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Investigation of the Establishment of the Prion State

朊病毒国家的建立调查

基本信息

批准号：
8250170
负责人：
IRINA L DERKATCH
金额：
$ 22.45万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8250170
关键词：
Investigation Establishment Prion State

项目摘要

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。朊病毒与其他与人类淀粉样变性相关的蛋白质的比较分析，以及真菌细胞功能的良性表观遗传修饰因子中朊病毒的鉴定表明，存在控制自我延续蛋白构象形成和消除的分子机制。揭示这些机制有望促进制定预防朊病毒疾病发病和传播的新战略。PI的长期研究目标是了解朊病毒的生物发生和生物学作用，而目前的研究目标是阐明朊病毒状态初始建立的机制。酵母的遗传分析是主要的方法。朊病毒的从头形成可能不是真正自发的第一个迹象来自于发现[PSI+]朊病毒仅在一个名为[PIN+]的朊病毒（用于[PSI+]诱导性）存在时才出现。在[PSI+]诱导过程中，多种朊病毒可以替代[PIN+]，而异源朊病毒同样可以促进[PSI+]以外的朊病毒的形成，这表明存在一种普遍的机制，通过这种机制，预先存在的朊病毒聚集会产生新的朊病毒。目的1是阐明这一机制。工作假设假设[PSI+]的形成是由[PSI+]和[PIN+]组成蛋白Sup35和Rnq1的朊病毒结构域中富含Q/ n的序列之间形成的短“极性拉链”引发的。通过对Rnq1的初始缺失分析，可以确定在Rnq1的多个富含Q/ n的区域中，哪一个参与了促进[PIN*]形成[PSI*]的过程。然后诱变将被定向到这些区域。筛选补偿性Sup35突变恢复中断播种将用于验证这一假设。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播种过程。此外，我们还将分析包含不同酵母菌Sup35结构域的朊病毒相互转化的效率，以检验播种模型是否适用于朊病毒的种间传播。目标二是鉴定新的朊病毒。策略(A)将测试满足先前确定的几种假定朊病毒标准的蛋白质是否确实能够呈现自我传播的构象。其中，Lsm4和Yck1分别参与mRNA加工和信号传导，它们成为朊病毒的能力有望促进我们对剪接、mRNA稳定性和信号转导的理解。Lsm4的研究也可能揭示脊髓性肌萎缩的机制。策略(B)涉及设计几个肽库。筛选这些肽形成或促进朊病毒形成的能力，以及随后的计算机数据分析将允许找到易于聚集的蛋白质的算法。这些蛋白本身可能不会引起可检测的朊病毒表型，但可能诱导危险的朊病毒。