ALS 相关的 FUS 突变使 G-四链体依赖性液-液相分离和液-固转变失调。,Journal of Biological Chemistry

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ALS 相关的 FUS 突变使 G-四链体依赖性液-液相分离和液-固转变失调。
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2021-10-06 , DOI: 10.1016/j.jbc.2021.101284
Akira Ishiguro ₁ , Jun Lu ₂ , Daisaku Ozawa ₃ , Yoshitaka Nagai ₃ , Akira Ishihama ₁

Affiliation

肌萎缩侧索硬化 (ALS) 是一种神经退行性疾病，其特征是运动神经元中蛋白质聚集体的积累。最近在 ALS 患者中发现的基因突变促进了对 ALS 复杂分子机制的研究。FUS（融合在肉瘤中）是一种代表性的 ALS 相关 RNA 结合蛋白 (RBP)，可特异性识别 G-四链体 (G4)-DNA/RNA。然而，从未研究过与 ALS 相关的 FUS 突变对 G4-RNA 结合活性和相位行为的影响。使用纯化的全长 FUS，我们分析了由与 G4 结合的多个功能模块组成的多域结构的分子机制。在这里，我们成功地观察到 FUS 冷凝物形成的液-液相分离 (LLPS) 和随后导致 FUS 聚集体形成的液固转变 (LST)。FUS 与 G4-RNA 的相互作用显着促进了这一过程。为了进一步研究，我们在多域结构中总共选择了 8 个具有代表性的 ALS 相关 FUS 突变体并纯化了这些蛋白质。所有 G4-RNA 识别缺陷的 ALS 相关 FUS 突变体都失去了 G4-RNA 依赖性 LLPS 和 LST 通路的调节，支持 G4-RNA 在该过程中的重要作用。值得注意的是，导致青少年 ALS 的 P525L 突变对 G4-RNA 结合和 FUS 聚集的影响最大。

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ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid-liquid phase separation and liquid-to-solid transition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid-liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.

更新日期：2021-10-05

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