Prion Cycle Regulation In Vivo

描述(申请人提供)：最近的进展表明，蛋白质物理状态的自我复制既指导了可传播海绵状脑病的发展和传播，也指导了低等真核生物中一些表型特征的遗传。这一新的生物学过程被称为普里恩假说，它预测一组独特的蛋白质具有在体内采用多种构象状态的能力，并产生不同的生理后果。由于一重一功能的蛋白质不能发挥历史上与核酸相关的角色，如传染性和遗传性，因此理解普恩蛋白质的结构如何受到限制，以允许相关表型的忠实繁殖，但保持足够的灵活性，以允许偶尔的状态转换，对于理解仅限蛋白质假说的生理后果至关重要。低等真核生物的蛋白循环为体内研究蛋白循环调控提供了易于实验处理的模型系统。例如，酿酒酵母的Sup35蛋白是翻译终止复合体的一个组成部分，其功能受Pron循环的可逆调节。在非Prion状态下，Sup35有助于有效的终止(psi-表型)，但在Prion形式下，Sup35‘S的活性受到损害，导致停止密码子通读([psi+]表型)。虽然[psi+]和[psi-]表型在很大程度上是稳定的，但它们会自发地相互转换(约1 cell/百万)，并可以被化学和分子刺激诱导进行定量转换。利用这个系统，我们将开始通过关注两个贡献因素来阐明在体内Pron形式近乎忠实地繁殖的分子机制：当不同的形式存在于同一细胞中时的相互作用和高效的Prion转换的反式调节。为此，我们将1)确定体内Pron变异优势的分子基础，2)阐明已知的Sup35/[PSI+]Pron周期调节子调控繁殖和表型转换的分子机制，3)筛选和表征新的Prion调节子。总而言之，这些研究将建立一个框架，以了解仅蛋白质的表型繁殖在蛋白质生物发生方面的作用。以往工作的坚实基础表明，从低等真核生物的普恩研究中获得的知识显然直接适用于我们对普恩机制及其在哺乳动物中的生理后果的理解。

DESCRIPTION (provided by applicant): Recent advances suggest that self-replication of protein physical states directs both the development and spread of the Transmissible Spongiform Encephalopathies and the inheritance of some phenotypic traits in lower eukaryotes. This novel biological process, known as the prion hypothesis, predicts that a unique group of proteins has the capacity to adopt multiple conformational states with distinct physiological consequences in vivo. Since one-fold-one-function proteins are unable to act in roles that have historically been linked to nucleic acids such as infectivity and inheritance, understanding how a prion protein's structure can be constrained to allow the faithful propagation of associated phenotypes but remain sufficiently flexible to allow occasional transitions in state is crucial to understanding the physiological consequences of the protein- only hypothesis. The prion cycles of lower eukaryotes provide experimentally tractable model systems for studying prion cycle regulation in vivo. For example, the Sup35 protein of S. cerevisiae is a component of the translation termination complex whose function is reversibly modulated by a prion cycle. In the non-prion state, Sup35 facilitates efficient termination (psi- phenotype), but in the prion form, Sup35's activity is compromised leading to stop codon read-through ([PSI+] phenotype). While the [PSI+] and [psi-] phenotypes are largely stable, they spontaneous interconvert (about 1 cell/million) and can be induced to quantitatively switch by chemical and molecular stimuli. Using this system, we will begin to elucidate the molecular mechanism underlying the near-faithful propagation of prion forms in vivo by focusing on two contributing factors: the interplay of distinct forms when present in the same cell and the trans regulators of efficient prion conversion. Toward this end, we will 1) determine the molecular basis of prion variant dominance in vivo, 2) elucidate the molecular mechanisms by which known regulators of the Sup35/[PSI+] prion cycle modulate propagation and phenotypic transitions, and 3) screen for and characterize novel prion regulators. Together, these lines of investigation will build a framework for understanding protein-only phenotypic propagation in terms of prion protein biogenesis. A strong foundation of previous work indicates that the knowledge gleaned from prion studies in lower eukaryotes is clearly and directly applicable to our understanding of prion mechanisms and their physiological consequences in mammals.