Understanding the sequence and structural determinants of phase behavior of ALS-causing proteins

总结 肌萎缩侧索硬化症（ALS）是一种危及生命的神经退行性疾病， 大脑和脊髓中运动神经元的退化。目前既没有治愈方法， 治疗以减缓进展。然而，最近新的遗传、生物化学和生物物理证据表明， 应激颗粒作为疾病发展的坩埚。应激颗粒是无膜细胞器，也称为 生物分子缩合物，其通过RNA结合蛋白的液-液相分离（LLPS）形成， 核糖核酸RNA结合蛋白的突变将液体样应激颗粒转化为固体内含物。长期的压力 颗粒组装可导致类似的效果。这些观察结果指出了新的治疗机会， 如果能够回答关于液体与固体组件性质的关键未决问题，我们将 因此，测试了基于上述观察的总体假设，即RNA结合突变 蛋白质改变了相分离的驱动力，液体冷凝物的动力学停滞， 缩合物促进蛋白质原纤维形成的能力。因此，我们建议的研究将集中于 RNA结合蛋白质相分离的物理学，特别是它们内在无序的低复杂性 这些结构域（LCD）足以介导相分离并且是疾病的典型位置 突变我们将使用hnRNPA 1的LCD作为ALS相关RNA类的原型成员， 结合蛋白，并将我们的研究也扩展到FUS的LCD。米塔格和帕普最近开发了 一个基于识别短暂的、有凝聚力的相互作用的粘着和间隔模型， 芳香族氨基酸残基作为提供相分离的主要驱动力。芳香族残基 是这个模型中的贴纸，垫片是连接贴纸的残留物。该模型使 作为温度函数的完全共存曲线的定量预测，重要的是， 这是我们对液晶显示器中相位分离编码的理解的概念性进步。免费 Mittag和Pappu的专业知识现在将结合生物物理实验，计算和 理论有以下三个具体目标：（1）通过量化 不同类型的贴纸和垫片之间的相互作用。(2)为了验证致病基因突变 在导致ALS的RNA结合蛋白的LCD内，引起动态停滞的相变。(3)揭开 侧链和主干之间的相互作用及其对LCD空间组织的贡献 在致密相内。我们的研究结果将能够定量预测ALS相关突变体的影响， 阶段行为。我们将清楚地了解序列特定的相图如何起作用 相分离和老化现象的动力学。我们将识别潜在的交互类型 液相和固相致密相。这些结果将对治疗干预有直接影响 防止可能由动态停止的相分离引起的功能中断。

Summary Amyotrophic lateral sclerosis (ALS) is a life-threatening, neurodegenerative disease that causes the degeneration of motor neurons in the brain and spinal cord. There are currently neither a cure nor effective treatments to slow progression. However, recent new genetic, biochemical and biophysical evidence implicates stress granules as crucibles for disease development. Stress granules are membraneless organelles, also called biomolecular condensates, which form via liquid-liquid phase separation (LLPS) of RNA-binding proteins and RNA. Mutations in RNA-binding proteins convert liquid-like stress granules into solid inclusions. Prolonged stress granule assembly can result in similar effects. These observations point to new opportunities for therapeutic interventions if key open questions regarding the nature of liquid vs. solid assemblies can be answered. We will thus test the overarching hypothesis, which is based on above observations, that mutations in RNA-binding proteins change the driving forces for phase separation, the dynamical arrest of the liquid condensates and the ability of the condensates to promote the formation of protein fibrils. Our proposed studies will thus focus on the physics of phase separation of RNA-binding proteins, specifically on their intrinsically disordered low-complexity domains (LCDs) that are sufficient for mediating phase separation and are the typical locations of disease mutations. We will use the LCD of hnRNPA1 as an archetypal member of the class of ALS-associated RNA- binding proteins and will extend our studies also to the LCD of FUS. Mittag and Pappu have recently developed a stickers-and-spacers model that is based on the identification of transient, cohesive interactions amongst aromatic amino acid residues as providing the main driving force for phase separation. The aromatic residues are the stickers in this model, the spacers are the residues that connect the stickers. The model enables the quantitative prediction of full coexistence curves as a function of temperature and, importantly, resulted in a conceptual advancement of our understanding of how phase separation is encoded in LCDs. The complimentary expertise of Mittag and Pappu will now bring to bear a combination of biophysical experiments, computation and theory on the following three specific aims: (1) To extend the stickers-and-spacers model by quantifying the interplay among different types of stickers and spacers. (2) To test the hypothesis that disease causing mutations within LCDs of ALS-causing RNA-binding proteins cause dynamically arrested phase transitions. (3) To uncover the interplay among sidechain and backbone interactions and their contributions to spatial organization of LCDs within dense phases. Our results will enable quantitative predictions of the effects of ALS-associated mutants on phase behavior. We will obtain a clear understanding of how sequence-specific phase diagrams contribute to the dynamics of phase separation and aging phenomena. We will identify the types of interactions underlying liquid-like and solid-like dense phases. These results will have a direct bearing on therapeutic interventions against the functional disruptions that are likely to be caused by dynamically arrested phase separation.