Structural basis of species-specific infectivities of two prion strains

描述（由申请人提供）：朊病毒是一种感染性蛋白质，可在人类、牛和其他哺乳动物中引起毁灭性神经退行性疾病（传染性脑病）。这些蛋白质的一个令人困惑的方面是，它们不仅形成一个单一的2-折叠丰富的构象，但一套相关的聚集构象，负责不同的朊病毒疾病。酵母中天然存在的朊病毒也表现出非常相似的行为。哺乳动物和酵母中不同朊病毒株的一个关键方面是它们具有穿越物种屏障的独特能力，这一点知之甚少，但具有广泛的生物学意义。本提案的目的是研究感染性朊病毒构象的结构差异如何控制它们建立和克服物种屏障的能力。不幸的是，目前不可能从重组哺乳动物朊病毒蛋白（PrP）形成高感染性朊病毒用于结构研究，但这对于酵母朊病毒是容易可能的。因此，我们建议研究朊病毒菌株之间的结构关系和种间屏障的酵母朊病毒Sup 35从S。cerevisiae（Sc）和C.白色念珠菌（Ca）。一个强大的朊病毒物种之间存在的障碍Sc和Ca Sup 35，但Sup 35嵌合体组成的域从两个物种可以克服这个障碍。使用肽微阵列，我们发现嵌合朊病毒克服这种障碍的能力是由两个一级序列的小元件（称为“识别元件”）编码的，每个元件来自每个酵母物种（Tessier & Lindquist，Nature，2007）。我们提出要检验的关键假设是，两种不同朊病毒株的种属特异性感染性是由于朊病毒识别元件相对于淀粉样蛋白核心的位置差异所致。对于特异性感染S.在酿酒酵母中，我们假设Sc Sup 35识别元件在淀粉样蛋白核心内（溶剂屏蔽），而Ca Sup 35识别元件在淀粉样蛋白核心外（溶剂暴露）或处于不胜任的非淀粉样蛋白构象（弱溶剂屏蔽），对于其他菌株构象，反之亦然。我们建议通过以下三个具体目标来检验这一假设：1）为了在细菌和酵母表达质粒中的Sup 35嵌合体的朊病毒结构域中产生单半胱氨酸突变体，2）为了评价单半胱氨酸突变体是否具有与野生型嵌合体相同的朊病毒性质，通过在体外和体内表征它们的朊病毒行为，和3）确定嵌合朊病毒的每种菌株构象中半胱氨酸的溶剂暴露程度。在这项研究中获得的数据将提供重要的结构信息的关键氨基酸序列的位置相对于朊病毒淀粉样蛋白核心的影响朊病毒的能力，以克服物种的障碍。此外，这些发现将通过提供不同哺乳动物朊病毒株如何跨越物种障碍的可检验假设，为哺乳动物朊病毒的研究提供信息。公共卫生相关性：传染性朊病毒从牛到人的传播继续威胁着我们的社会。朊病毒形成多种感染性菌株构象，具有通过未知机制克服物种障碍的独特能力。这项拟议的研究旨在破译这种机制，长期目标是合理开发治疗方法，以预防和逆转种间朊病毒传播。

DESCRIPTION (provided by applicant): Prions are infectious proteins that cause devastating neurodegenerative diseases (transmissible encephalopathies) in humans, cattle and other mammals. A puzzling aspect of these proteins is that they form not only a single 2-sheet rich conformation, but a suite of related aggregated conformers that are responsible for different prion diseases. Naturally occurring prions in yeast also display very similar behavior. A key aspect of different prion strains in both mammals and yeast is that they have unique capacities for traversing species barriers, which is poorly understood but has broad biological importance. The goal of this proposal is to investigate how differences in the structure of infectious prion conformers govern their ability to establish and overcome species barriers. Unfortunately, it is currently not possible to form highly infectious prions from recombinant mammalian prion protein (PrP) for structural studies, but this is readily possible for yeast prions. Therefore, we propose to investigate the structural relationship between prion strains and species barriers for the yeast prion Sup35 from S. cerevisiae (Sc) and C. albicans (Ca). A robust prion species barrier exists between Sc and Ca Sup35, but a Sup35 chimera composed of domains from both species can overcome this barrier. Using peptide microarrays we discovered that the capacity of chimeric prions to overcome this barrier is encoded by two small elements of primary sequence (named "recognition elements"), one from each yeast species (Tessier & Lindquist, Nature, 2007). The key hypothesis we propose to test is that the species-specific infectivities of two different prion strains are due to differences in the location of the prion recognition elements relative to the amyloid core. For the chimeric strain specific for infecting S. cerevisiae, we hypothesize that the Sc Sup35 recognition element is within the amyloid core (solvent shielded) while the Ca Sup35 recognition element is either outside the amyloid core (solvent exposed) or in an incompetent, non-amyloid conformation (weakly solvent shielded), and vice versa for the other strain conformation. We propose to test this hypothesis through the following three specific aims: 1) To generate single cysteine mutants in the prion domain of the Sup35 chimera in both bacterial and yeast expression plasmids, 2) To evaluate if single cysteine mutants have the same prion properties as the wild-type chimera by characterizing their prion behavior both in vitro and in vivo, and 3) To determine the degree of solvent exposure of cysteines in each strain conformation of the chimeric prion. The data obtained in this study will provide important structural information on how the locations of key amino acid sequences relative to the prion amyloid core impact the capacities of prions to overcome species barriers. Moreover, these findings will inform investigations of mammalian prions by providing testable hypotheses of how different mammalian prion strains traverse species barriers. PUBLIC HEALTH RELEVANCE: The transmission of infectious prions from cattle to humans continues to threaten our society. Prions form multiple infectious strain conformations that have unique capacities to overcome species barriers through an unknown mechanism. This proposed research aims to decipher this mechanism with the long-term goal of rationally developing therapeutics to prevent and reverse interspecies prion transmission.