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: 9980949
• 负责人: David Michael Mueller
• 金额: $ 42.55万
• 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/9980949
• 关键词: ATP Synthesis Pathway; Adolescent; Binding; 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. !

据估计，真核细胞中 20-30% 的开放阅读框编码膜蛋白和 据估计，60% 的药物与膜蛋白结合。毫不奇怪，对当前的统计分析 药物表明疏水性是药物靶点常见的 8 种特性之一。然而， 膜蛋白的高分辨率结构数量相对较少，且结构数量较少 与膜蛋白结合的疏水性分子的数量甚至更小。该提案有 2 个不同的 项目唯一的共同点是所研究的蛋白质是膜蛋白和 参与细胞的关键功能。第一个项目涉及酵母线粒体 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， 并且没有与嵌入膜的 Fo 结构域结合的抑制剂。我们建议扩大这些 研究提供了在各种反应中间体中被抑制或捕获的 ATP 合成酶的结构。 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 利用处于扭曲状态的转子捕获中间体。因此，我们的方法可以捕获 反应中间体。抑制剂的使用将有助于捕获中间体并确定它们的模式 绑定。第二个项目是对酵母 Yhc3p 的研究，它是人类 Cln3p 的同源物，存在于人类中， 患有青少年神经退行性疾病巴顿（Batten）有缺陷。而 Cln3 于 1995 年被报道为 幼年型巴顿病基因有缺陷，但该蛋白的功能仍不清楚。基于 根据同源性和其他数据，我们假设 Cln3p 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 YHC3 受到严格调控 并相信通过识别调控，我们将了解它在细胞中的重要性。最后，我们有 开发了 Yhc3p 的过表达系统和纯化方案，我们将用它来研究 功能并开始 X 射线衍射研究的结晶试验。这些项目虽然各不相同，但效果都很好 在我们的专业知识范围内，将为理解膜蛋白奠定必要的基础 健康、疾病以及药物发现的目标。 ！