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集体，可能代表了一种古老的蛋白质折叠，是朊病毒（感染性蛋白）传播的分子工具。朊病毒感染被认为是通过有核聚合传播的，因此聚合朊病毒“种子”固定了具有相同氨基酸序列的正常单体蛋白，并将其转化为朊病毒。朊病毒状态从预先存在的淀粉样聚合物到新动员的蛋白质分子的传播是一个严格的序列特异性过程。所谓的“物种屏障”阻止了朊病毒状态的传播，即使是在密切相关的蛋白质之间。如果这一屏障被克服，就会导致朊病毒跨物种传播。该项目的总体目标是揭示在低水平蛋白质序列分化下控制朊病毒物种屏障的分子机制。朊病毒的跨物种传播包括将一种不同的蛋白质固定在异聚物中（共聚集），并通过其破碎使新产生的淀粉样蛋白繁殖，产生新的“种子”。高度分化的朊病毒域之间的物种屏障是由于它们无法共同聚集。据推测，在低水平的序列分化下，异种朊病毒结构域可以被纳入到异聚物中，但无法获得构象状态，这将允许在异种宿主中有效地分裂和产生新的朊病毒“种子”。该项目旨在验证这一假设。具体研究目的如下：1)研究不同朊病毒域的体外交叉播种能力；2)鉴定导致异源朊病毒物种屏障和繁殖缺陷的序列差异；3)探讨细胞环境对外源朊病毒繁殖和物种屏障的影响。密切相关的酵母菌种的朊病毒蛋白将被研究。这些蛋白质之间的变异水平与哺乳动物系统中物种屏障和跨物种朊病毒传播现象中观察到的差异范围相对应。将采用体外和体内方法的结合，然后对结果进行直接比较。朊病毒存在于哺乳动物、酵母和其他真菌中。它们控制着酵母菌和其他真菌的非孟德尔性状的遗传，并为“结构遗传”提供了一种机制，可能在进化中起着重要作用。本研究结果将有助于制定预防密切相关动物间朊病毒跨物种传播的策略，并可能对了解结构遗传中蛋白质-蛋白质相互作用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，由一个由男性和女性组成的多元化团队进行。该项目将为本科生和研究生提供综合课程，学生将通过其博士项目为其做出贡献。PI的实验室参与了多机构基础和应用分子进化中心（FAME），该中心促进了与化学和生物进化相关的各种主题的跨学科研究和教育。本项目构建的除酿酒酵母外的其他酵母菌的遗传标记菌株将有助于进一步研究这些生物。

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.