TRANSGENIC STUDIES OF MUTANT PRP GENES

突变 PRP 基因的转基因研究

• 批准号: 6565180
• 负责人: STANLEY B PRUSINER
• 金额: $ 25.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565180
• 关键词: enzyme linked immunosorbent assay; genetic strain; genetically modified animals; laboratory mouse; mutant; northern blottings; prions; protein isoforms; scrapie; tissue mosaicism; western blottings

Prion diseases are disorders of protein conformation. The pathogenic form of the prion protein, PrP/sc, is distinguished from its cellular counterpart, PrP/C, by a marked increase in the degree of beta-sheet content and a decrease in the proportion of alpha-helix. Although progress has been made in efforts to determine the three-dimensional structure of PrP/c, considerable work remains. Even more challenging is determining the structure of PrP/sc, which is extremely insoluble in the native state. The insolubility of PrP/sc has hindered attempts to elucidate the complete molecular structure of the infectious prion. To gain insight into the structural transitions which feature in PrP/sc formation, we plan to create mutations in PrP which will facilitate structural studies of PrP/sc. Our goal is to create novel mutations which lower the activation energy barrier for spontaneous formation of infectious prions de novo, especially using recombinant PrP in an in vitro reaction. By using computational methods, we have shown that it is possible to design highly pathogenic mutations in PrP which cause disease with unprecedented rapidity. Preliminary studies indicate that transmissible prions may be formed de novo, and spectroscopic studies have shown that the mutant protein adopts a beta-sheet conformation under conditions where the wild- type protein is predominantly alpha-helical. Of major importance, the beta-sheet conformer formed in vitro is soluble and preliminary NMR studies have been initiated. In a complementary approach, we have identified a mutated PrP/c molecule of 106 residues that can be converted into a protein that closely resembles PrP/sc when expressed in scrapie- infected mammalian cells. This truncated PrP/sc-like molecule is soluble in the presence of low concentrations of ionic detergent, facilitating purification of the protein for structure determination. In this proposal, we describe experimental studies which exploit these findings and extend out knowledge or PrP/sc formation. Such investigations in concert with those outlined in Projects 2, 3 and 4 should allow us to determine the complete molecular structure for a prion.

朊病毒疾病是蛋白质构象的紊乱。致病形式 朊病毒蛋白PrP/sc与其细胞内 对应物，PrP/C，通过β-折叠程度的显著增加 含量和α-螺旋比例的降低。虽然进展 已经在努力确定的三维结构， PrP/C，仍有大量工作要做。更具挑战性的是确定 PrP/sc的结构，其在天然状态下极不溶。的 PrP/sc的不溶性阻碍了试图阐明完整的 感染性朊病毒的分子结构为了深入了解 PrP/sc形成中的结构转变，我们计划 在PrP中产生突变，这将有助于对 我们的目标是创造新的突变，降低激活 传染性朊病毒从头自发形成的能量屏障， 特别是在体外反应中使用重组PrP。通过使用 计算方法，我们已经表明，有可能设计高度 PrP中的致病性突变导致前所未有的疾病 速度初步研究表明，传染性朊病毒可能是 新形成，光谱研究表明，突变体 蛋白质在野生型的条件下采用β折叠构象， 型蛋白主要是α-螺旋。最重要的是， 体外形成的β-折叠构象异构体是可溶的， 研究已经开始。作为一种补充方法，我们 鉴定了一种106个残基的突变PrP/c分子， 转化成一种在羊瘙痒症中表达时与PrP/sc非常相似的蛋白质， 感染的哺乳动物细胞这种截短的PrP/sc样分子是可溶性的 在低浓度的离子去污剂存在下， 纯化用于结构测定的蛋白质。在这项提案中， 我们描述了利用这些发现的实验研究， 知识或PrP/SC形成。此类调查与 项目2、项目3和项目4中概述的这些内容应使我们能够确定 朊病毒的完整分子结构