MOLECULAR SITES OF NEUROSTEROID BINDING

神经类固醇结合的分子位点

• 批准号: 8610535
• 负责人: DOUGLAS F COVEY
• 金额: $ 40.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8610535
• 关键词: Action Potentials; Affinity; Alkynes; Anesthetics; Azides; Base Sequence; Behavior; Binding; Binding Proteins; Binding Sites; Biological; Biology; Biotin; Brain; Cells; Chemicals; Chemistry; Copper; Coupled; Custom; Data; Detection; Development; Diazomethane; Epilepsy; Epitopes; Future; GABA-A Receptor; Gel; General Anesthesia; Geometry; Goals; Imagery; Investigation; Ions; Label; Ligands; Location; Manuals; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Mental Depression; Methods; Molecular; Molecular Models; Moods; Neurons; Peptides; Pharmaceutical Preparations; Play; Positioning Attribute; Protein Databases; Protein Isoforms; Proteins; Proteome; Proteomics; Rattus; Reagent; Research; Resolution; Role; Sampling; Sensitivity and Specificity; Site; Solutions; Steroids; Stress; Structure; Synthesis Chemistry; Techniques; Tissues; Trypsin; Vertebral column; Work; adduct; analog; base; brain tissue; chemical cleavage; cycloaddition; gamma-Aminobutyric Acid; liquid chromatography mass spectrometry; molecular modeling; molecular site; neuroprotection; neurosteroids; novel; prevent; public health relevance; receptor; sedative; stereochemistry; tool

Project Description Neurosteroid analogues produce general anesthesia by binding to GABA-A receptors and enhancing neuronal inhibition in the brain. The number of binding sites for neurosteroids on GABA-A receptors and their molecular localization has been incompletely defined and is based on indirect methods. Neurosteroids are also endogenous mediators of brain development and function whose sites of action remain undefined. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on GABA-A receptors. Novel "click chemistry"-tagged, neurosteroid analogue photolabeling reagents will be developed and used in conjunction with isotopically tagged, "click chemistry" bifunctional linkers, to purify photolabeled proteins and peptides, providing the sensitivity and specificity to use high resolution mass spectrometry to detect and sequence low-abundance photolabeled peptides in biological samples. These techniques will also be applied as a discovery tool to identify additional proteins in brain that have neurosteroid binding sites. The Project has three specific aims: In Aim 1, novel neurosteroid analogue photolabeling reagents containing an alkyne group and isotopically-tagged, bifunctional linker molecules containing both an azide group and an affinity tag will be synthesized for use in "click chemistry"-based visualization and purification of photolabeled proteins and peptides (Aims 2 and 3). The isotopic tag will provide a "signature" for specific mass spectrometric detection of photolabeled peptides. Neurosteroid analogues will be synthesized that differ in stereochemistry, mechanism of photolabeling and the location of the diazirine photolabeling moiety on the neurosteroid backbone. In Aim 2 the number and structure of the neurosteroid binding sites on a defined GABA-A receptor (¿1¿3) will be assessed by photolabeling with the neurosteroid analogues prepared in Aim 1. Incorporating this photolabeling data in a molecular model of the receptor will define the geometry of the binding site(s). Aim 2 will also quantitatively analyze which GABA-A receptor subunits are photolabeled by neurosteroids in native brain tissue. In Aim 3, photolabeling will be used to identify additional proteins that have neurosteroid binding sites. Whole brain homogenates will be photolabeled with neurosteroid analogue photolabeling reagents and the labeled proteins will be identified either: 1) by using "click chemistry"-based enrichment of photolabeled proteins in conjunction with global proteomic methods or; 2) by using bifunctional fluorescent linkers to track the photolabeled proteins through electrophoretic separation, and then identifying the proteins using gel-based mass spectrometry. The proposed work is expected to identify the sites of neurosteroid binding on GABA-A receptors and to discover new neurosteroid binding proteins. These data will provide a structural template for future synthetic chemistry and suggest novel mechanisms of neurosteroid action and potential pharmacological targets.

项目描述 神经类固醇类似物通过与 GABA-A 受体结合并增强全身麻醉作用 GABA-A 受体上神经类固醇的结合位点数量和大脑中的神经元抑制。 它们的分子定位尚未完全确定，并且基于间接方法。 也是大脑发育和功能的内源性介质，其作用部位仍不清楚。 该项目将使用光标记技术来定义 GABA-A 上神经类固醇结合的精确位点 将开发新型“点击化学”标记的神经类固醇类似物光标记试剂。 并与同位素标记的“点击化学”双功能接头结合使用，以纯化光标记的 蛋白质和肽，提供使用高分辨率质谱分析的灵敏度和特异性 这些技术将检测和测序生物样品中的低丰度光标记肽。 也可用作发现工具来识别大脑中具有神经类固醇结合的其他蛋白质 该项目有三个具体目标： 在目标 1 中，新型神经类固醇类似物光标记试剂含有炔基和 含有叠氮基团和亲和标签的同位素标记的双功能连接分子将被 合成用于基于“点击化学”的光标记蛋白质的可视化和纯化 同位素标签将为特定的质谱检测提供“签名”。 将合成立体化学不同的神经类固醇类似物， 光标记机制以及二氮丙啶光标记部分在神经类固醇上的位置 在目标 2 中，确定 GABA-A 上神经类固醇结合位点的数量和结构。 受体 (¿1¿3) 将通过目标 1 中制备的神经类固醇类似物进行光标记来评估。 将这些光标记数据纳入受体的分子模型中将定义受体的几何形状 目标 2 还将定量分析哪些 GABA-A 受体亚基被光标记。 在目标 3 中，将使用光标记来识别天然脑组织中的神经类固醇。 具有神经类固醇结合位点。全脑匀浆将用神经类固醇类似物进行光标记。 光标记试剂和标记的蛋白质将通过以下任一方式进行识别：1) 通过使用基于“点击化学”的 结合全局蛋白质组学方法富集光标记蛋白质，或者； 双功能荧光接头通过电泳分离追踪光标记蛋白质，以及 然后使用凝胶质谱法鉴定蛋白质。 拟议的工作预计将确定神经类固醇在 GABA-A 受体上的结合位点，并 发现新的神经类固醇结合蛋白将为未来的合成提供结构模板。 化学并提出神经类固醇作用的新机制和潜在的药理学靶点。