Molecular Sites of Neurosteroid Binding

神经类固醇结合的分子位点

基本信息

批准号：
10357845
负责人：
ALEX S. EVERS
金额：
$ 58.98万
依托单位：
WASHINGTON UNIVERSITY
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：
2014-05-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10357845
关键词：
ART protein Affect Affinity Allopregnanolone Amino Acids Anesthetics Antidepressive Agents Antiepileptic Agents Baculoviruses Behavior Binding Binding Sites Biochemical Biological Assay Biology Brain Cell surface Cells Chloride Channels Data Development Docking GABA Receptor Goals Label Laboratories Libraries Ligand Binding Ligands Mass Spectrum Analysis Measures Mediating Methods Molecular Molecular Target Muscimol Mutate Mutation Neuroprotective Agents Nootropic Agents Outcome Pharmaceutical Chemistry Pharmaceutical Preparations Photoaffinity Labels Positioning Attribute Pregnanolone Production Protein Chemistry Protein Isoforms Proteins Reagent Research Site Sterols Surface Synapses System Techniques Therapeutic Therapeutic Agents Transferase Transmembrane Domain Vertebral column Viral Work analog gamma-Aminobutyric Acid glycosylation insight molecular modeling molecular site mutant nervous system development neuronal excitability neurosteroids novel novel therapeutics pharmacophore positive allosteric modulator pregnenolone sulfate prevent protein expression receptor receptor expression receptor function sedative simulation stoichiometry trafficking

项目摘要

Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anti-depressants, anesthetics and neuro-protectants. The principal molecular target of neurosteroids is the GABAA receptor. We have shown that there are multiple, subunit- specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the effects of neurosteroids on receptor expression and function. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on the most abundant forms of synaptic and extrasynaptic GABAA receptors. Novel neurosteroid analogue photolabeling reagents will be developed and used to determine the number of neurosteroid binding sites on each subunit, to identify the binding sites and to determine the orientation of the neurosteroids in these sites. To achieve these goals we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down, intact protein and native MS. We will then mutate amino acids in each of the identified binding sites and use functional readouts to determine which binding sites mediate the various effects of neurosteroids on GABAA receptor expression and function. The Project has two specific aims: In Aim 1, a viral expression system (BacMam) will be used to express large quantities of synaptic (α1β2γ2) and extrasynaptic (α4β3δ) GABAA receptors and a suite of neurosteroid analogue photolabeling reagents will be synthesized in which the photolabeling moieties are placed at various positions around the neurosteroid backbone. The expressed receptors will be used in conjunction with the photolabeling reagents to determine the number of labeling sites on each subunit using intact protein MS and on each pentameric receptor using native MS. The specific amino acids modified by photolabeling will be identified using middle-down MS. The photolabeling data will then be used in conjunction with molecular modeling and docking to determine the preferred orientation of neurosteroids in each of the binding pockets and to identify critical residues which may be necessary for neurosteroid binding or effect. In Aim 2 receptors will be expressed in which these critical residues are mutagenized. The mutant receptors will be used in functional assays (channel gating, trafficking of receptors to the surface and modulation of orthosteric ligand binding) to determine the contribution of each binding site to neurosteroid action(s). Neurosteroid labeling and orientation will be then be assessed in the mutated receptors (as in Aim 1) to determine how the mutations alter neurosteroid binding and/or effect. The data from these studies will provide insight into the specific molecular interactions underlying neurosteroid actions at each of its binding sites on GABAA receptors. These data will provide a structural template for development of subunit- and function-specific neurosteroid analogues as well as novel insights into neurosteroid biology.

神经类固醇是神经元令人兴奋和神经系统发育的重要调节剂作为抗抑郁药，麻醉和神经保护剂的巨大治疗潜力。校长神经类固醇的分子靶标是GABAA接收器。我们已经表明有多个亚基 - 神经类固醇在GABAA受体上的特定结合位点，每种受体都有助于神经素的受体表达和功能。该项目将使用光标记技术来定义神经类固醇的精确部位在最丰富的突触和抗肿瘤外GABAA上结合受体。新型的神经类固醇类似标记试剂将开发并用于确定每个亚基上的神经类固醇结合位点的数量，以识别结合位点并确定这些位点的神经类固醇的方向。为了实现这些目标，我们将利用最先进的蛋白质化学和表达技术与尖端质谱法（MS）方法结合使用，包括中向下，完整的蛋白质和天然MS。然后，我们将在每个已鉴定的氨基酸中突变结合位点并使用功能读数来确定哪些结合位点介导 GABAA受体表达和功能上的神经蛋白酶。该项目有两个具体的目标：在AIM 1中，病毒表达系统（BACMAM）将用于表达大量突触（α1β2γ2）和外突触（α4β3δ）GABAA受体和一套神经类固醇模拟光标记试剂将会合成的，将光标记部分放置在神经类固醇周围的各种位置骨干。表达的接收器将与光标记试剂一起使用以确定使用完整的蛋白质MS和每个五聚体接收器上的每个亚基上标记位点的数量本机MS。将使用中倒数MS鉴定通过光标记修饰的特定氨基酸。这然后，光标记数据将与分子建模和对接结合使用，以确定每个结合口袋中神经类固醇的优选方向，并确定可能对于神经类固醇的结合或作用是必要的。在AIM 2中将表达这些关键残基被诱变。突变接收器将用于功能分析（通道门控，贩运受体对表面的受体和直角配体结合的调节）以确定每个人的贡献与神经类动物作用的结合位点。然后将在突变的接收器（如目标1所示），以确定突变如何改变神经类固醇的结合和/或效应。这些研究的数据将提供有关神经类固醇基础的特定分子相互作用的洞察力在GABAA接收器上的每个绑定位点的操作。这些数据将为结构模板提供亚基和功能特异性神经类似类似物的发展以及对神经类似生物学。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Roles for Anesthesiologists in the Future of Medicine in the United States.

DOI：
10.1213/ane.0000000000005852
发表时间：
2022-02-01
期刊：
Anesthesia and analgesia
影响因子：
5.7
作者：
Evers AS;Wiener-Kronish JP
通讯作者：
Wiener-Kronish JP

Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.

DOI：
10.1021/acs.analchem.6b05003
发表时间：
2017-02-21
期刊：
Analytical chemistry
影响因子：
7.4
作者：
Budelier MM;Cheng WW;Bergdoll L;Chen ZW;Abramson J;Krishnan K;Qian M;Covey DF;Janetka JW;Evers AS
通讯作者：
Evers AS