MOLECULAR ANALYSIS OF YEAST PRIONS

感染性蛋白质（朊病毒），最初被假定用于解释一组可传播的海绵状病毒， 目前已知真菌（包括酵母[PSI+]和[URE 3]状态）和高等生物体中的许多表观遗传元件是TSE的基础。虽然哺乳动物PrP和真菌朊病毒蛋白在氨基酸序列上是不相关的，但它们的朊病毒状态具有共同的结构特征，这使它们成为一类错误折叠的蛋白质（淀粉样蛋白），导致广泛的非感染性疾病。酵母的简单遗传学，以及从纯物质中重新产生具有感染性形式的蛋白质的能力，极大地帮助我们理解了朊病毒遗传的原理以及细胞因子在促进和抑制朊病毒复制中的作用。还有很多东西需要了解， 细胞辅助因子的特殊作用。尽管如此，哺乳动物和酵母朊病毒现象之间的机制相似点构象为基础的感染和遗传的普遍特征，涉及有序的β折叠丰富的淀粉样蛋白聚集体的繁殖。这些现象包括普遍存在的传递障碍，其抑制甚至密切相关的朊病毒蛋白之间的传递，以及朊病毒菌株，其中由相同蛋白组成的感染性颗粒引起一系列不同的可遗传朊病毒状态。本项目的目标是利用酵母系统的简易性以及在体外重建[PSI+]朊病毒形成的能力，阐明朊病毒生物学在结构、生物物理和生物化学性质方面的这些普遍特征的机制，这些特征是富含β-折叠的蛋白质聚集体和作用于这些物种的细胞因子。最后，我们预计，我们在研究蛋白质错误折叠成朊病毒构象的机制中所取得的许多方法、实验和理论上的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，我们将重点研究以下三个具体目标：（1）朊病毒的生长和复制机制。我们正在定义Sup 35朊病毒纤维捕获和催化Sup 35正常可溶形式转化的机制。我们也将重建和机械分析的过程中，细胞因子，如分子伴侣切断预制Sup 35纤维，从而允许复制的朊病毒元件。（二） 朊病毒元件的解剖和设计。我们将扩展解剖方法，我们成功地定义了Sup 35和New 1朊病毒结构域的模块化架构，以探索其他已知的真菌朊病毒，包括Ure 2，Rnq 1和Het-S。然后，我们将使用这些研究阐明的原理在酵母中创建新的朊病毒元件，包括基于Abeta，α-突触核蛋白和哺乳动物PrP的元件。(3)朊病毒株的结构基础分析。我们将使用一种综合的方法，结合冷冻电子显微镜（cryo-EM），定点自旋标记（SDSL），结合电子顺磁共振（ESR）， 共振（EPR）光谱与建模和突变研究一起来定义导致不同朊病毒株的四级和/或三级结构差异。

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.