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实验室研究神经固醇和胆固醇与整体膜的结合相互作用 蛋白质，目的是识别蛋白质功能立体声调制的特定结合事件。 我们的主要重点是氨基丁酸A型受体（GABAA-）的神经类固醇调节 R）。神经类固醇是神经元令人兴奋和神经系统发育的重要调节剂 作为麻醉药，抗抑郁药和神经保护剂的巨大治疗潜力。我们已经显示了 GABAA受体上的神经类固醇有多种亚基特异性结合位点 有助于神经类固醇的功能作用。在拟议的研究中，我们将使用光标记 定义主要类别神经类固醇与最丰富的精确位点的技术 突触和突触外GABAA受体的形式，并确定每个受体的功能意义 通过评估靶向氨基酸取代对NS调节GABAA-的影响，确定结合位点 R电流。为了确定光标记的结果，我们将利用最先进的蛋白质化学和表达 技术与尖端质谱法（MS）方法结合使用，包括中向下和 完整的蛋白质MS。将获得高分辨率低分辨率 - 电子显微镜结构，以识别 新型NS结合位点的原子细节，并研究似乎稳定的结合相互作用 当前结构未捕获的构象。然后，基于荧光的结合测定将用于 测量各种NS对已识别结合位点的位点特异性亲和力。这些测定将是 适应停止流量的荧光法以确定结合的状态依赖性和读取器的状态 格式以筛选特定地点激动剂和拮抗剂。我们的NS计划的长期目标是 开发和使用特定位点的NS配体来探测特定NS结合位点和GABAA-R亚型的作用 内源性NS的行为影响以及NS镇静剂和麻醉药的作用机理。 我们还使用了胆固醇 - 动物呈光标记来识别特定的结合位点 介导脂质cramblase，NHTMEM16和胆固醇调节的胆固醇抑制 MTOR1由溶酶体膜蛋白GPR155。 NHTMEM16和GPR155都有两个特定的 每个蛋白质单体胆固醇结合位点，我们使用靶向氨基酸取代 了解每个站点的功能作用。我们还正在开发基于荧光的结合测定法 测量这些位点的胆固醇亲和力和固醇特异性。新颖的胆固醇结合位点出现了新 膜蛋白功能的小分子变构调节剂的靶标和我们拥有的工具 开发的广泛适用于识别其他胆固醇调节蛋白质和 筛选特定于位点的配体。