Mechanism of liquid phase homeostasis of prion-like RNA binding proteins

朊病毒样RNA结合蛋白的液相稳态机制

• 批准号: 9809312
• 负责人: Priya R. Banerjee
• 金额: $ 23.75万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9809312
• 关键词: Address; Age of Onset; Aging; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Arginine; Behavior; Binding; Biological Models; Biology; C-terminal; Cations; Cells; Charge; Coupled; Development; Dipeptides; Disease; Educational Models; Electrostatics; Equilibrium; Family; Fluorescence Microscopy; Fluorescence Spectroscopy; Frontotemporal Dementia; Future; Glycine; Goals; Homeostasis; Huntington Disease; Lead; Link; Liquid substance; Mediating; Membrane; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; N-terminal; Neurodegenerative Disorders; Neutrons; Parkinson Disease; Pathologic; Pattern; Phase; Phase Transition; Physical condensation; Physics; Play; Polymers; Polyphosphates; Proteins; Public Health; RNA; RNA Binding; RNA-Binding Proteins; Research; Role; Science; Solid; Solubility; Stress; Structure; System; Testing; amyloid formation; base; flexibility; frontotemporal lobar dementia-amyotrophic lateral sclerosis; in vivo; inhibitor/antagonist; link protein; polyanion; prion-like; protein aggregation; proteostasis; self assembly; single molecule; stress granule; tau Proteins; theories

Project Summary/Abstract Age-onset neurodegenerative diseases, hallmarked by liquid-to-solid protein phase transition, represent a major public health burden and are rapidly growing worldwide. Therefore, understanding the molecular bases by which soluble protein molecules transform into pathological aggregates is absolutely crucial to ameliorate protein homeostasis in cells during stress and aging. RNA-binding proteins containing a prion-like domain (termed hereafter as prion-like RBPs) form reversible liquid condensates by phase separation in a healthy cell, but undergo aggregation in degenerating cells as in frontotemporal dementia (FTD), multisystem proteinopathy, and amyotrophic lateral sclerosis (ALS). This raises two critical questions: (a) what is the role of liquid phase condensation in pathological aggregation of prion-like RBPs, and (b) what is the mechanism of liquid phase homeostasis of prion-like RBPs? Besides a prion-like domain, many RBPs also contain an arginine-rich low complexity domain (R-rich LCD), but its role in their phase separation/aggregation behavior is less clear. The goals of this proposed research are to (a) systematically evaluate the roles of liquid-liquid phase separation in the co-aggregation of prion-like domain and R-rich LCD, and (b) examine how cellular polyanions promote the stability of phase separated condensates against protein aggregation, at the single-molecule level. The PI will test the hypotheses that the R-rich LCDs act as a nucleator for prion-like RBP aggregation within the phase separated condensate, whereas polyanions, such as RNA and polyphosphate, binding to R-rich LCD critically regulates this effect. At the molecular level, R-rich LCDs bind to prion-like sequences by multi-pronged cation-π interactions, whereas polyanion binding is conferred by a combination of long-range electrostatic and short- range charge regulated attraction. Since the range and strength of electrostatic interactions (~ 1/r; long-range) are greater than the cation-π interactions (~ 1/r3; short-range), we envision that RNA/polyphosphate will effectively counteract the “nucleator” function of R-rich LCDs and promote liquid -phase homeostasis of prion- like RBPs. To test these ideas, an integrated research strategy will be employed that encompasses a powerful combination of quantitative fluorescence microscopy, single-molecule fluorescence spectroscopy, small-angle neutron scattering, and polymer physics-based theories. Results of this project are expected to provide a unified view of the molecular mechanism of liquid-to-solid phase transition of prion-like RBPs and how RNA/polyphosphate binding regulates this devastating transformation. Our results are expected to be generally applicable to other disease-linked protein systems, such as tau phase separation in Alzheimer’s disease (AD). By uncovering how cellular polyanions, such as RNA and polyphosphate, promote liquid phase homeostasis and counteract aggregation, we envision future development of molecular agents that will serve as inhibitors targeting LCD-mediated aberrant phase transition.

项目概要/摘要 以液体到固体蛋白质相变为标志的年龄发病的神经退行性疾病是一种主要的疾病 公共卫生负担在全球范围内迅速增长。因此，了解其分子基础 可溶性蛋白质分子转化为病理性聚集体对于改善蛋白质绝对至关重要 压力和衰老过程中细胞的稳态。含有朊病毒样结构域的 RNA 结合蛋白（称为 下文称为朊病毒样 RBP）通过在健康细胞中相分离形成可逆液体冷凝物，但是 在退化细胞中发生聚集，如额颞叶痴呆 (FTD)、多系统蛋白病和 肌萎缩侧索硬化症（ALS）。这就提出了两个关键问题：(a) 液相的作用是什么 类朊病毒RBP病理性聚集的凝结，以及(b)液相的机制是什么 朊病毒样 RBP 的稳态？除了类似朊病毒的结构域外，许多 RBP 还含有富含精氨酸的低 复杂域（R-rich LCD），但其在相分离/聚集行为中的作用尚不清楚。这 这项研究的目标是 (a) 系统地评估液-液相分离在 朊病毒样结构域和富含 R 的 LCD 的共聚集，以及 (b) 检查细胞聚阴离子如何促进 在单分子水平上相分离冷凝物对蛋白质聚集的稳定性。 PI 将 测试以下假设：富含 R 的 LCD 充当相内类朊病毒 RBP 聚集的成核剂 分离冷凝物，而聚阴离子，如 RNA 和多磷酸盐，与富含 R 的 LCD 关键结合 调节这种效应。在分子水平上，富含 R 的 LCD 通过多管阳离子-π 与朊病毒样序列结合 相互作用，而聚阴离子结合是由长程静电和短程静电的组合赋予的。 范围收费调节吸引力。由于静电相互作用的范围和强度（​​~ 1/r；长程） 大于阳离子-π 相互作用（~ 1/r3；短程），我们设想 RNA/多磷酸盐将 有效抵消富含R的LCD的“成核剂”功能，促进朊病毒的液相稳态 像 RBP 一样。为了检验这些想法，将采用综合研究策略，其中包含强大的 结合定量荧光显微镜、单分子荧光光谱、小角度 中子散射和基于聚合物物理的理论。该项目的结果预计将提供一个统一的 类朊病毒RBP的液固相变分子机制的观点以及如何 RNA/多磷酸盐结合调节这种毁灭性的转变。我们的结果预计一般 适用于其他与疾病相关的蛋白质系统，例如阿尔茨海默病 (AD) 中的 tau 相分离。 通过揭示细胞聚阴离子（例如 RNA 和聚磷酸盐）如何促进液相稳态和 抵消聚集，我们设想未来开发将作为靶向抑制剂的分子制剂 LCD 介导的异常相变。