Prion Cycle Regulation In Vivo

描述（由申请人提供）：最近的进展表明，蛋白质物理状态的自我复制指导了传染性海绵状脑病的发展和传播以及低等真核生物中某些表型性状的遗传。这种新的生物学过程被称为朊病毒假说，它预测了一组独特的蛋白质具有在体内采用具有不同生理后果的多种构象状态的能力。由于单倍单功能蛋白无法发挥历史上与核酸相关的作用，如感染性和遗传性，因此了解朊病毒蛋白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蛋白质唯一假说的生理后果至关重要。低等真核生物的朊病毒循环为研究体内朊病毒循环调控提供了实验上易处理的模型系统。例如，S.酿酒酵母是翻译终止复合物的组分，其功能由朊病毒循环可逆地调节。在非朊病毒状态下，Sup 35促进有效终止（PSI-表型），但在朊病毒形式下，Sup 35的活性受损，导致终止密码子通读（[PSI+]表型）。虽然[PSI+]和[PSI-]表型在很大程度上是稳定的，但它们自发相互转化（约1个细胞/百万），并且可以通过化学和分子刺激诱导定量转换。使用这个系统，我们将开始阐明潜在的分子机制，近忠实的传播朊病毒形式在体内集中在两个促成因素：不同形式的相互作用时，在同一个细胞和有效的朊病毒转换的反式调节。为此，我们将1）确定体内朊病毒变体优势的分子基础，2）阐明已知的Sup 35/[PSI+]朊病毒周期调节剂调节繁殖和表型转变的分子机制，3）筛选和表征新型朊病毒调节剂。总之，这些线的调查将建立一个框架，了解蛋白质只有表型繁殖朊病毒蛋白的生物合成。以前的工作的坚实基础表明，从朊病毒在低等真核生物的研究中收集的知识是明确和直接适用于我们的理解朊病毒的机制及其在哺乳动物中的生理后果。

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.