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）： 朊病毒是蛋白质同工型，通过将相同维持基因编码的正常蛋白质转化为朊病毒形式来自我繁殖。 PrP 蛋白易于聚集的朊病毒亚型与哺乳动物和人类致命和无法治愈的传染性神经退行性疾病有关。其中一些疾病非常重要，例如“疯牛病”（可传播给人类），它在欧洲造成了重大损害，最近在美国发现。朊病毒疾病的动物模型系统昂贵且费力，使得大规模体内筛选抗朊病毒药物基本上不可能。在酵母中，哺乳动物 PrP 可以形成聚集体，从而获得朊病毒的一些生化特征。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 近端结构域具有朊病毒特性。该结构域被替换为编码 PrP 部分的小鼠序列。在酵母中，这种名为 PrP-Sup35MC 的嵌合蛋白的瞬时过量产生会导致 Sup35 部分功能丧失，导致翻译终止缺陷（在专门设计的菌株中很容易检测到）并伴有聚集。 PrP-Sup35MC 在有丝分裂中保持部分非功能状态，类似于全尺寸 Sup35 的内源性朊病毒亚型的某些模式。假设 PrP-Sup35MC 聚集体是由于 PrP 区域转变为朊病毒状态而产生的。这一假设将得到检验。此外，由于聚集的 PrP-Sup35MC 肯定至少类似于朊病毒的某些模式，因此它将用作大规模筛选抵抗朊病毒聚集的蛋白质、肽和化学物质的强大工具。这项研究将补充美国国立卫生研究院父母资助的目标之一，即鉴定影响朊病毒的哺乳动物伴侣，并且还可能导致鉴定新的潜在抗朊病毒疗法。