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

遗传特征通常通过遗传物质(DNA或RNA)的转移从一代传到下一代。Prion是以特定和受控的方式聚集的蛋白质，仅通过这种聚集的蛋白质因子的转移就可以导致可遗传的特征。普恩是一种感染性蛋白质，至少在动物身上是这样，它可以引起疾病。普里恩现象具有巨大的科学价值，原因有很多。聚集是蛋白质的一个基本属性，所有蛋白质都会聚集，特别是当它们被破坏时。然而，Prion的聚集是自我传播的。Prion聚集体能够招募正常蛋白质，改变其形状，并迫使其形成新的聚集体。近年来，由于牛海绵状脑病在牛中的流行，以及随后在英国人口中出现的变异型克雅氏病，Prion得到了广泛的研究。然而，这些基于蛋白质的遗传特征并不局限于动物，在面包酵母中已经发现了一些普鲁恩。事实证明，理解Pron现象如何工作的基本分子细节是困难的，至少在一定程度上是因为没有关于Pron聚集体的形状和结构的详细信息，或者关于单个蛋白质在聚集时如何改变形状的详细信息。由于酵母菌比小鼠等动物模型系统更容易在实验中操作，因此在酵母中发现了Pron特征，这提供了一个令人兴奋的科学机会。酵母蛋白更容易被分离和大量生产，这是结构分析所需的。这些蛋白质的变化更容易进行，从而使分子的不同部分对自我传播聚集过程的贡献得以理解。因此，取得的研究进展更快、更具成本效益。因此，酵母蛋白是研究基于蛋白的疾病的分子基础的理想模型系统。Ure2p是一种酵母蛋白，正常情况下可以帮助酵母调节如何利用环境中的营养。然而，Ure2p表现出类似普里恩的行为，因为它可以从正常的、活跃的形式转化为不活跃的纤维，在这些纤维中，正常的功能丧失了。在我们的初步工作中，我们已经生长了Ure2p纤维，并利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三维结构。我们现在希望极大地提高这一结构的分辨率。我们还将解决较小的Ure2p组件的结构，它似乎是制造纤维的积木。这将使我们能够看到已知的可溶性形式的结构如何适应纤维和组装中间体的结构，这反过来将帮助我们理解Ure2p的可溶性形式必须改变其形状以并入普里恩纤维的方式。因此，我们的实验计划将提供有关Pron自蔓延聚集的分子基础的基本信息

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