Investigation of the Establishment of the Prion State

朊病毒国家的建立调查

• 批准号: 7093954
• 负责人: IRINA L DERKATCH
• 金额: $ 27.04万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093954
• 关键词: atomic force microscopy; atrophy; conformation; gene deletion mutation; mathematical model; model design /development; molecular assembly /self assembly; mutant; peptide library; plasmids; prions; protein protein interaction; protein structure function; site directed mutagenesis; spongiform encephalopathy; virus genetics; virus protein

DESCRIPTION (provided by applicant): Prions cause tremendous concern as the causative agents of lethal neurodegenerative disorders such as CJD and BSE. Comparative analysis of prions and other proteins linked to human amyloidosis, and the identification of prions among benign epigenetic modifiers of cellular functions in fungi suggest the existence of molecular mechanisms governing the formation and elimination of self-perpetuating protein conformations. Revealing these mechanisms is expected to facilitate the development of new strategies for preventing the onset and transmission of prion diseases. The long-term goal of PI's research is to understand the biogenesis and biological role of prions, whereas the objective of the current proposal is to elucidate the mechanism of initial establishment of prion state. Genetic analysis in yeast is the major approach. The first indication that the de novo formation of prions may not be truly spontaneous came from the finding that the [PSI+] prion appears only in the presence of a prion named [PIN+] (for [PSI+] inducibility). The demonstrations that various prions can substitute for [PIN+] during [PSI+] induction and that formation of prions other than [PSI+] can be similarly facilitated by heterologous prions suggest that there is a general mechanism through which pre-existing prion aggregates seed novel prions. Aim 1 is to elucidate this mechanism. The working hypothesis postulates that the formation of [PSI+] is initiated by the formation of a short "polar zipper" between Q/N-rich sequences in the prion domains of [PSI+] and [PIN+]-composing proteins, Sup35 and Rnq1. The initial deletion analysis of Rnq1 will determine, which of multiple Q/N-rich regions of Rnq1 are involved in the facilitation of the [PSI*] formation by [PIN*]. Then mutagenesis will be directed to these region(s). Screening for compensatory Sup35 mutations that restore disrupted seeding will be used validate the hypothesis. The conclusions of in vivo studies will be verified in vitro with bacterially expressed proteins, and atomic force microscopy will be used to monitor the seeding process. Also, the efficiency of interconversion of prions encompassing Sup35 prion domains from different Saccharomycetes species will be analyzed to test if the seeding model is applicable to prion transmission between species. Aim 2 is to identify new prions. Strategy (A) will test whether the proteins that satisfy several previously established criteria for presumptive prions can indeed take on self-propagating conformations. Among them, Lsm4 and Yck1 play roles in mRNA processing and signaling, respectively, and their ability to become prions is expected to advance our understanding of splicing, mRNA stability and signal transduction. Lsm4 studies may also shed light on the mechanism of spinal muscular atrophy. Strategy (B) involves designing several libraries of peptides. Screening these peptides for the ability to form prions or promote prion formation, and subsequent computer analysis of data will allow finding an algorithm for aggregation-prone proteins. Such proteins may not cause detectable prion phenotypes themselves but may induce dangerous prions.

描述（由申请人提供）：朊病毒作为致死性神经退行性疾病（如CJD和BSE）的病原体引起了极大的关注。朊病毒和其他蛋白质与人类淀粉样变性的比较分析，并在真菌中的细胞功能的良性表观遗传修饰剂的朊病毒的鉴定表明存在的分子机制，管理自我永存的蛋白质构象的形成和消除。揭示这些机制有望促进预防朊病毒疾病发病和传播的新策略的发展。PI研究的长期目标是了解朊病毒的生物起源和生物学作用，而目前的建议的目标是阐明朊病毒状态最初建立的机制。酵母中的遗传分析是主要的方法。第一个表明朊病毒的从头形成可能不是真正自发的迹象来自于发现[PSI+]朊病毒仅在名为[PIN+]（[PSI+]诱导蛋白）的朊病毒存在时出现。在[PSI+]诱导过程中，各种朊病毒可以取代[PIN+]，并且异源朊病毒可以类似地促进[PSI+]以外的朊病毒的形成，这表明存在一种通用机制，通过该机制，预先存在的朊病毒聚集并播种新的朊病毒。目的1是阐明这一机制。工作假说假定[PSI+]的形成是由[PSI+]和[PIN+]组成蛋白质Sup 35和Rnq 1的朊病毒结构域中富含Q/N序列之间形成短的“极性拉链”引发的。Rnq 1的初始缺失分析将确定Rnq 1的多个Q/N富集区中的哪些参与[PIN*]促进[PSI*]形成。然后诱变将针对这些区域。将使用恢复中断的播种的补偿性Sup 35突变的筛选来验证该假设。体内研究的结论将在体外用细菌表达的蛋白质进行验证，原子力显微镜将用于监测接种过程。此外，将分析包含来自不同放线菌属物种的Sup 35朊病毒结构域的朊病毒相互转化的效率，以测试接种模型是否适用于朊病毒在物种之间的传播。目标2是识别新的朊病毒。策略（A）将测试满足先前建立的几个假定朊病毒标准的蛋白质是否真的可以呈现自传播构象。其中，Lsm 4和Yck 1分别在mRNA加工和信号传导中发挥作用，它们成为朊病毒的能力有望推进我们对剪接、mRNA稳定性和信号转导的理解。lsm 4的研究也可能揭示脊髓性肌萎缩症的机制。策略（B）涉及设计几个肽文库。筛选这些肽形成朊病毒或促进朊病毒形成的能力，随后对数据进行计算机分析，将有助于找到易聚集蛋白质的算法。这些蛋白质本身可能不会引起可检测到的朊病毒表型，但可能诱导危险的朊病毒。