Neurosteroid and Cholesterol Binding to Integral Membrane Proteins

神经类固醇和胆固醇与整合膜蛋白的结合

• 批准号: 10623887
• 负责人: ALEX S. EVERS
• 金额: $ 55.28万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623887
• 关键词: Affinity; Agonist; Amino Acid Substitution; Aminobutyric Acids; Anesthetics; Antidepressive Agents; Behavioral; Binding; Binding Sites; Biological; Biological Assay; Cholesterol; Cryoelectron Microscopy; Dependence; Event; FRAP1 gene; Fluorescence; Goals; Integral Membrane Protein; Laboratory Study; Ligands; Lipids; Mass Spectrum Analysis; Measures; Mediating; Membrane Proteins; Methods; Molecular Conformation; Neuroprotective Agents; Protein Chemistry; Proteins; Reader; Research; Resolution; Role; Signaling Molecule; Signaling Protein; Site; Specificity; Sterols; Structure; Synapses; Techniques; Therapeutic; Work; antagonist; cholesterol analog; gamma-Aminobutyric Acid; insight; monomer; nervous system development; neuronal excitability; neurosteroids; novel; programs; protein expression; protein function; protein phosphatase inhibitor-2; receptor; screening; sedative; small molecule; tool

Project Summary The Evers laboratory studies the binding interactions of neurosteroids and cholesterol with integral membrane proteins, with the aim of identifying the specific binding events underlying sterol modulation of protein function. Our major focus is on neurosteroid (NS) modulation of -aminobutyric acid type A receptors (GABAA- R). Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anesthetics, anti-depressants and neuro-protectants. We have shown that there are multiple, subunit-specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the functional effects of neurosteroids. In the proposed research, we will use photolabeling techniques to define the precise sites at which the major classes of neurosteroids bind on the most abundant forms of synaptic and extra-synaptic GABAA receptors and determine the functional significance of each identified binding site by assessing the effect of targeted amino acid substitutions on NS modulation of GABAA- R currents. To identify photo-labeled residues we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down and intact protein MS. High-resolution cryogenic-electron microscopy structures will be obtained to identify the atomic details of novel NS binding sites and to investigate binding interactions that appear to stabilize conformations not captured in current structures. Fluorescence-based binding assays will then be used to measure the site-specific affinity of various NS for the identified binding sites. These assays will be adapted to stopped-flow fluorimetry to determine the state-dependence of binding and to a plate reader format to screen for site-specific agonists and antagonists. The long term goal of our NS program is to develop and use site-specific NS ligands to probe the role of specific NS binding sites and GABAA-R subtypes in the behavioral effects of endogenous NS and the mechanisms of action of NS sedatives and anesthetics. We have also used cholesterol-analogue photolabeling to identify specific binding sites that mediate cholesterol inhibition of the lipid scramblase, nhTMEM16, and cholesterol modulation of mTOR1 by the lysosomal membrane protein GPR155. Both nhTMEM16 and GPR155 have two specific cholesterol binding sites per protein monomer and we are using targeted amino acid substitution to understand the functional role of each site. We are also developing fluorescence-based binding assays to measure cholesterol affinity and sterol specificity for these sites. Novel cholesterol binding sites present new targets for small molecule allosteric modulators of membrane protein function and the tools we have developed are widely applicable to identifying binding sites on other cholesterol-modulated proteins and screening for site-specific ligands.

项目摘要 EVERS实验室研究神经类固醇和胆固醇与整体膜的结合作用 蛋白质，目的是确定固醇调节蛋白质功能的特定结合事件。 我们主要关注神经类固醇(NS)对-氨基丁酸A型受体(GABA-A-R)的调节作用。 r)。神经类固醇是神经元兴奋性和神经系统发育的重要调节剂 作为麻醉剂、抗抑郁剂和神经保护剂，具有巨大的治疗潜力。我们已经展示了 在GABAA受体上有多个亚基特异的神经类固醇结合位点，每一个都是 有助于神经类固醇的功能效应。在拟议的研究中，我们将使用光标记法 确定主要种类的神经类固醇与最丰富的 突触和突触外GABAA受体的形式及其功能意义 通过评估靶向氨基酸取代对GABAA-NS调制的影响来确定结合部位 R电流。为了鉴定光标记残基，我们将利用最先进的蛋白质化学和表达 与尖端质谱学(MS)方法相结合的技术，包括中下和 完整的蛋白质MS将获得高分辨率的低温电子显微镜结构来鉴定 新的NS结合位点的原子细节并研究似乎稳定的结合相互作用 当前结构中没有捕捉到的构象。然后将使用基于荧光的结合分析来 测量不同NS对已鉴定结合位点的特异性亲和力。这些化验结果将是 适用于停流荧光法以确定结合的状态依赖性和平板阅读器 筛选特定部位的激动剂和拮抗剂的格式。我们NS计划的长期目标是 开发和使用特定位点的NS配体来探索特定的NS结合位点和GABAA-R亚型的作用 内源性NS的行为效应以及NS镇静剂和麻醉药的作用机制。 我们还使用胆固醇类似物光标记来识别特定的结合部位 介导胆固醇抑制脂质混杂酶，nhTMEM16，和胆固醇调节 MTOR1由溶酶体膜蛋白GPR155表达。NhTMEM16和GPR155都有两个特定的 每个蛋白质单体的胆固醇结合位点，我们正在使用定向氨基酸替代来 了解每个站点的功能角色。我们还在开发基于荧光的结合分析，以 测量这些部位的胆固醇亲和力和固醇专一性。新的胆固醇结合部位呈现新的 膜蛋白功能小分子变构调节剂的靶点和我们拥有的工具 已开发的方法广泛适用于识别其他胆固醇调节蛋白上的结合位点和 筛选特定位点的配体。