MOLECULAR ANALYSIS OF YEAST PRIONS

感染性蛋白质(普恩)，最初被认为是为了解释一组可传播的海绵状 脑病(TSE)，现在已知是真菌(包括酵母[PSI+]和[URE3]状态)以及高等生物体中的一些表观遗传因素的基础。虽然哺乳动物PrP和真菌PrP蛋白在氨基酸序列上是不相关的，但它们的Prion状态具有共同的结构特征，使它们属于一类错误折叠的蛋白质(淀粉样蛋白)，导致广泛的非传染性疾病。酵母的简便遗传学，以及从纯物质中产生从头开始的、具有感染性的蛋白质的能力，极大地帮助了我们理解Pron遗传的原理以及细胞因子在促进和抑制Prion复制中的作用。关于这一点，还有很多需要了解的地方 细胞辅因子的特殊作用。尽管如此，哺乳动物和酵母蛋白现象之间的机制相似之处指向了基于构象的感染和遗传的普遍特征，涉及有序的富含β-折叠的淀粉样蛋白聚集体的繁殖。这些现象包括无处不在的传播障碍的存在，这阻碍了即使是密切相关的普恩蛋白之间的传输，以及普恩病毒株，其中由相同蛋白质组成的感染颗粒导致了一系列不同的可遗传的普恩状态。本项目的目标是利用酵母系统的易用性以及在体外重建[PSI+]Pron的能力，从富含β-折叠的蛋白质聚集体的结构、生物物理和生化特性以及作用于此类物种的细胞因素方面阐明Prion生物学的这些普遍特征的机制。最后，我们预计，我们在研究蛋白质错误折叠为PrP构象的机制方面取得的许多方法、实验和理论进展将直接适用于哺乳动物PrP的相关研究。为了实现上述目标，我们将重点关注以下三个具体目标：(1)Pron的生长和复制机制。我们正在确定Sup35蛋白纤维捕获和催化正常可溶形式Sup35转化的机制。我们还将重新构建和机械分析分子伴侣等细胞因子切断Sup35纤维的过程，从而允许Pron元件的复制。(2) Pron元件的剖析和设计。我们将扩展我们成功地定义Sup35和New1Prion结构域的解剖方法来探索其他已知的真菌Prion，包括Ure2，Rnq1和Het-S的那些。然后，我们将使用这些研究阐明的原理在酵母中创建新的Pron元件，包括基于Abeta、α-突触核蛋白和哺乳动物PrP的元件。(3)PrP菌株的结构基础分析。我们将使用一种综合的方法，结合低温电子显微镜(CRYO-EM)、定点自旋标记(SDSL)和电子顺磁 共振(EPR)波谱结合建模和突变研究，以确定导致不同Pron菌株的第四和/或第三级结构差异。

Infectious proteins (prions), originally postulated to explain a set of transmissible spongiform encephalopathies (TSEs), are now known to underlie a number of epigenetic elements in fungi (including the yeast [PSI+] and [URE3] states) and perhaps in higher organisms. Although mammalian PrP and fungal prion proteins are unrelated in amino acid sequence, their prion states share common structural features that place them in a class of misfolded proteins (amyloids) responsible for a broad range of noninfectious diseases. The facile genetics of yeast together with the ability to create de novo, infectious forms of proteins from pure material have greatly aided our understanding of the principles of prion inheritance as well as the role of cellular factors in facilitating and inhibiting prion replication. Much remains to be learned about the specific role of cellular cofactors. Nonetheless, mechanistic parallels between the mammalian and yeast prion phenomena point to universal features of conformation-based infection and inheritance involving propagation of ordered beta-sheet-rich amyloid protein aggregates. These phenomena include the ubiquitous presence of transmission barriers, which inhibit transmission between even closely related prion proteins, and prion strains wherein infectious particles composed of the same protein give rise to a range of different heritable prion states. The goal of the present project is to take advantage of the facile nature of yeast systems together with the ability to reconstitute [PSI+] prion formation in vitro to elucidate the mechanisms of these universal features of prion biology in terms of the structural, biophysical and biochemical properties of beta-sheet-rich protein aggregates and the cellular factors that act on such species. Finally, we anticipate that many of the methodological, experimental, and theoretical advances we are making in studying the mechanisms by which proteins misfold into prion conformations will be directly applicable to related studies on mammalian PrP. To accomplish the above goals, we will focus on the following three Specific Aims: (1) Mechanism of prion growth and replication. We are defining the mechanism by which Sup35 prion fibers capture and catalyze the conversion of normal soluble forms of Sup35. We will also reconstitute and mechanistically analyze the process by which cellular factors such as molecular chaperones sever preformed Sup35 fibers, thereby allowing replication of the prion element. (2) Dissection and design of prion elements. We will extend the dissection approach we used successfully to define the modular architecture of the Sup35 and New1 prion domains to explore other known fungal prions including those of Ure2, Rnq1 and Het-s. We will then use the principles elucidated by these studies to create novel prion elements in yeast including ones based on Abeta, alpha-synuclein and mammalian PrP. (3) Analysis of the structural basis of prion strains. We will use a comprehensive approach combining cryoelectron microscopy (cryo-EM), site-directed spin labeling (SDSL) in conjunction with electron paramagnetic resonance (EPR) spectroscopy together with modeling and mutational studies to define the quaternary and/or tertiary structural differences responsible for the different prion strains.