Phenotypic detection of mouse PrP aggregation in yeast

描述(由申请人提供)： Prion是一种蛋白质异构体，通过将由相同维持基因编码的正常蛋白质转化为Prion形式来自我繁殖。PrP蛋白的易于聚集的PrP蛋白亚型与哺乳动物和人类的致命和不可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”(可传染给人类)，它在欧洲造成重大破坏，最近在美国发现。Prion病的动物模型系统既昂贵又费力，这使得大规模的体内筛选反Prion药物基本上是不可能的。在酵母中，哺乳动物PrP可以形成聚集体，获得Pron的某些生化特征。这表明酵母可以作为研究PrP聚集的模型。利用酵母翻译终止因子Sup35建立了PrP在酵母中聚集的表型分析方法。Sup35的N-近端结构域具有蛋白性质。这个结构域被编码PrP部分的鼠标序列所取代。在酵母中，这种名为PrP-Sup35MC的嵌合蛋白的瞬时过量生产导致Sup35的部分功能丧失，导致翻译终止缺陷(在特殊设计的菌株中很容易检测到)并伴随聚集。PrP-Sup35MC的部分非功能状态维持在有丝分裂中，类似于全尺寸Sup35的内源性PrP-Sup35亚型的某些模式。假设PrP-Sup35MC聚集体是由于PrP区域转换为普里子态而产生的。这一假设将得到检验。此外，由于聚集的PrP-Sup35MC至少类似于Prion的某些模式，它将被用作大规模筛选抗Prion聚集的蛋白质、多肽和化学物质的有力工具。这项研究将补充NIH父母拨款的目标之一，涉及识别影响Prion的哺乳动物伴侣，并可能导致识别新的潜在的反Prion治疗方法。

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.