Molecular Genetics Of Scrapie Pathogenesis

传染性海绵状脑病（Transmissible spongiform encephalopathies，TSEs）是一组罕见的神经退行性疾病，包括人类的克雅氏病（Creutzfeldt-Jakob disease，CJD）、绵羊的瘙痒病、牛海绵状脑病（bovine spongiform encephalopathies，BSE）和骡鹿和麋鹿的慢性消耗病（chronic wasting diseases，CWD）。TSE传染性可跨越物种屏障。BSE在英国感染人类的事实和CWD在美国可能具有类似作用的担忧强调了了解TSE发病机制和开发有效治疗方法的重要性。TSE疾病的感染因子被称为朊病毒，主要由正常的蛋白酶敏感朊病毒蛋白PrP-sen的异常重折叠的蛋白酶抗性形式（PrP-res或PrPSc）组成。感染的易感性可受PrP-sen和PrP-res之间的氨基酸同源性的影响，而来自不同朊病毒株的PrP-res分子之间的结构差异被认为编码菌株表型。我的研究涉及朊病毒疾病在分子水平和致病水平上的许多不同方面。特别是，我的实验室专注于：1）识别朊病毒感染期间发生的最早事件，2）精确定义PrP-res形成发生的不同细胞区室，3）确定朊病毒株的分子基础，4）开发有效的朊病毒疗法。 朊病毒疾病是蛋白质折叠的疾病。因此，确定PrP-res的结构不仅对于理解正常宿主蛋白如何具有感染性至关重要，而且对于确定不同的PrP-res结构是否负责编码不同的朊病毒株也至关重要。 大多数蛋白质结构技术需要高度纯化的蛋白质。 然而，PrP-res制剂被多种其它蛋白质污染（摩尔，R.A.，Timmes，A.，Wilmarth，P.A.，和Priola，S.A. （2010年）。高度富集的22 l和钱德勒小鼠瘙痒症朊病毒蛋白制备物的比较分析。 Proteomics 10：2858-2869），这可能混淆PrP-res结构的分析。 在2011年，我们利用LC-MS/MS Nanospray离子阱质谱仪和改进的PrP-res纯化技术来研究与PrP-res共纯化的其他分子是否有助于来自不同朊病毒菌株的PrP-res的结构分析。 我们的研究结果表明，非PrP蛋白有助于α螺旋，环/转，和β折叠结构，以前已经完全归因于PrP-res。此外，由于我们改进的PrP-res纯化在某些情况下可以去除大于99%的主要非PrP污染物，我们的数据表明，α螺旋，环/转，和β折叠二级结构，仍然是来自PrP-res本身。 我们的研究是第一个利用相结合的蛋白质组学/蛋白质结构的方法来分析PrP-res构象。 2011年，我们完成了分析小鼠急性朊病毒感染的初步研究的体内工作，目前正在分析数据。 我们还启动了体外研究，其特征在于在小鼠和人朊病毒感染的初始阶段PrP-res与细胞的相互作用。

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective therapeutics. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrP-res or PrPSc) of the normal, protease-sensitive prion protein, PrP-sen. Susceptibility to infection can be influenced by amino acid homology between PrP-sen and PrP-res while differences in structure between PrP-res molecules from different prion strains are believed to encode strain phenotypes. My studies address many different aspects of prion diseases at both the molecular and pathogenic level. In particular, my laboratory focuses on: 1) identifying the earliest events which occur during prion infection, 2) precisely defining the different cellular compartments where PrP-res formation occurs, 3) determining the molecular basis of prion strains and, 4) development of effective prion therapeutics. Prion diseases are diseases of protein folding. Determining the structure of PrP-res is therefore critical for understanding not only how a normal host protein can become infectious but also for determining whether or not different PrP-res structures are responsible for encoding different prion strains. Most protein structure techniques require highly purified protein. However, PrP-res preparations are contaminated with multiple other proteins (Moore, R.A., Timmes, A., Wilmarth, P.A., and Priola, S.A. (2010). Comparative profiling of highly enriched 22l and Chandler mouse scrapie prion protein preparations. Proteomics 10: 2858-2869) which might confound the analysis of PrP-res structure. In 2011, we utilized a LC-MS/MS Nanospray Ion Trap Mass Spectrometer and an improved PrP-res purification technique to study whether or not other molecules that co-purify with PrP-res can contribute to the structural analysis of PrP-res from different prion strains. Our results show that non-PrP proteins do contribute to alpha helical, loop/turn, and beta sheet structures that previously had been solely attributed to PrP-res. Furthermore, since our improved PrP-res purification can in some cases remove greater than 99% of the major non-PrP contaminants, our data suggest that the alpha helical, loop/turn, and beta sheet secondary structure that remains are derived from PrP-res itself. Our study is the first to utilize a combined proteomics/protein structure approach for analyzing PrP-res conformation. In 2011, we completed in vivo work for the initial studies analyzing acute prion infection in mice and are currently in the process of analyzing the data. We also initiated in vitro studies characterizing the interaction of PrP-res with the cell during the initial stages of both mouse and human prion infection.