Structure and mechanism of the mitochondrial ATP synthase and Batten Disease gene product, Cln3p

线粒体 ATP 合酶和巴顿病基因产物 Cln3p 的结构和机制

• 批准号: 10388683
• 负责人: David Michael Mueller
• 金额: $ 4.92万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388683
• 关键词: 18 year old; Adolescent; Agreement; Authorization documentation; Award; Binding; Biochemistry; Budgets; CLN3 gene; Cell Extracts; Cells; Conflict (Psychology); Contracts; Cryoelectron Microscopy; Crystallization; Crystallography; Data; Direct Costs; Disease; Drug Targeting; Ensure; Environment; Enzymes; Eukaryotic Cell; Floor; Fractionation; Funding; Genes; Genetic Diseases; Goals; Grant; Health; Homologous Gene; Human; Human Resources; Hydrophobicity; Institutes; Integral Membrane Protein; Investigation; Laboratories; Link; Lysosomes; Maintenance; Membrane; Membrane Proteins; Mitochondria; Mitochondrial Diseases; Mitochondrial Proton-Translocating ATPases; Modeling; National Institute of General Medical Sciences; Neurodegenerative Disorders; Notification; Open Reading Frames; Pharmaceutical Preparations; Postdoctoral Fellow; Preparation; Process; Productivity; Property; Proteins; Reaction; Reactive Oxygen Species; Regulation; Reporting; Research; Research Personnel; Resolution; Running; Scheme; Services; Spielmeyer-Vogt Disease; Statistical Data Interpretation; Structure; System; TimeLine; Training; United States National Institutes of Health; Universities; Vacuole; Variant; Work; X ray diffraction analysis; Yeasts; base; cost; design; drug discovery; experimental study; falls; gene product; inhibitor/antagonist; instrument; instrumentation; member; overexpression; prevent; professor; protein function; safety net; small molecule

Research Strategy Abstract An estimated 20-30% of all open reading frames in the eukaryotic cell encode a membrane protein and an estimated 60% of all drugs bind to a membrane protein. Not surprisingly, statistical analysis of the current drugs indicate that hydrophobicity is one of 8 properties that is common in a drug target. However, the number of high-resolution structures of membrane proteins is relatively small and the number of structures of hydrophobic molecules bound to membrane proteins is even smaller. This proposal has 2 disparate projects whose only commonality is that the proteins under investigation are membrane proteins and involved in critical functions in the cell. The first project entails the yeast mitochondrial ATP synthase of which we have solved the first near atomic model of the entire monomeric enzyme and we did this, with and without inhibitors bound to the membrane embedded, Fo domain. We propose to expand these studies to provide structures of the ATP synthase inhibited or trapped in various reaction intermediates. Our approach is unique in that we have linked 2 subunits of the ATP synthase and this allows us to capture intermediates with the rotor in a twisted state. As such, our approach allows for the capture of reaction intermediates. The use of inhibitors will serve to trap intermediates and identify their mode of binding. The second project is studies on yeast Yhc3p, a homologue of human Cln3p, which is in humans, is defective in the juvenile neurodegenerative disease, Batten. While, Cln3 was reported in 1995 as the gene defective for the juvenile form of Batten disease, the function of this protein is still unknown. Based on homology and other data, we hypothesize that Cln3p is a small molecule transporter whose function is tied to the formation of oxygen radicals. We have evidence that the yeast gene, YHC3, is tightly regulated and believe that by identifying the regulation, we will understand its importance in the cell. Lastly, we have developed an over-expression system and purification scheme for Yhc3p, which we will use to study the function and start crystallization trials for x-ray diffraction studies. While disparate, these projects fall well within our expertise and will add to the base necessary for the understanding of membrane proteins in health, disease, and as targets for drug discovery.

研究策略 抽象的 估计真核细胞中所有开放式阅读框的20-30％编码膜蛋白和一个 估计所有药物中有60％与膜蛋白结合。毫不奇怪，对电流的统计分析 药物表明疏水性是在药物靶标中常见的8种特性之一。但是， 膜蛋白的高分辨率结构数量相对较少，结构数量 与膜蛋白结合的疏水分子甚至更小。该提议有2个不同的 唯一共同点的项目是正在研究的蛋白质是膜蛋白和 参与细胞中的关键功能。第一个项目需要酵母线粒体ATP合酶 我们已经解决了整个单体酶的第一个近原子模型，我们做到了这一点 并且没有结合到膜嵌入的FO结构域的抑制剂。我们建议扩大这些 提供ATP合酶结构的研究抑制或捕获在各种反应中间体中。 我们的方法是独一无二的，因为我们已经链接了ATP合酶的2个亚基，这使我们能够 捕获与转子处于扭曲状态的中间体。因此，我们的方法允许捕获 反应中间体。使用抑制剂将有助于捕获中间体并确定其模式 结合。第二个项目是研究酵母YHC3P，这是人类CLN3P的同源物，在人类中， 在少年神经退行性疾病中有缺陷。而1995年据报道CLN3为 基因缺乏淡淡疾病的少年形式，该蛋白质的功能仍然未知。基于 关于同源性和其他数据，我们假设CLN3P是一个小分子转运蛋白，其功能是 与氧自由基的形成有关。我们有证据表明酵母基因YHC3受到严格调节 并相信，通过确定法规，我们将了解其在细胞中的重要性。最后，我们有 为YHC3P开发了一种过表达的系统和纯化方案，我们将用于研究 用于X射线衍射研究的功能并开始结晶试验。虽然不同，但这些项目却很好 在我们的专业知识中，将增加理解膜蛋白所必需的基础 健康，疾病和作为药物发现的靶标。