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: 10220997
• 负责人: David Michael Mueller
• 金额: $ 42.7万
• 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/10220997
• 关键词: ATP Synthesis Pathway; Adolescent; Binding; CLN3 gene; Cells; Coupling; Crystallization; Data; Disease; Drug Targeting; Enzymes; Eukaryotic Cell; Genes; Goals; Health; Homologous Gene; Human; Hydrophobicity; Investigation; Ions; Link; Membrane; Membrane Proteins; Mitochondrial Proton-Translocating ATPases; Modeling; Neurodegenerative Disorders; Open Reading Frames; Pharmaceutical Preparations; Property; Proteins; Reaction; Reactive Oxygen Species; Regulation; Reporting; Resolution; Scheme; Spielmeyer-Vogt Disease; Statistical Data Interpretation; Structural Protein; Structure; System; X ray diffraction analysis; Yeasts; base; drug discovery; falls; gene product; inhibitor/antagonist; overexpression; protein function; small molecule

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. !

真核细胞中所有开放阅读框的估计20-30%编码膜蛋白和膜蛋白。 估计60%的药物与膜蛋白结合。毫不奇怪，目前的统计分析显示， 药物表明疏水性是药物靶标中常见的8种性质之一。但 膜蛋白的高分辨率结构的数量相对较少， 疏水分子与膜蛋白结合的比例甚至更小。该提案有两个不同的 这些项目的唯一共同点是，所研究的蛋白质是膜蛋白， 参与细胞的关键功能。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， 而没有抑制剂结合到膜嵌入的Fo结构域。我们建议扩大这些 研究提供ATP合酶的结构抑制或捕获在各种反应中间体。 我们的方法是独特的，因为我们连接了ATP合酶的2个亚基，这使我们能够 在转子处于扭转状态的情况下捕获中间体。因此，我们的方法允许捕获 反应中间体抑制剂的使用将用于捕获中间体并确定其降解模式。 约束力第二个项目是对酵母Yhc 3 p的研究，Yhc 3 p是人类Cln 3 p的同源物，人类Cln 3 p存在于人类中， 在青少年神经退行性疾病中有缺陷巴顿而Cln 3在1995年被报道为 由于Batten病幼年型的基因缺陷，这种蛋白质的功能仍然未知。基于 根据同源性和其他数据，我们假设Cln 3 p是一种小分子转运蛋白，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YHC 3受到严格的调控， 并相信通过识别调控，我们将了解其在细胞中的重要性。最后，我们有 建立了Yhc 3 p的过表达系统和纯化方案，我们将用它来研究Yhc 3 p的表达。 功能和开始结晶试验，用于X射线衍射研究。尽管这些项目各不相同， 在我们的专业知识，并将增加必要的基础上了解膜蛋白， 健康，疾病，以及作为药物发现的目标。!