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

摘要 肌萎缩侧索硬化症(ALS)是一种危及生命的神经退行性疾病，可导致 大脑和脊髓中运动神经元的变性。目前既没有治愈的方法，也没有有效的方法 延缓病情发展的治疗方法。然而，最近新的遗传、生化和生物物理证据表明 应激颗粒是疾病发展的熔炉。应激颗粒是无膜细胞器，也称为 生物分子凝聚体，通过RNA结合蛋白和蛋白质的液液相分离(LLP)形成 核糖核酸。RNA结合蛋白的突变将液体状应激颗粒转化为固体包裹体。长期应激 颗粒组装也会产生类似的效果。这些观察指出了治疗的新机会 如果关于液体组件与固体组件的性质的关键开放问题可以得到回答，则可以进行干预。我们会 从而检验基于上述观察结果的压倒一切的假设，即RNA结合中的突变 蛋白质改变了相分离的驱动力，液体冷凝物的动态停止，以及 凝结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结合蛋白的相分离物理学，特别是关于其内在无序的低复杂性 足以调节相分离的区域(LCD)，是疾病的典型位置 突变。我们将使用hnRNPA1的LCD作为ALS相关RNA类的原型成员- 并将我们的研究扩展到FUS的LCD。Mittag和Pappu最近开发了 一种贴纸和间隔物模型，该模型基于识别相互之间的瞬时、内聚相互作用 芳香族氨基酸残基是相分离的主要驱动力。芳香残留物 在这个模型中是贴纸，间隔是连接贴纸的残基。该模型使 作为温度函数的完全共存曲线的定量预测，并且重要的是，导致了 我们对相分离是如何在LCD中编码的理解的概念性进展。免费赠品 Mittag和Pappu的专业知识现在将结合生物物理实验、计算和 关于以下三个具体目标的理论：(1)通过量化 不同类型的贴纸和垫片之间的相互作用。(2)检验疾病导致突变的假设 在肌萎缩侧索硬化症的液晶显示器内，引起肌萎缩侧索硬化症的RNA结合蛋白引起动态停止的相变。(三)揭开 侧链和骨架相互作用及其对液晶显示器空间组织的贡献 在稠密的相中。我们的结果将使对ALS相关突变的影响进行定量预测成为可能 关于相态行为。我们将清楚地了解特定于序列的相图如何起作用 相分离和老化现象的动力学。我们将确定潜在的交互类型 类液体和类固体致密相。这些结果将直接影响到治疗干预。 以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.