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

遗传物质（DNA或RNA）的转移通常从一代传递到下一代。王室是以特定和受控的方式聚集的蛋白质，仅通过该聚合蛋白质因子的转移而引起可遗传的特征。王室是传染病，至少在动物中会引起疾病。由于许多原因，这种王室现象具有巨大的科学兴趣。聚集是蛋白质的基本特性，所有蛋白质都可以做到，尤其是当它们受损时。然而，王室的聚集是自我传播的。 Prion骨料能够募集正常蛋白质，改变其形状并将其迫使其成新聚集体。近年来，由于牛的牛海绵状脑病流行，以及随后在英国人口中的变异creutzfeldt-jakob病出现，因此对王室进行了深入研究。但是，这些基于蛋白质的遗传性状不仅限于动物，而且在酿酒酵母的面包师酵母糖酵母中发现了许多prion。了解prion现象的工作方式的基本分子细节已被证明是困难的，至少部分是因为没有有关prion骨聚集体形状和结构的详细信息，或者在单个蛋白质聚集时如何改变形状。酵母中的prion特征的存在提供了令人兴奋的科学机会，因为酵母比小鼠等动物模型系统更容易在实验上操纵。酵母蛋白更容易分离，并以结构分析所需的大量数量产生。这些蛋白质的变化更容易做出，从而使分子的不同部分对自我传播聚集的过程做出了贡献。因此，所取得的研究进度更快，更具成本效益。因此，酵母菌是研究基于王室疾病的分子基础的理想模型系统。 URE2P是一种酵母蛋白，通常可以帮助酵母调节它们如何从环境中使用营养。但是，URE2P显示出类似prion的行为，因为它可以从其正常的活性形式转换为丢失正常函数的无效纤维。在我们的初步工作中，我们使用了低分辨率的ure2p纤维，并使用冷冻电子显微镜和图像处理以低分辨率解决了其3D结构。现在，我们希望显着改善该结构的分辨率。我们还将解决URE2P较小组件的结构，该组件似乎是制造纤维的构件。这将使我们能够研究可溶性形式的已知结构如何拟合到纤维和组装中间体的结构中，这反过来又有助于我们理解URE2P的可溶形式必须改变其形状以将其形状结合到prion纤维中。因此