Structure-function Analysis of Bestrophins

黄斑黄蛋白的结构功能分析

• 批准号: 8867696
• 负责人: Tingting Yang
• 金额: $ 10.58万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8867696
• 关键词: Adult; Basic Science; Binding; Biological; Biological Assay; Blindness; C-terminal; Cell membrane; Cells; Chloride Channels; Chloride Ion; Chlorides; Crystallization; Crystallography; Defect; Disease; Drosophila melanogaster; Electrophysiology (science); Elements; Event; Eye; Eye diseases; Flowers; Functional disorder; Future; Genes; Goals; Heart; Homologous Gene; Homologous Protein; Human; In Vitro; Ion Channel; Ions; Klebsiella pneumonia bacterium; Knowledge; Light; Link; Lipid Bilayers; Mediating; Mentors; Methods; Modeling; Molecular; Muscle Contraction; Mutation; Pathology; Pathway interactions; Phase; Physiological; Physiology; Play; Proteins; Radial; Research; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Role; Shapes; Site-Directed Mutagenesis; Structural Models; Structure; Testing; Therapeutic; Transmembrane Domain; Vitelliform macular dystrophy; Work; base; career; design; disease-causing mutation; inhibitor/antagonist; insight; mutant; novel; patch clamp; protein expression; public health relevance; research study; trafficking

 DESCRIPTION (provided by applicant): Many physiological events, such as heart beat and muscle contraction, are orchestrated by the cellular concentrations of certain ions, which flow in and out of the cell through designated structures on the cell membrane called ion channels. Dysregulation of ion channel/flux is clinically linked to many diseases. The human bestrophin-1 (hBest1) is a chloride (Cl-) conducting channel highly expressed in eyes. So far, over 120 distinct mutations of hBest1 have been identified to associate with multiple kinds of eye diseases that cause vision loss. Although many hBest1 disease-causing mutants have been documented as defective Cl- channels, how this defect pathologically results in eye disorders remains elusive. Moreover, it is largely unclear how the Cl- channel function of hBest1 is regulated/activated. Therefore, it is of both biological and biomedical significance to thoroughly understand the structure and function of hBest1. The goal of this proposal is to elucidate the working mechanisms of hBest1 by crystallography and electrophysiology from the structural and functional aspects, respectively. Importantly, a recently obtained structure model of a bacterial bestrophin homolog (KpBest, from Klebsiella pneumoniae) provides novel clues and a structural basis for the research aims in this proposal. To be specific, in Aim 1, the two putative ion permeation gates on bestrophins, as suggested by the KpBest structure model, will be examined in both KpBest and hBest1; in Aim 2, the hypothesis that ATP directly interacts and activates bestrophins will be tested by co-crystallizing ATP and KpBest, and further functionally examined with both KpBest and hBest1; in Aim 3, the crystal structure of eukaryotic bestrophin(s) and/or hBest1 will be obtained using the KpBest structure as a search model. During the K99 phase (Aim 1, and parts of Aims 2 and 3), I will be mentored by Dr. Wayne Hendrickson, a leader in the field of protein crystallography. This work will shed new light on how disease-causing hBest1 mutations influence the structure and function of the channel to contribute to eye diseases, and provide valuable insight for structure-based specific channel activator/inhibitor design in the future.

 描述（由申请人提供）：许多生理事件，如心跳和肌肉收缩，是由某些离子的细胞浓度协调的，这些离子通过细胞膜上称为离子通道的指定结构流入和流出细胞。离子通道/通量的失调在临床上与许多疾病相关。人脑啡肽-1（hBest 1）是一种在眼睛中高度表达的氯离子（Cl-）传导通道。到目前为止，已经确定了超过120种不同的hBest 1突变与导致视力丧失的多种眼科疾病有关。虽然许多hBest 1致病突变体已被证明是有缺陷的Cl-通道，但这种缺陷如何在病理上导致眼部疾病仍然是难以捉摸的。此外，很大程度上还不清楚hBest 1的Cl-通道功能是如何调节/激活的。因此，深入研究hBest 1的结构和功能具有重要的生物学和生物医学意义。 本研究的目的是通过晶体学和电生理学分别从结构和功能方面阐明hBest 1的工作机制。重要的是，最近获得的细菌雌激素同系物（KpBest，从肺炎克雷伯氏菌）的结构模型提供了新的线索和结构基础的研究目标，在这个建议。具体而言，在目标1中，将在KpBest和hBest 1中检验KpBest结构模型所建议的两个推定的雌激素离子渗透门;在目标2中，将通过共结晶ATP和KpBest来检验ATP直接相互作用并激活雌激素的假设，并进一步用KpBest和hBest 1检验功能;在目的3中，将使用KpBest结构作为搜索模型获得真核雌激素和/或hBest 1的晶体结构。 在K99阶段（目标1，以及目标2和3的一部分），我将由蛋白质晶体学领域的领导者韦恩亨德里克森博士指导。这项工作将揭示致病hBest 1突变如何影响通道的结构和功能，从而导致眼部疾病，并为未来基于结构的特异性通道激活剂/抑制剂设计提供有价值的见解。