Molecular Targets of Neurosteroid Anti-depressant Action

项目说明 神经活性类固醇(NAS)是神经元兴奋性的重要调节剂，已被证明具有治疗作用 作为麻醉剂和快速起效的抗抑郁药的功效。而NAS的麻醉作用是通过 通过GABAA受体，其抗抑郁作用的机制(S)尚不清楚。NAS功能 调节NMDA受体的活性，并特异性地结合几种可能有助于 他们的抗抑郁作用。值得注意的是，NAS也定位于跨高尔基膜，这表明它们是 或者选择性地运输到高尔基体，或者在高尔基体中具有丰富的选择性结合伙伴。这样做的目的是 项目是表征可能的抗抑郁蛋白靶标上的NAS结合位点，并识别新的 细胞内NAS结合蛋白可能与NAS的抗抑郁作用有关。要实现这些目标 目标，我们将利用NAS模拟光亲和标记与最先进的蛋白质化学和 表达技术和尖端的质谱仪(MS)方法。埃弗斯实验室已经研发出 这些方法并成功地应用于描绘NAS与VDAC、β结合的分子细节。 GABAA受体上的微管蛋白和异构体特异性位点。用于该项目的新型光亲和标记试剂将 在化学核心中合成。我们标签研究的结果将是识别新的NAS NAS结合位点中的结合蛋白和突变，使这些靶标的作用得以表征 NAS抗抑郁生理和行为效应中的蛋白质(项目2和3)。该项目有 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(NAMS和PAM)。GluN1/GluN2B NMDA受体将被标记 对于NAM和PAM，NAS光亲和性类似物和每个亚基上的标记位点数将是 用完整的蛋白质MS测定，经过光标记试剂修饰的特定氨基酸将是 使用中下层MS鉴定分子模型将与光标记数据结合使用， 预测NAS结合或作用所必需的关键残基。在Aim 2A中，我们将执行 无偏搜索以确定完整海马神经元中NAS结合蛋白的“蛋白质组” 用“点击化学”标记的亲和标记试剂来丰富结合蛋白，然后以LC-MS/MS为基础 蛋白质鉴定。在Aim 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/运输NAS的蛋白质，并可能改变细胞器膜和 调节细胞应激反应。已确定的NAS结合的生理和行为效应 参与细胞应激反应通路(AIM 2A)和NAS/脂质运输(AIM 2B)的蛋白质将是 使用CRISPR淘汰法进行评估(项目2和3)。

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).