Mechanism of prion species barrier at short phylogenetic distances in a yeast model

淀粉样蛋白是高度有序的自种子纤维蛋白聚合体，可能代表着一个古老的蛋白质折叠，并作为一种分子工具来繁殖具有感染性的蛋白质--普恩。普恩病毒的感染被认为是通过成核聚合传播的，因此聚合性普鲁恩病毒“种子”固定了相同氨基酸序列的正常单体蛋白，并将其转化为普恩病毒。从先前存在的淀粉样蛋白聚合物到新动员的蛋白质分子的Prion状态的传递是一个严格的序列特定的过程。所谓的“物种屏障”阻止了普里恩状态的传播，甚至在密切相关的蛋白质之间也是如此。如果这一障碍被克服，就会导致普恩病毒的跨物种传播。该项目的总体目标是揭示在蛋白质序列差异较低的水平上控制Prion物种屏障的分子机制。普恩病毒的跨物种传播包括将发散的蛋白质固定到杂聚体中(共聚集)，并通过其碎裂来繁殖新产生的淀粉样蛋白，产生新的“种子”。高度不同的Pron结构域之间的物种障碍是由于它们不能共同聚集。它假设，在低水平的序列分歧，异源的Prion结构域可以被结合到杂多的聚合物中，但不能获得构象状态，这将允许有效的片断和生产新的Pron“种子”在异源宿主。该项目旨在检验这一假设。具体的研究目标如下：1)研究发散的Prion结构域在体外的交叉播种能力；2)确定导致异源Prion的物种屏障和缺陷繁殖的序列差异；3)研究细胞环境在异源Prion繁殖和物种屏障中的作用。我们将研究与其亲缘关系密切的酵母菌中的普里恩蛋白。这些蛋白质之间的变异程度与在哺乳动物系统中观察到的物种屏障和跨物种Pron传递现象中观察到的分歧范围相对应。将采用体外和体内方法相结合的方法，然后对结果进行直接比较。在哺乳动物、酵母菌和其他真菌中都可以发现普里恩。它们控制着酵母和其他真菌中非孟德尔特征的遗传，并为“结构遗传”提供了一种机制，可能在进化中发挥了重要作用。这项研究的结果将有助于制定预防密切相关动物之间普恩病毒跨物种传播的策略，并可能对理解结构遗传中蛋白质-蛋白质相互作用的特异性的控制机制具有深远的意义。该研究项目在很大程度上依赖于研究生和本科生的参与，并由不同的男性和女性团队进行。该项目将是面向本科生和研究生的综合课程，他们将通过博士项目为该项目做出贡献。该研究所的实验室参与了多机构基础和应用分子进化中心(FAME)，该中心促进与化学和生物进化相关的各种主题的跨学科研究和教育。在该项目过程中构建的不同于酿酒酵母的新的遗传标记菌株将有助于推进对这些生物的进一步研究。

Amyloids are highly ordered self-seeded fibrous protein aggregates possibly representing an ancient protein fold and serving as a molecular tool for propagation of prions, which are infectious proteins. Prion infection is thought to spread via nucleated polymerization, so that polymeric prion "seeds" immobilize a normal monomeric protein of the same amino acid sequence and convert it into a prion. Transmission of the prion state from a pre-existing amyloid polymer to the newly mobilized protein molecules is a strictly sequence-specific process. The so-called "species barrier" prevents transmission of the prion state even between closely related proteins. If the barrier is overcome, this results in a cross-species prion transmission. The overall goal of this project is to uncover the molecular mechanisms that control the prion species barrier at low levels of protein sequence divergence. Cross-species prion transmission involves immobilization of a divergent protein into heteropolymers (co-aggregation), and propagation of the newly generated amyloid via its fragmentation, producing new "seeds". The species barrier between highly divergent prion domains is due to their inability to co-aggregate. It is hypothesized that at low levels of sequence divergence, a heterologous prion domain can be incorporated into the heteropolymers, but is unable to acquire the conformational state which would allow for efficient fragmentation and production of new prion "seeds" in the heterologous host. The project is designed to test this hypothesis. Specific Research Objectives are as follows: 1) to study cross-seeding capabilities of the divergent prion domains in vitro; 2) to identify the sequence differences that are responsible for the species barrier and defective propagation of the heterologous prions; 3) to investigate the role of cellular environment in the propagation of heterologous prions and the species barrier. Prion proteins of the closely related yeast species will be studied. Level of variation among these proteins corresponds to the range of divergence observed in the species barrier and cross-species prion transmission phenomena in the mammalian systems. A combination of both in vitro and in vivo approaches will be employed, followed by a direct comparison of the results. Prions can be found in mammals, yeast, and other fungi. They control inheritance of non-Mendelian traits in yeast and other fungi, and provide a mechanism for the "structural inheritance," possibly playing an important role in evolution. Results of this research will contribute to development of the strategies for prevention of cross-species prion transmission among closely related animals, and may have far-reaching implications for understanding the mechanisms controlling the specificity of protein-protein interactions involved in structural inheritance. The research project relies heavily on the participation of graduate and undergraduate students, and is being carried out by a diverse team of males and females. The project will be integrated courses for undergraduares and graduate students, who will contribute to it through their doctoral projects. The PI's laboratory participates in the multi-institutional Center for Fundamental and Applied Molecular Evolution (FAME), which promotes interdisciplinary research and education on various topics related to chemical and biological evolution. New genetically marked strains of the Saccharomyces species other than S. cerevisiae, constructed in the course of this project, will help to advance further studies of these organisms.