Phenotypic detection of mouse PrP aggregation in yeast

酵母中小鼠 PrP 聚集的表型检测

• 批准号: 6926788
• 负责人: YURY O CHERNOFF
• 金额: $ 4.8万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6926788
• 关键词: Commonwealth of Independent States; chemical aggregate; chimeric proteins; fungal genetics; fungal proteins; genetic library; heat shock proteins; meiosis; molecular chaperones; phenotype; prions; protein protein interaction

DESCRIPTION (provided by applicant): Prions are protein isoforms that propagate themselves by converting the normal protein, encoded by the same maintenance gene, into a prion form. The aggregation-prone prion isoform of PrP protein is implicated in fatal and incurabe infectious neurodegenerative diseases in mammals and humans. Some of these diseases are of huge importance, for example "mad cow" disease (transmissible to humans) which caused significant damage in Europe and was recently detected in US. Animal model systems for prion diseases are expensive and laborious, making large-scale in vivo screening of antiprion agents essentially impossible. In yeast, mammalian PrP can form aggregates that acquire some biochemical characteristics of a prion. This suggests that yeast could be used as a model to study PrP aggregation. Yeast translation termination factor Sup35 was used to develop a phenotypic assay for PrP aggregation in yeast. The N-proximal domain of Sup35 possesses prion properties. This domain was substituted with a mouse sequence, coding for the portion of PrP. In yeast, transient overproduction of such a chimeric protein, named PrP-Sup35MC, induced partial loss of function of the Sup35, leading to the translation termination defect (easily detectable in the specially designed strains) and accompanied by aggregation. Partially non-functional state of PrP-Sup35MC is maintained in mitotic divisions, resembling some patterns of the endogenous prion isoform of full-size Sup35. It is hypothesized that PrP-Sup35MC aggregates are generated due to conversion of the PrP region into a prion state. This hypothesis will be tested. In addition, as aggregated PrP-Sup35MC certainly resembles at least some patterns of a prion, it will be used as a powerful tool for large-scale screening of the proteins, peptides and chemicals counteracting prion aggregation. This research will complement one of the aims of the parental NIH grant, dealing with identification of the mammalian chaperones that influence prions, and may also lead to identification of the new potential anti-prion treatments.

描述（由申请人提供）： prion是蛋白质同工型，可以通过将同一维持基因编码的正常蛋白转化为prion形式来传播自身。 PRP蛋白的容易发生prion同工型与哺乳动物和人类的致命和无关神经退行性疾病有关。其中一些疾病非常重要，例如“疯牛”疾病（人类传播），这在欧洲造成了重大损害，最近在美国被发现。动物模型的prion疾病既昂贵又费力，这使得对抗抗原剂的体内大规模筛查本质上是不可能的。在酵母中，哺乳动物PRP可以形成聚集体，从而获得一些prion的生化特征。这表明酵母可以用作研究PRP聚集的模型。酵母翻译终止因子SUP35用于开发酵母中PRP聚集的表型测定。 Sup35的N-耐Ximal结构域具有prion特性。该域用小鼠序列取代，编码PRP的一部分。在酵母中，这种嵌合蛋白（称为PRP-SUP35MC）的瞬时过量生产引起了SUP35的部分功能损失，从而导致翻译终止缺陷（在特殊设计的菌株中易于检测），并伴有聚集。 PRP-SUP35MC的部分非功能状态保持在有丝分裂的状态，类似于全尺寸SUP35的内源性prion同工型的一些模式。假设PRP-SUP35MC聚集体是由于PRP区域转化为Prion态而产生的。该假设将进行检验。此外，由于汇总的PRP-SUP35MC肯定至少类似于一些prion的模式，因此它将用作大规模筛选蛋白质，肽和化学物质来抵消prion practregation的强大工具。这项研究将补充父母NIH赠款的目的之一，处理影响王室的哺乳动物伴侣的识别，并可能导致对新的潜在反派治疗的识别。