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

项目摘要 拟议的研究将在分子细节上确定poly（A）结合蛋白Pab 1 相分离以在应力期间形成水凝胶组件。细胞压力导致进化上保守的， 泥质适应性地层应力颗粒。Pab 1是应力颗粒的定义标记，Pab 1的相位 分离先于应力颗粒形成。多种RNA结合蛋白相分离的失调 与主要神经退行性疾病相关的病理性蛋白质聚集有关。的 相分离的机理是一个积极研究的领域。关键的障碍是缺乏易处理的体外 RNA结合蛋白的生物相关相分离模型。 我们已经突破了这个障碍。在已发表的研究中（Wallace et al.，Cell 2015; Riback等人，Cell 2017）， 我们已经确定Pab 1在生理应激反应中相分离成水凝胶液滴 干扰水凝胶液滴的形成会破坏芽殖酵母在热环境中存活的能力， 饥饿的压力。先前没有描述的系统结合应力触发的相分离过程， 胁迫期间的表型后果，以及在生理条件下重建相分离的能力。 浓度和条件。 Pab 1/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物，使其成为一种重要的应激蛋白。 有前途的模型，用于确定应力诱导相分离的分子基础。我们将研究Pab 1的 使用体外和体内组合，在多个尺度上进行相分离，包括RNA的影响 方法来确定如何编码这种独特的相位分离过程。 我们将测试具体的假设，如RNA结合之间的静电相互作用的参与， 结构域和IDR之间的疏水相互作用，使用突变方法结合氢 交换质谱（HX-MS）、NMR、动态光散射和小角X射线散射。我们 进一步开发中尺度测定，其提供关于Pab 1相互作用如何影响 所得到的组件的性质（粘度、成核与生长、相变）被认为 有助于其生物学功能。该项目需要两名互补调查员的专门知识。 Drummond带来了他在Pab 1和压力生物学方面的丰富经验，而Sosnick则带来了他在以下方面的专业知识： 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.