MOLECULAR SITES OF NEUROSTEROID BINDING

项目说明 神经类固醇类似物通过与GABA-A受体结合并增强 大脑中的神经元抑制。神经类固醇在GABA-A受体上的结合位点数和 它们的分子定位还不完全确定，主要基于间接方法。神经类固醇 也是大脑发育和功能的内源性调节因子，其作用部位尚不确定。 该项目将使用光标记技术来确定神经类固醇与GABA-A结合的精确位置 感受器。新型“点击化学”标记的神经类固醇类似物光标记试剂将被开发 并与同位素标记的、“点击化学”双功能连接物一起使用，以纯化光标记物 蛋白质和多肽，提供了使用高分辨率质谱仪进行 检测生物样品中的低丰度光标记肽并对其进行排序。这些技术将 也可以作为一种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 网站。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 同时包含叠氮基和亲和标记的同位素标记的双官能团连接物分子将是 合成用于基于“点击化学”的光标记蛋白质的可视化和纯化 多肽(目标2和3)。同位素标记物将为特定的质谱学检测提供“签名” 光标记的多肽。神经类固醇类似物将被合成在立体化学上不同的， 神经类固醇的光标记机理及二氮杂光标记部分的定位 我的脊梁。在目标2中，已定义的GABA-A上的神经类固醇结合位点的数目和结构 受体(？1？3)将通过在AIM 1中制备的神经类固醇类似物的光标记进行评估。 将这种光标记数据结合到受体的分子模型中将定义 结合部位(S)。AIM 2还将定量分析哪些GABA-A受体亚单位被光标记 天然脑组织中的神经类固醇。在目标3中，光标记将被用来识别更多的蛋白质 有神经类固醇结合部位。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被鉴定为：1)使用基于点击的化学 结合全球蛋白质组学方法或2)通过使用 通过电泳法分离跟踪光标记蛋白的双功能荧光连接物，以及 然后用基于凝胶的质谱学鉴定蛋白质。 这项拟议的工作有望确定神经类固醇与GABA-A受体的结合部位，并 发现新的神经类固醇结合蛋白。这些数据将为未来的合成提供结构模板 并提出了神经类固醇作用的新机制和潜在的药理靶点。

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.