Monahan Zachary, Ryan Veronica H, Janke Abigail M, Burke Kathleen A, Rhoads Shannon N, Zerze Gül H, O'Meally Robert, Dignon Gregory L, Conicella Alexander E, Zheng Wenwei, Best Robert B, Cole Robert N, Mittal Jeetain, Shewmaker Frank, Fawzi Nicolas L
Department of Pharmacology and Molecular Therapeutics, Uniformed Services University, Bethesda, MD, USA.
Neuroscience Graduate Program, Brown University, Providence, RI, USA.
EMBO J. 2017 Oct 16;36(20):2951-2967. doi: 10.15252/embj.201696394. Epub 2017 Aug 8.
Neuronal inclusions of aggregated RNA-binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation-prone, yeast prion-like, low sequence-complexity domain (LC) is known to be targeted for phosphorylation. Here we map and in-cell phosphorylation sites across FUS LC We show that both phosphorylation and phosphomimetic variants reduce its aggregation-prone/prion-like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self-interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity. Hence, post-translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.
肉瘤融合蛋白（FUS）聚集形成的神经元内含物是肌萎缩侧索硬化症（ALS）和额颞叶痴呆亚型的标志。有趣的是，FUS具有几乎不带电荷、易于聚集、类似酵母朊病毒、低序列复杂性的结构域（LC），已知该结构域会发生磷酸化。在此，我们绘制了FUS LC的体外和细胞内磷酸化位点图谱。我们发现，磷酸化和模拟磷酸化变体均降低了其易于聚集/类似朊病毒的特性，在存在RNA或盐的情况下破坏了FUS的相分离，并降低了FUS的聚集倾向。核磁共振波谱表明，FUS LC磷酸化后仍保留其内在无序结构；然而，模拟磷酸化会减少瞬时结构域折叠和自相互作用。此外，我们表明模拟磷酸化的FUS可减少人和酵母细胞模型中的聚集，并可改善FUS相关的细胞毒性。因此，翻译后修饰可能是细胞控制生理组装并防止病理性蛋白质聚集的一种机制，这提示了一条适合药物调节的潜在治疗途径。
