预测 NAS 结合或效应可能必需的关键残基，我们将在目标 2A 中执行一个操作。使用照片进行无偏搜索以识别完整海马神经元中 NAS 结合蛋白的“蛋白质组” 带有“点击化学”标签的亲和标记试剂可富集结合蛋白，然后是基于 LC-MS/MS 的在目标 2B 中，我们将进行重点蛋白质组学搜索来鉴定胞质神经元。选择性结合/运输 NAS 并可能改变细胞器膜脂质组成的蛋白质调节细胞应激反应已确定的 NAS 结合的生理和行为效应。参与细胞应激反应途径（目标 2A）和 NAS/脂质运输（目标 2B）的蛋白质将被使用 CRISPR 敲除方法进行评估（项目 2 和 3）。