Biochemical and genetic analysis of prion formation

细胞已经进化出一种复杂而必要的蛋白质机制，称为分子伴侣，以确保新合成的多肽的正确折叠。许多疾病，包括阿尔茨海默氏症、帕金森氏症多谷氨酰胺重复障碍(如亨廷顿病)以及涉及感染性蛋白质(普恩)的疾病，都与蛋白质错误折叠密切相关，这一事实突显了正确蛋白质折叠的重要性。这种错误折叠事件经常导致一种特定类型的蛋白质聚集体的形成，称为淀粉样纤维。直到最近，由于缺乏简便的遗传或生化系统来研究蛋白质的形成和类普里子的繁殖，人们一直无法理解为什么一些蛋白质会在构象上经历自我繁殖的致病变化。随着酵母的[URE3]和[PSI+]状态的发现，这种情况得到了很大的改善，这是内源蛋白质类病毒聚集的结果。我的实验室利用[PSI+]现象来研究内源蛋白质。我的实验室利用[PSI]现象来研究Pron的形成和传播机制。目前的建议旨在建立一套通用的工具，用于研究体内蛋白质构象中类普里子、自传播的变化。特别是，我们将寻找新的Prion，以努力确定在生物过程中通常如何出现类Prion聚集体。此外，将结合酵母中的遗传分析和体外生化研究来鉴定和表征来源于哺乳动物PrP蛋白的致病多肽(PrP89-143)的性质，使其能够采用富含构象的β-折叠。然后将使用培养的神经母细胞瘤细胞和小鼠等模型系统来探索这种丰富的β-折叠形式的形成与致病性之间的关系。这些努力将极大地促进与这项计划项目赠款的合作。例如，我们的突变分析将指导和帮助指导Wemmer和Pine的结构研究(项目2)以及Cohen的化学和计算方法(项目1)。另一方面，我们在酵母中发现的生理学意义的分析将在很大程度上依赖于Prusiner和DeArmond团队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.