ROLE OF PRION PROTEIN BIOGENESIS IN PATHOGENESIS OF SCRAPIE

近年来，在解剖分子结构方面取得了进展， 朊病毒蛋白（PrP）的生物合成。 一种新的拓扑序列， 被称为停止转移效应子（STE），它指导新生PrP进入细胞内。 无细胞系统的双重跨膜或分泌 拓扑结构已被识别。 在这些拓扑命运之间的选择 依赖于胞质因子的存在。 一个 具有两种拓扑形式特征的中间体已经被识别 in vivo. 交替拓扑命运和快速ER的途径 可能发生的退化已经确定。 然而， 这些事件对羊瘙痒症的影响尚不清楚。 我们建议探讨 这一新的拓扑序列在羊瘙痒病发病机制中的作用。 的STE 序列将被诱变，突变对PrP的影响 生物起源和羊痒病发病机制进行了研究。 突变体改变了 PrP生物发生的步骤将通过无细胞转录来选择- 连锁翻译和非洲爪蟾卵母细胞显微注射。 其中一些将 用于识别受体和分子伴侣， PrP相互作用。 选定的突变体将在转基因小鼠中进行研究， 为了确定不寻常事件之间是否存在关系， 朊病毒生物发生和羊瘙痒病的发病机制。 最后，突变体 转染到N2 a细胞中将用于探测分子的作用。 伴侣蛋白对PrP生物发生和羊瘙痒病感染参数的影响。

In recent years, progress has been made in dissecting the molecular events of prion protein (PrP) biogenesis. A novel topogenic sequence, termed the Stop Transfer Effector (STE), which directs nascent PrP in cell-free systems to either a doubly transmembrane or a secretory topology, has been identified. The choice between these topologic fates was shown to depend on the presence of a cytosolic factor. An intermediate with features of both topologic forms has been identified in vivo. Pathways by which alternate topologic fates and rapid ER degradation may occur have been identified. However, the relationship of these events to scrapie remains unknown. We propose to explore the role of this novel topogenic sequence in scrapie pathogenesis. The STE sequence will be mutagenized and the effects of mutations on PrP biogenesis and scrapie pathogenesis investigated. Mutants which alter steps in PrP biogenesis will be selected by cell-free transcription- linked translation and Xenopus oocyte microinjection. Some of these will be used to identify receptors and molecular chaperones with which nascent PrP interacts. Selected mutants will be studied in transgenic mice in order to determine if a relationship exists between unusual events in prion biogenesis and the pathogenesis of scrapie. Finally mutants transfected into N2a cells will be used to probe the effect of molecular chaperones on PrP biogenesis and parameters of scrapie infection.