TRANSGENIC STUDIES OF MUTANT PRP GENES

Pron病是一种蛋白质构象紊乱。致病形式 PrP/sc是PrP/sc蛋白的一部分，区别于它的细胞 与之对应的PrP/C，β-Sheet程度显著增加 含量和α-螺旋比例下降。虽然进步 已经做出了努力来确定其三维结构 关于PRP/c，仍有大量工作要做。更具挑战性的是确定 PrP/sc的结构，在原生状态下是极不溶解的。这个 PrP/sc的不溶性阻碍了对完整的 传染性普恩病毒的分子结构。要深入了解 在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PrP的结构研究 Prp/sc.我们的目标是创造新的突变，降低激活 新生传染性病毒的自发形成的能量屏障， 尤其是在体外反应中使用重组PrP。通过使用 计算方法，我们已经证明了高度设计的可能性 PrP致病突变导致疾病史无前例 速度快。初步研究表明，可传播的普恩可能是 从新形成，光谱研究表明，突变体 蛋白质在以下条件下采用β-折叠构象- 类型的蛋白质主要是α-螺旋。最重要的是， 体外形成的β-折叠构象是可溶的和初步的核磁共振 研究工作已经启动。在一个互补的方法中，我们有 确定了一个106个残基的突变PrP/c分子，可以转化为 转化成一种在瘙痒病中表达时与PrP/sc非常相似的蛋白质- 被感染的哺乳动物细胞。这种截短的prp/sc类分子是可溶的。 在低浓度离子洗涤剂存在的情况下，促进 纯化后的蛋白质用于结构测定。在这份提案中， 我们描述了利用这些发现并扩展的实验研究 我们的知识或PrP/Sc形成。这样的调查与 项目2、3和4中概述的那些应该使我们能够确定 一个普恩病毒的完整分子结构。

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.