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，从哺乳动物中神经退行性疾病的出现、进展和传播，到真菌中独特性状的非孟德尔遗传。根据这一观点，当朊病毒蛋白采用另一种物理状态并在该形式自我复制时持续存在时，就会出现朊病毒相关表型。这种自我复制是由可折叠的朊病毒蛋白组装成聚集体介导的，聚集体模板将其他形式的蛋白质转化为类似状态。朊病毒蛋白利用其构象灵活性的固有能力是建立不同表型的核心事件，但在活细胞的背景下，将这一过程扩展到实践成为一个多步骤的努力。蛋白质质量控制途径、朊蛋白生物发生和细胞生物学都可以修饰朊蛋白在体内的错误折叠，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中的一个明显空白，限制了我们将蛋白质错误折叠机制与体内疾病机制联系起来的能力。由于朊病毒相关表型的出现、传播和逆转必然涉及蛋白质状态的变化，这些力量必须集中在调节朊病毒形式之间转变的事件上。本研究的长期目标是通过了解朊病毒蛋白动力学及其细胞环境之间的相互作用，阐明朊病毒蛋白作为疾病和遗传因素的分子机制。该应用程序的总体目标是确定朊病毒蛋白的物理特性如何调节朊病毒聚集动力学以创建不同的表型。核心假设是，朊病毒蛋白内的特定序列元素会影响分子伴侣对其的识别和/或加工，从而形成一个连续的动态系统，允许不同表型的出现和持续存在，但偶尔也会相互转换。在实验可处理的酿酒葡萄球菌Sup35/[PSI+]朊病毒强有力的初步数据指导下，这一假设将通过三个特定目标进行验证：1)确定Sup35序列变异改变朊病毒繁殖的分子机制，2)确定过量Hsp104导致朊病毒丢失的分子机制，以及3)确定Sup35/[PSI+]朊病毒表型的分子基础。这些拟议的研究具有创新性，因为它们使用了独特的实验和数学分析相结合的方法来暂时连接朊病毒蛋白物理和功能状态的转变。该研究为探索朊病毒蛋白错误折叠与其生理后果之间的关系提供了一个新的动态框架，具有重要意义。鉴于朊病毒序列和从酵母到人类的错误折叠途径的显著相似性，这些观察结果将广泛适用于由这些迷人的蛋白质调节的广泛的生物事件。

DESCRIPTION (provided by applicant): The prion hypothesis provides an explanation for a diverse collection of previously inexplicable phenomena, ranging from the appearance, progression and spread of neurodegenerative disease in mammals to the non- Mendelian inheritance of unique traits in fungi. According to this idea, prion-associated phenotypes arise when a prion protein adopts an alternative physical state and persist when that form self-replicates. This self- replication is mediated by the assembly of alternatively folded prion protein into aggregates, which template the conversion of other forms of the protein to a like state. The inherent ability of prion proteins to harness their conformational flexibility is a central event in establishing distinct phenotypes, but the extension of this process to practice becomes a multistep endeavor within the context of a living cell. Protein quality control pathways, prion protein biogenesis, and cell biology all modify prion protein misfolding in vivo to create transmissible changes in physiology. A molecular understanding of how these forces intersect is a clear gap in current knowledge, limiting our ability to correlate protein misfolding mechanisms in vitro and disease mechanisms in vivo. As the appearance, spread and reversal of prion-associated phenotypes necessarily involve changes in protein state, these forces must converge on events that regulate transitions between prion forms. The long-term goal of this research is to elucidate the molecular mechanisms allowing prion proteins to act as elements of disease and heritability by developing an understanding of the interplay between prion protein dynamics and its cellular context. The overall objective of this application is to determine how the physical characteristics of prion proteins modulate prion aggregate dynamics to create distinct phenotypes. The central hypothesis is that specific sequence elements within prion proteins impact their recognition and/or processing by molecular chaperones, creating a continuum of dynamic systems that allow distinct phenotypes to appear and persist but also to occasionally interconvert. Guided by strong preliminary data using the experimentally tractable 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 prion propagation, 2) Determine the molecular mechanism by which excess Hsp104 leads to prion loss, and 3) Determine the molecular basis of the Sup35/[PSI+] prion phenotype. These proposed studies are innovative because they use a unique combination of experimental and mathematical analyses to temporally link transitions in prion protein physical and functional state. The proposed research is significant because it will provide a new and dynamic framework for exploring the relationship between prion protein misfolding and its physiological consequences. Given the remarkable similarity of prion sequences and misfolding pathways from yeast to man, these observations will be broadly applicable to the wide range of biological events regulated by these fascinating proteins.