Biochemical and genetic analysis of prion formation

细胞已经进化出一种复杂而重要的蛋白质机制，称为分子伴侣，以确保新产生的多肽正确折叠。许多疾病，包括阿尔茨海默氏病、帕金森氏病、多聚谷氨酰胺重复障碍（例如，亨廷顿氏病）和涉及感染性蛋白（朊病毒）的疾病，都与蛋白质错误折叠密切相关，这一事实强调了正确蛋白质折叠的重要性。这种错误折叠事件通常导致形成一种特殊类型的蛋白质聚集体，称为淀粉样纤维。直到最近，由于缺乏研究其形成和朊病毒样繁殖的简易遗传或生化系统，理解为什么一些蛋白质在构象上发生自繁殖致病性变化的努力一直受到阻碍。随着发现酵母的[URE 3]和[PSI+]状态是由内源性蛋白质的朊病毒样聚集引起的，这种情况得到了极大的改善。我的实验室利用[PSI+]现象来研究内源性蛋白。我的实验室已经利用[PSI]现象来研究朊病毒形成和繁殖的机制。本建议的目的是建立一套通用的工具，研究朊病毒样，自我传播的蛋白质构象在体内的变化。特别是，我们将寻找新的朊病毒，以确定朊病毒样聚集体在生物过程中是如何发生的。此外，酵母中的遗传分析和体外生物化学研究的组合将用于鉴定和表征源自哺乳动物PrP蛋白的致病肽（PrP 89 -143）的特性，所述特性允许其采用富含构象的β折叠。然后将使用模型系统如培养的神经母细胞瘤细胞和小鼠探索这种富含β折叠的形式的形成与致病性之间的关系。这些努力将极大地促进了与本计划项目赠款的合作。例如，我们的突变分析将同时受到Wemmer和Pine（项目2）的结构研究以及Cohen（项目1）的化学和计算方法的指导。另一方面，对我们在酵母中发现的生理意义的分析将在很大程度上取决于Prusiner和DeArmond小组在转基因动物生产和分析方面的专业知识。

Cells have evolved a sophisticated and essential machinery of proteins called molecular chaperones to ensure the proper folding of newly made polypeptides. The importance of correct protein folding is underscored by the fact that a number of diseases, including Alzheimer's, Parkinson's polyglutamine repeat disorders (e.g. Huntington's Disease), and those involving infectious proteins (prions), are intimately associated with protein misfolding. Such misfolding events often lead to formation of a specific type of protein aggregate termed amyloid fibers. Efforts to understand why some proteins undergo self-propagating pathogenic changes in conformation have been hampered until recently because of the lack of a facile genetic or biochemical system for studying their formation and prion-like propagation. This situation has improved greatly with the finding that the [URE3] and [PSI+] states of yeast result from the prion-like aggregation of endogenous proteins. My laboratory has taken advantage of the [PSI+] phenomenon to study the endogenous proteins. My laboratory has taken advantage of the [PSI] phenomenon to study the mechanism of prion formation and propagation. The present proposal aims to establish a general set of tools for studying prion-like, self-propagating changes in protein conformations in vivo. In particular, we will search for novel prions in an effort to determine how generally prion-like aggregates occur in biological processes. In addition, a combination of genetic analyses in yeast and in vitro biochemical studies will be employed to identify and characterize properties of a pathogenic peptide (PrP89-143), derived from the mammalian PrP protein, that allow it to adopt a beta-sheet rich in conformation. The relationship between formation of this beta-sheet rich form and pathogenicity will then be explored using model systems such as cultured neuroblastoma cells and mice. These efforts will be greatly facilitated by the collaborations made possible with this program project grant. For example, one the one human our mutational analysis will both be guided by and help guide the structural studies of Wemmer and Pine (Project 2) and the chemical and computational approaches of Cohen (Project 1). On the other hand, analysis of the physiological significance of our findings in yeast will depend heavily on the expertise of the Prusiner and DeArmond groups in the production and analysis of transgenic animals.