Kitamura Keiji, Tsukui Itta, Sasaki Fuka, Shiramasa Yutaro, Arayama Miyu, Morishita Manato, Oshima Ayano, Kitazawa Soichiro, Kameda Tomoshi, Kitahara Ryo
Graduate School of Pharmacy, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
J Mol Biol. 2025 Sep 1;437(17):169295. doi: 10.1016/j.jmb.2025.169295. Epub 2025 Jun 16.
Fused in sarcoma (FUS) is an RNA-binding protein, the aberrant aggregation of which is linked to amyotrophic lateral sclerosis (ALS). Liquid-liquid phase separation (LLPS) of FUS facilitates functional condensate formation and can drive pathological aggregation under certain conditions. The aggregation-inhibitory effects of ATP, a key cellular hydrotrope, have been reported for multiple proteins; however, how ATP, present at approximately 1-12 mM concentrations in cells, regulates LLPS and amyloid fibril formation remains unclear. Therefore, we investigated how ATP modulates the LLPS behavior and aggregation of FUS and its ALS-linked variants, R495X and P525L. ATP destabilized both normal LLPS and aberrant high-pressure LLPS (HP-LLPS), with a relatively strong inhibitory effect on HP-LLPS. Pressure-jump experiments demonstrated that ATP reduced the irreversible aggregation propensity of HP-LLPS, particularly in ALS variants that exhibited enhanced aggregation compared to that by wild-type FUS. Molecular dynamic simulations further revealed that the triphosphate and adenosine moieties of ATP synergistically disrupted intermolecular interactions that were crucial for phase separation, leveraging its amphipathic properties. Notably, ATP concentrations within the physiological range (1-12 mM) significantly inhibited FUS aggregation, suggesting a protective role in cellular environments. These results indicate that decreased intracellular ATP levels may exacerbate aberrant phase transitions of FUS, contributing to ALS onset. This study underscores the potential of ATP as a therapeutic modulator of protein phase separation and aggregation, providing valuable insights into the molecular mechanisms of ALS. Our findings open new avenues for targeting ATP-regulated pathways for treating neurodegenerative disorders.
肉瘤融合蛋白（FUS）是一种RNA结合蛋白，其异常聚集与肌萎缩侧索硬化症（ALS）有关。FUS的液-液相分离（LLPS）促进功能性凝聚物形成，并在某些条件下可驱动病理性聚集。ATP作为一种关键的细胞促溶剂，对多种蛋白质的聚集抑制作用已有报道；然而，细胞内浓度约为1-12 mM的ATP如何调节LLPS和淀粉样原纤维形成仍不清楚。因此，我们研究了ATP如何调节FUS及其与ALS相关变体R495X和P525L的LLPS行为和聚集。ATP使正常LLPS和异常的高压LLPS（HP-LLPS）均不稳定，对HP-LLPS的抑制作用相对较强。压力跳跃实验表明，ATP降低了HP-LLPS的不可逆聚集倾向，特别是在与野生型FUS相比表现出增强聚集的ALS变体中。分子动力学模拟进一步揭示，ATP的三磷酸和腺苷部分协同破坏了对相分离至关重要的分子间相互作用，利用了其两亲性特性。值得注意的是，生理范围内（1-12 mM）的ATP浓度显著抑制FUS聚集，表明在细胞环境中具有保护作用。这些结果表明，细胞内ATP水平降低可能会加剧FUS的异常相变，导致ALS发病。本研究强调了ATP作为蛋白质相分离和聚集治疗调节剂的潜力，为ALS的分子机制提供了有价值的见解。我们的发现为靶向ATP调节途径治疗神经退行性疾病开辟了新途径。
