Structural basis of species-specific infectivities of two prion strains

描述(申请人提供)：Pron是一种传染性蛋白质，在人类、牛和其他哺乳动物中会导致毁灭性的神经退行性疾病(传播性脑病)。这些蛋白质的一个令人费解的方面是，它们不仅形成一个单一的两页丰富的构象，而且形成一套相关的聚集构象，这些构象与不同的Pron疾病有关。酵母中自然产生的普恩也表现出非常相似的行为。哺乳动物和酵母中不同的Pron菌株的一个关键方面是它们具有独特的穿越物种障碍的能力，这一点人们知之甚少，但具有广泛的生物学意义。这项提议的目的是调查传染性普恩构象的结构差异如何支配它们建立和克服物种障碍的能力。不幸的是，目前还不可能从重组哺乳动物普恩蛋白(PrP)中形成高感染性的普恩蛋白用于结构研究，但对于酵母普恩蛋白来说，这是很容易实现的。因此，我们建议研究来自酿酒酵母(SC)和白色念珠菌(CA)的酵母Pron Sup35的Pron菌株之间的结构关系和物种屏障。Sc和CaSup35之间存在强大的Prion物种障碍，但由两个物种的结构域组成的Sup35嵌合体可以克服这一障碍。利用多肽微阵列，我们发现嵌合普鲁恩克服这一障碍的能力是由两个初级序列的小元素(称为“识别元素”)编码的，每个酵母物种一个(Tessier&Lindquist，自然，2007)。我们建议检验的关键假设是，两个不同的Pron菌株的物种特异性感染性是由于Prion识别元件相对于淀粉样蛋白核心的位置不同所致。对于感染酿酒酵母的嵌合菌株，我们假设SC Sup35识别元件在淀粉样蛋白核心内(溶剂屏蔽)，而Ca Sup35识别元件要么在淀粉样蛋白核心外(暴露在溶剂中)，要么处于不起作用的非淀粉样构象(弱溶剂屏蔽)，对于另一株菌株构象反之亦然。我们建议通过以下三个具体目标来验证这一假说：1)在细菌和酵母表达载体中在Sup35嵌合体的Prion结构域中产生单个半胱氨酸突变体，2)通过表征它们在体外和体内的Prion行为来评估单个半胱氨酸突变体是否具有与野生型嵌合体相同的Prion特性，以及3)确定半胱氨酸在嵌合Prion构象的每个菌株中的溶剂暴露程度。这项研究中获得的数据将提供重要的结构信息，说明关键氨基酸序列相对于Prion淀粉样蛋白核心的位置如何影响Prion克服物种障碍的能力。此外，这些发现将通过提供关于不同哺乳动物Pron菌株如何跨越物种障碍的可测试假说，为哺乳动物Prion的研究提供信息。公共卫生相关性：传染性普恩病毒从牛到人的传播继续威胁着我们的社会。Pron形成多种感染性菌株构象，这些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一机制，长期目标是合理开发治疗方法，以防止和逆转普恩病毒的跨物种传播。

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.