Determining the molecular basis of adaptive stress-triggered protein phase separation

项目摘要 拟议的研究将从分子细节上确定Poly(A)结合蛋白Pab1的机制(S) 在应力过程中相分离以形成水凝胶组件。细胞压力导致进化上保守的， 推测为适应性地层应力颗粒。Pab1是应激颗粒的定义标志，Pab1‘S期 分离先于应力颗粒的形成。多个RNA结合蛋白的相分离失调 与主要神经退行性疾病相关的病理性蛋白质聚集有关。这个 相分离机理(S)是一个活跃的研究领域。关键的障碍是缺乏可处理的体外培养。 通过RNA结合蛋白进行生物相关相分离的模型。 我们已经突破了这一障碍。在已发表的研究中(Wallace等人，Cell 2015；Riback等人，Cell 2017)， 我们已经确定了Pab1在生理应激反应中相分离成水凝胶液滴。 条件，干扰水凝胶液滴的形成扰乱了萌芽酵母在高温下生存的能力 和饥饿压力。先前没有描述的系统结合了应力触发的相分离过程， 应激过程中的表型后果，以及在生理状态下重建相分离的能力 浓度和条件。 Pab1/Poly(A)结合蛋白在真核生物中是保守的，是应激颗粒的核心标志，使其成为 确定应力诱导相分离的分子基础的有前途的模型。我们将学习帕布里的S 多尺度的相分离，包括RNA的影响，使用体外和体内的组合 确定这一独特的相分离过程如何编码的方法。 我们将测试特定的假设，例如RNA结合之间的静电相互作用 结合氢的突变方法，研究IDR之间的结构域和疏水相互作用 交换质谱仪(HX-MS)、核磁共振、动态光散射和小角X射线散射。我们 随着进一步发展中尺度分析，它提供了关于Pab1相互作用如何影响的丰富信息 由此产生的组件的性质(粘度、成核与生长、相变)被认为是 有助于发挥其生物学功能。该项目需要两名互为补充的调查人员的专业知识。 德拉蒙德带来了他在Pab1和压力生物学方面的丰富经验，而索斯尼克则带来了他在 蛋白质折叠和化学。

Project Summary The proposed studies will determine in molecular detail the mechanism(s) by which poly(A)-binding protein Pab1 phase-separates to form hydrogel assemblies during stress. Cellular stresses cause the evolutionarily conserved, putatively adaptive formation stress granules. Pab1 is a defining marker of stress granules, and Pab1's phase separation precedes stress granule formation. Dysregulation of phase separation of multiple RNA-binding proteins is linked to pathological protein aggregation associated with major neurodegenerative disorders. The mechanism(s) of phase separation is an area of active inquiry. The key barrier has been a lack of tractable in vitro models of biologically relevant phase separation by an RNA-binding protein. We have broken through this barrier. In published studies (Wallace et al., Cell 2015; Riback et al., Cell 2017), we have established that Pab1 phase-separates into hydrogel droplets in response to physiological stress conditions, and that interfering with hydrogel droplet formation disrupts budding yeast's ability to survive thermal and starvation stress. No previously described system combines a stress-triggered phase separation process, phenotypic consequences during stress, and the ability to reconstitute phase separation at physiological concentrations and conditions. Pab1/poly(A)-binding protein is conserved across eukaryotes and is a core marker of stress granules, making it a promising model for determining the molecular basis of stress-induced phase separation. We will study Pab1's phase separation at multiple scales, including the influence of RNA, using a combination in vitro and in vivo approaches to determine how this unique phase separation process is encoded. We will test specific hypotheses, such as the involvement of electrostatic interactions between RNA-binding domains and hydrophobic interactions between IDRs, using mutational approaches combined with hydrogen exchange mass spectrometry (HX-MS), NMR, dynamic light scattering, and small-angle X-ray scattering. We with further develop a mesoscale assay which provides rich information about how Pab1 interactions influence the properties of the resulting assemblies (viscosity, nucleation versus growth, changes in phase) thought to contribute to its biological functions. The project requires the expertise of two complementary investigators. Drummond brings his extensive experience in Pab1 and stress biology while Sosnick brings his expertise in protein folding and chemistry.