University Health Network, Princess Margaret Cancer Centre , University of Toronto , TMDT 4-305, 101 College Street , Toronto , ON M5G 1L7 , Canada.
Department of Medical Biophysics, Princess Margaret Cancer Centre , University of Toronto , TMDT 4-305, 101 College Street , Toronto , ON M5G 1L7 , Canada.
Biochemistry. 2019 Feb 12;58(6):590-607. doi: 10.1021/acs.biochem.8b00842. Epub 2018 Dec 13.
Intraneuronal aggregation of TDP-43 is seen in 97% of all amyotrophic lateral sclerosis cases and occurs by a poorly understood mechanism. We developed a simple in vitro model system for the study of full-length TDP-43 aggregation in solution and in protein droplets. We found that soluble, YFP-tagged full-length TDP-43 (yTDP-43) dimers can be produced by refolding in low-salt HEPES buffer; these solutions are stable for several weeks. We found that physiological electrolytes induced reversible aggregation of yTDP-43 into 10-50 nm tufted particles, without amyloid characteristics. The order of aggregation induction potency was K < Na < Mg < Ca, which is the reverse of the Hofmeister series. The kinetics of aggregation were fit to a single-step model, and the apparent rate of aggregation was affected by yTDP-43 and NaCl concentrations. While yTDP-43 alone did not form stable liquid droplets, it partitioned into preformed Ddx4N1 droplets, showing dynamic diffusion behavior consistent with liquid-liquid phase transition, but then aggregated over time. Aggregation of yTDP-43 in droplets also occurred rapidly in response to changes in electrolyte concentrations, mirroring solution behavior. This was accompanied by changes to droplet localization and solvent exchange. Exposure to extracellular-like electrolyte conditions caused rapid aggregation at the droplet periphery. The aggregation behavior of yTDP-43 is controlled by ion-specific effects that occur at physiological concentrations, suggesting a mechanistic role for local electrolyte concentrations in TDP-43 proteinopathies.
TDP-43 在所有肌萎缩性侧索硬化症病例中的 97%中都存在细胞内聚集，其发生机制尚不清楚。我们开发了一种简单的体外模型系统，用于研究全长 TDP-43 在溶液中和蛋白液滴中的聚集。我们发现，通过在低盐 HEPES 缓冲液中重折叠可以产生可溶性、YFP 标记的全长 TDP-43（yTDP-43）二聚体；这些溶液可以稳定数周。我们发现，生理电解质诱导 yTDP-43 可逆聚集形成 10-50nm 的丛生颗粒，没有淀粉样特征。聚集诱导能力的顺序为 K < Na < Mg < Ca，与豪夫迈斯特系列相反。聚集的动力学符合单步模型，聚集的表观速率受 yTDP-43 和 NaCl 浓度的影响。虽然 yTDP-43 本身不能形成稳定的液滴，但它可以分配到预先形成的 Ddx4N1 液滴中，表现出与液-液相转变一致的动态扩散行为，但随着时间的推移会聚集。yTDP-43 在液滴中的聚集也会迅速响应电解质浓度的变化，反映出溶液行为。这伴随着液滴定位和溶剂交换的变化。暴露于类似细胞外的电解质条件会导致液滴边缘迅速聚集。yTDP-43 的聚集行为受生理浓度下发生的离子特异性效应控制，表明局部电解质浓度在 TDP-43 蛋白病中具有机制作用。
