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

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

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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）是令人兴奋的神经元的重要调节剂作为麻醉和迅速起作用的抗抑郁药的功效。而NAS的麻醉作用是介导的通过GABAA接收器，其抗抑郁作用的机制仍然未知。 NAS功能调节NMDA接收器的活性，并专门结合几种可能有助于的细胞内蛋白对他们的抗抑郁作用。值得注意的是，NAS还本地化于跨性高尔基膜，表明它们是选择性地运输到高尔基体，或在高尔基体中具有丰富的选择性结合伴侣。目标的目标项目旨在表征NAS结合位点上推定的抗抑制蛋白靶标的并识别新颖的细胞内NAS结合蛋白可能有助于NAS的抗抑郁作用。实现这些目标，我们将与最先进的蛋白质化学和表达技术和尖端的质谱法（MS）方法。 Evers实验室已经发展这些方法并成功地应用它们来描述NAS与VDAC，β-的分子细节 GABAA受体上的微管蛋白和同工型特异性位点。该项目的新型照片亲和力标签试剂将在化学核心中合成。我们的标签研究的输出将是新型NAS的识别 NAS结合位点的结合蛋白和突变，使这些目标的作用能够表征 NAS的抗抑郁药生理和行为作用中的蛋白质（项目2和3）。该项目有两个具体的目标：在AIM 1中，我们将确定并表征NA的NA的结合位点，这些NA是负和正的 - NMDA受体的变构调节剂（NAM和PAMS）。 Glun1/glun2b NMDA受体将被标记使用NAM和PAM NAS照片亲和力类似物以及每个亚基上的标签位点的数量使用完整的蛋白质MS确定。由光标记试剂修饰的特定氨基酸将是使用中向下MS确定。分子建模将与光标记数据一起使用，预测NAS结合或效果可能需要的关键残差。在AIM 2A中，我们将执行无偏见的搜索，以使用照片 - 具有“点击化学”标签的亲和力标记试剂以富集结合蛋白，其次是基于LC-MS/MS 蛋白质鉴定。在AIM 2B中，我们将执行重点的蛋白质组学搜索以识别胞质神经元选择性结合/转运NA并可能改变细胞器膜的脂质组成的蛋白质和调节细胞应激反应。 NAS结合的身体和行为影响参与细胞应力反应途径（AIM 2A）和NAS/脂质转运（AIM 2B）的蛋白质将是使用CRISPR敲除方法进行评估（项目2和3）。