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

项目总结/摘要 以液体到固体的蛋白质相变为特征的慢性神经退行性疾病，代表了一种主要的 公共卫生负担，并在全球范围内迅速增长。因此，了解分子基础， 可溶性蛋白质分子转化为病理性聚集体对于改善蛋白质是绝对关键 在压力和衰老过程中保持细胞内稳态。含有朊病毒样结构域的RNA结合蛋白（称为 以下称为朊病毒样RBP）在健康细胞中通过相分离形成可逆液体冷凝物，但 在退化细胞中发生聚集，如额颞叶痴呆（FTD）、多系统蛋白质病， 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 在朊病毒样RBP的病理性聚集中的冷凝，以及（B）液相的机制是什么 朊病毒样RBPs的体内平衡除了朊病毒样结构域外，许多RBP还含有富含精氨酸的低 复杂结构域（富R LCD），但其在相分离/聚集行为中的作用不太清楚。的 本研究的目的是（a）系统地评价液-液相分离在 朊病毒样结构域和富R 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，相分离的缩合物对蛋白质聚集的稳定性。PI将 测试富含R的LCD作为朊病毒样RBP在相内聚集的成核剂的假设 分离的冷凝物，而聚阴离子，如RNA和聚磷酸盐，结合到富R液晶临界 调节这种效果。在分子水平上，富R液晶通过多分支阳离子-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。 射程电荷调节引力。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 有效地抵消了富含R的LCD的“成核剂”功能，并促进朊病毒的液相稳态， 就像限制性商业惯例为了检验这些想法，将采用一种综合研究战略，其中包括一个强大的 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学的理论。该项目的成果预计将提供一个统一的 朊病毒样RBP液-固相变的分子机制以及如何 RNA/多磷酸盐结合调节这种破坏性的转化。我们的结果预计将普遍 适用于其他疾病相关的蛋白质系统，如阿尔茨海默病（AD）中的tau相分离。 通过揭示细胞聚阴离子，如RNA和多磷酸盐，如何促进液相稳态， 对抗聚集，我们设想未来的分子制剂的发展，将作为抑制剂靶向 LCD介导的异常相变。

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.