Defining the molecular pathway for yeast prion fibril assembly using cryo-electron microscopy

使用冷冻电子显微镜定义酵母朊病毒原纤维组装的分子途径

• 批准号: BB/E01433X/1
• 负责人: Neil Ranson
• 金额: $ 48.57万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE01433X%2F1
• 关键词: Defining; molecular; pathway; yeast; prion

Inherited traits are normally passed from one generation to the next by the transfer of genetic material (DNA or RNA). Prions are proteins that aggregate in a specific and controlled manner, and that can cause inheritable traits solely by the transfer of this aggregated protein factor. Prions are infectious proteins that, at least in animals, can cause disease. The prion phenomenon is of huge scientific interest for many reasons. Aggregation is a fundamental property of proteins and all proteins do it, especially when they are damaged. The aggregation of prions however is self-propagating. Prion aggregates are able to recruit normal protein, change its shape and force it into new aggregates. Prions have been the studied intensively in recent years because of the bovine spongiform encephalopathy epidemic in cattle, and the subsequent appearance of variant Creutzfeldt-Jakob disease in the UK's human population. However, these protein-based inheritable traits are not confined to animals, and a number of prions have been found in the bakers yeast Saccharomyces cerevisiae. Understanding the basic molecular details of how the prion phenomenon works has proved difficult, at least in part because there is no detailed information on the shape and structure of prion aggregates, or on how the individual proteins change shape as they aggregate. The presence of prion traits in yeast provides an exciting scientific opportunity, as yeasts are very much easier to manipulate experimentally than animal model systems such as mice. Yeast proteins are more readily isolated and produced in the large quantities required for structural analysis. Changes in those proteins are more easily made, allowing the contribution that the different parts of the molecule make to the process of self-propagating aggregation to be understood. The research progress made is therefore faster and more cost-effective. Yeast prions are therefore an ideal model system to study the molecular basis of prion-based disorders. Ure2p is a yeast protein that normally functions to help yeast regulate how they use nutrients from their environment. However, Ure2p shows prion-like behaviour in that it can convert from its normal, active form, into inactive fibres in which the normal function is lost. In our preliminary work, we have grown Ure2p fibres and solved their 3D structure at low resolution using cryo-electron microscopy and image processing. We now wish to dramatically improve the resolution of this structure. We will also solve the structure of a smaller assembly of Ure2p which appears to be the building block from which the fibres are made. This will enable us to look at how the known structure of the soluble form fits into the structure of both the fibres and the assembly intermediate, which will in turn help us to understand the ways in which the soluble form of Ure2p must change its shape to be incorporated into a prion fibre. Our programme of experiments will therefore provide fundamental information on the molecular basis of self-propagating aggregation by prions

遗传性状通常通过遗传物质（DNA或RNA）的传递从一代传递到下一代。朊病毒是一种以特定和受控方式聚集的蛋白质，仅通过这种聚集的蛋白质因子的转移就可以引起遗传性状。朊病毒是一种传染性蛋白质，至少在动物中，可以引起疾病。由于许多原因，朊病毒现象引起了巨大的科学兴趣。聚集是蛋白质的基本特性，所有的蛋白质都会聚集，尤其是当它们被破坏的时候。然而，朊病毒的聚集是自我繁殖的。朊病毒聚集体能够吸收正常蛋白质，改变其形状并迫使其形成新的聚集体。近年来，由于牛海绵状脑病的流行，以及随后在英国人群中出现的变异型克雅氏病，朊病毒得到了深入的研究。然而，这些以蛋白质为基础的遗传特征并不局限于动物，并且在面包酵母中发现了许多朊病毒。了解朊病毒现象如何运作的基本分子细节已被证明是困难的，至少部分原因是没有关于朊病毒聚集体的形状和结构的详细信息，也没有关于单个蛋白质在聚集体时如何改变形状的详细信息。酵母中朊病毒特征的存在提供了一个令人兴奋的科学机会，因为酵母比动物模型系统（如小鼠）更容易在实验中操作。酵母蛋白更容易分离和大量生产用于结构分析。这些蛋白质的变化更容易产生，从而使分子的不同部分对自我繁殖聚集过程的贡献得以理解。因此，研究进展更快，成本效益更高。因此，酵母朊病毒是研究基于朊病毒的疾病分子基础的理想模型系统。Ure2p是一种酵母蛋白，通常帮助酵母调节它们如何利用环境中的营养物质。然而，Ure2p表现出类似朊病毒的行为，它可以从正常的活性形式转化为失去正常功能的非活性纤维。在我们的初步工作中，我们已经培养了Ure2p纤维，并使用低温电子显微镜和图像处理技术在低分辨率下解决了它们的3D结构。我们现在希望大大提高这一结构的分辨率。我们还将解决一个较小的Ure2p组件的结构，它似乎是制造纤维的基石。这将使我们能够了解已知的可溶形式的结构如何适应纤维和组装中间体的结构，这将反过来帮助我们了解可溶形式的Ure2p必须改变其形状以并入朊病毒纤维的方式。因此，我们的实验计划将提供有关朊病毒自我繁殖聚集的分子基础的基本信息