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 神经类似类似物通过与GABA-A受体结合并增强产生全身麻醉 大脑中的神经元抑制作用。 GABA-A受体上神经类固醇的结合位点的数量和 它们的分子定位未完全定义，并基于间接方法。神经类似 也是脑发育和功能的内源性介体，其作用部位仍然不确定。 该项目将使用光标记技术来定义神经类固醇在GABA-A上的精确位点 受体。新颖的“点击化学”标记，神经类固醇模拟光标记试剂将被开发 并与同位素标记的“单击化学”双功能连接器一起使用，以纯化光标记 蛋白质和胡椒粉，提供了使用高分辨率质谱的灵敏度和特异性 在生物学样品中检测和序列低估光片的辣椒。这些技术会 还可以用作发现具有神经类固醇结合的大脑中其他蛋白质的发现工具 站点。该项目具有三个特定的目标： 在AIM 1中，新型的神经类固醇模拟光标记试剂，其中包含炔烃和 同位素标记的双功能连接器分子包含叠氮化物组和亲和力标签 合成用于“点击化学”的可视化和光贴蛋白的纯化和纯化 肽（目标2和3）。同位素标签将为特定的质谱检测提供“签名” 神经类似类似物将被合成，在立体化学上不同， 光标记的机理和重氮嗪光标记部分在神经类固醇上的位置 骨干。在AIM 2中，神经类固醇结合位点的数量和结构在定义的GABA-A上 受体（€1�3）将通过在AIM 1中制备的神经类似类似物来评估。 将此光标记数据纳入接收器的分子模型将定义 绑定位点。 AIM 2还将定量分析哪些GABA-A受体亚基由 天然脑组织中的神经类似。在AIM 3中，将使用光标记来识别其他蛋白质 具有神经类固醇结合位点。整个脑匀浆将用神经类似于神经类似 光标记试剂和标记的蛋白质将被识别：1）使用基于“单击化学”的 将光标记蛋白与全球蛋白质组学方法结合富集； 2）使用 双功能荧光接头，通过电泳分离跟踪光标记蛋白，并 然后使用基于凝胶的质谱法识别蛋白质。 提出的工作有望鉴定神经类固醇对GABA-A受体的结合和与 发现新的神经类固醇结合蛋白。这些数据将为将来的合成提供结构模板 化学并提出了神经类固醇作用和潜在药物靶标的新型机制。