Molecular Targets of Neurosteroid Anti-depressant Action

神经类固醇抗抑郁作用的分子靶点

• 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
• 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

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，具有已证实的治疗作用。 作为麻醉剂和快速作用的抗抑郁药的功效。虽然NAS的麻醉作用是通过 由于GABAA受体的作用，其抗肿瘤作用的机制仍不清楚。NAS功能 调节NMDA受体的活性，并特异性结合几种可能有助于 他们的反腐败行动。值得注意的是，NAS也定位于trans-Golgi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伴侣。这个的目标 项目是表征推定的抗CD 4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的抗肿瘤作用。实现这些 目标，我们将利用NAS类似物光亲和标记与最先进的蛋白质化学和 表达技术和尖端质谱（MS）方法。埃弗斯实验室开发了 这些方法，并成功地将它们应用于描绘NAS结合VDAC，β- 微管蛋白和GABAA受体上的同种型特异性位点。用于该项目的新型光亲和标记试剂将 在化学中心合成。我们的标签研究的结果将是鉴定新的NAS 结合蛋白和NAS结合位点中的突变，使得能够表征这些靶点的作用。 蛋白质在NAS的抗衰老生理和行为效应中的作用（项目2和3）。该项目 两个具体目标： 在目标1中，我们将识别和表征NAS的负性和正性结合位点， NMDA受体的变构调节剂（NAM和PAM）。将标记GluN 1/GluN 2B NMDA受体 与NAM和PAM NAS光亲和类似物，每个亚基上的标记位点的数量将是 使用完整蛋白质MS测定。将通过光标记试剂修饰的特定氨基酸 将使用分子建模，结合光标记数据， 以预测NAS结合或作用可能必需的关键残基。在目标2A中，我们将执行 无偏见的搜索，以确定“蛋白质组”的NAS结合蛋白在完整的海马神经元使用照片， 亲和标记试剂与“点击化学”标签富集结合蛋白，然后进行基于LC-MS/MS的 蛋白质鉴定在目标2B中，我们将进行集中的蛋白质组学搜索，以鉴定胞浆神经元 选择性结合/转运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。鉴定的NA结合的生理和行为效应 参与细胞应激反应途径（Aim 2A）和NAS/脂质转运（Aim 2B）的蛋白质将被 使用CRISPR敲除方法进行评估（项目2和3）。