Biochemical and genetic analysis of prion formation

朊病毒形成的生化和遗传分析

• 批准号: 6578751
• 负责人: JONATHAN S. WEISSMAN
• 金额: $ 28.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6578751
• 关键词: chemical aggregate; conformation; fungal genetics; genetically modified animals; laboratory mouse; molecular assembly /self assembly; molecular chaperones; molecular pathology; prions; protein folding; protein isoforms; protein structure function; tissue /cell culture; yeasts

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.

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，包括阿尔茨海默病、帕金森病多谷氨酰胺重复障碍（如亨廷顿病）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。这种错误折叠事件经常导致形成一种称为淀粉样纤维的特定类型的蛋白质聚集体。直到最近，由于缺乏一个简单的遗传或生化系统来研究蛋白质的形成和朊病毒样的繁殖，了解为什么一些蛋白质在构象上经历自我繁殖的致病变化的努力一直受到阻碍。酵母的[URE3]和[PSI+]状态是由内源性蛋白的朊病毒样聚集引起的，这一发现大大改善了这种情况。我的实验室利用[PSI+]现象来研究内源性蛋白质。我的实验室利用[PSI]现象来研究朊病毒的形成和传播机制。目前的建议旨在建立一套通用的工具来研究朊病毒样，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，以确定在生物过程中一般朊病毒样聚集体是如何发生的。此外，将采用酵母遗传分析和体外生化研究相结合的方法来鉴定和表征一种致病肽（PrP89-143）的特性，该肽来源于哺乳动物PrP蛋白，使其能够采用丰富构象的β -片。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富含β -薄片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与本计划项目赠款可能的合作。例如，一个人的突变分析将被Wemmer和Pine的结构研究（项目2）和Cohen的化学和计算方法（项目1）所指导并帮助指导。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。