Prion Cycle Regulation In Vivo

描述（由申请人提供）：朊病毒假说为一系列以前无法解释的现象提供了解释，从哺乳动物神经退行性疾病的出现、进展和传播到真菌独特性状的非孟德尔遗传。根据这一观点，朊病毒相关表型出现在朊病毒蛋白采用另一种物理状态时，并在该形式自我复制时持续存在。这种自我复制是由可折叠的朊病毒组装成聚集体介导的，聚集体将其他形式的蛋白质转化为类似的状态。朊病毒利用其构象灵活性的能力是建立不同表型的中心事件，但这一过程在活细胞的背景下成为一个多步骤的努力。蛋白质质量控制途径、朊病毒生物发生和细胞生物学在体内修饰朊病毒折叠，从而在生理上产生可传播的变化。对这些作用力如何相交的分子理解是当前知识的空白，限制了我们在体外将朊病毒折叠机制与其体内生理后果联系起来的能力。由于朊病毒相关表型之间的转变必然涉及蛋白质状态的变化，这些力量必须集中在体内调节朊病毒动态的事件上。本研究的长期目标是阐明影响朊病毒折叠产生生理上可传递变化的细胞机制。该应用程序的总体目标是确定朊病毒折叠途径、细胞质量控制和细胞生物学的其他方面结合起来产生朊病毒相关表型的分子机制。我们的方法是确定Sup35序列、构象和表达水平的变化改变朊病毒在体内繁殖的途径。核心假设是，Sup35的物理特性和丰度调节了分子伴侣识别和/或处理朊病毒形式的效率，从而允许不同的表型出现和持续存在，但也偶尔相互转换。在实验示踪表Sup35/[PSI+]朊病毒的强大初步数据的指导下，这一假设将通过三个特定目标进行验证：1)确定Sup35序列变异改变[PSI+]繁殖的分子机制，2)确定过量Hsp104导致[PSI+]损失的分子机制，以及3)确定Sup35聚集产生[PSI+]表型的分子机制。这些拟议的研究具有创新性，因为它们使用了实验和数学分析的独特组合来测试朊病毒相关表型的新动态模型。提出的研究意义重大，因为它解决了朊病毒折叠的细胞调控，这是一个鲜为人知但重要的因素，允许朊病毒假说在酵母中产生基于蛋白质的表观遗传元件。在实验中可处理的酵母系统中获得的这一知识有可能提供新的假设，可以在涉及朊病毒机制的更复杂系统中进行测试。

DESCRIPTION (provided by applicant): The prion hypothesis provides an explanation for a collection of previously inexplicable phenomena, ranging from the appearance, progression and spread of mammalian neurodegenerative disease to the non-Mendelian inheritance of unique traits in fungi. According to this idea, prion-associated phenotypes arise when a protein, known as a prion, adopts an alternative physical state and persist when that form self-replicates. This self- replication is mediated by the assembly of alternatively folded prions into aggregates, which template the con- version of other forms of the protein to a like state. The ability of prions to harness their conformational flexibil- ity is a central event in establishing distinct phenotypes, but this process becomes a multistep endeavor within the context of a living cell. Protein quality control pathways, prion biogenesis, and cell biology modify prion folding in vivo to create transmissible changes in physiology. A molecular understanding of how these forces intersect is a gap in current knowledge, limiting our ability to correlate prion folding mechanisms in vitro and their physiological consequences in vivo. As transitions between prion-associated phenotypes necessarily in- volve changes in protein state, these forces must converge on events that regulate prion dynamics in vivo. The long-term goal of this research is to elucidate the cellular mechanisms that influence prion folding to pro- duce transmissible changes in physiology. The overall objective of this application is to determine the molecu- lar mechanisms through which the prion folding pathway, cellular quality control, and other aspects of cell biol- ogy combine to create prion-associated phenotypes. Our approach is to determine the pathways through which variations in Sup35 sequence, conformation, and expression levels alter prion propagation in vivo. The central hypothesis is that the physical characteristics and abundance of Sup35 temper the efficiency with which mo- lecular chaperones recognize and/or process the prion form, thereby allowing distinct phenotypes to arise and persist but also to occasionally interconvert. Guided by strong preliminary data using the experimentally trac- table Sup35/[PSI+] prion of S. cerevisiae, this hypothesis will be tested through three specific aims: 1) Deter- mine the molecular mechanism by which sequence variants of Sup35 alter [PSI+] propagation, 2) Determine the molecular mechanism by which excess Hsp104 leads to [PSI+] loss, and 3) Determine the molecular mechanism by which Sup35 aggregation creates the [PSI+] phenotype. These proposed studies are innovative because they use a unique combination of experimental and mathematical analyses to test a new and dynamic model for prion-associated phenotypes. The proposed research is significant because it addresses the cellular regulation of prion folding, a poorly understood but significant factor that allows the prion hypothesis to create protein-based epigenetic elements in yeast. This knowledge, gained in the experimentally tractable yeast sys- tem, has the potential to provide new hypotheses that can be tested in more complex systems where a prion mechanism has been implicated.