TAR DNA-binding protein 43 (TDP-43) is an RNA-regulating protein that carries out many cellular functions through liquid-liquid phase separation (LLPS). The LLPS of TDP-43 is mediated by its C-terminal low-complexity domain (TDP43-LCD) corresponding to the region 267–414. In neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia, pathological inclusions of the TDP-43 are found that are rich in the C-terminal fragments of ∼25 and ∼35 kDa, of which TDP43-LCD is a part. Thus, understanding the assembly process of TDP43-LCD is essential, given its involvement in the formation of both functional liquid-like assemblies and solid- or gel-like pathological aggregates. Here, we show that the solution pH and salt modulate TDP43-LCD LLPS. A gradual reduction in the pH below its isoelectric point of 9.8 results in a monotonic decrease of TDP43-LCD LLPS due to charge-charge repulsion between monomers, while at pH 6 and below no LLPS was observed. The addition of heparin to TDP43-LCD solution at pH 6, at a 1:2 heparin-to-TDP43-LCD molar ratio, promotes TDP43-LCD LLPS, while at higher concentration, it disrupts LLPS through a reentrant phase transition. Upon incubation at pH 6, TDP43-LCD undergoes gelation without phase separation. However, in the reentrant regime in the presence of a high heparin concentration, it forms thick amyloid aggregates that are significantly more SDS resistant than the gel. The results indicate that the material nature of the TDP43-LCD assembly products can be modulated by heparin which is significant in the context of liquid-to-solid phase transition observed in TDP-43 proteinopathies. Our findings are also crucial in relation to similar transitions that could occur due to alteration in the molecular level interactions among various multivalent biomolecules involving other LCDs and RNAs.

TAR DNA 结合蛋白 43 (TDP-43) 是一种 RNA 调节蛋白，通过液-液相分离 (LLPS) 执行许多细胞功能。TDP-43 的 LLPS 由其对应于区域 267-414 的 C 端低复杂性结构域 (TDP43-LCD) 介导。在神经退行性疾病肌萎缩侧索硬化症和额颞叶痴呆中，发现 TDP-43 的病理包涵体富含~25 和~35 kDa 的 C 端片段，TDP43-LCD 是其中的一部分。因此，鉴于 TDP43-LCD 参与功能性液体状组装体和固体或凝胶状病理聚集体的形成，了解 TDP43-LCD 的组装过程至关重要。在这里，我们展示了溶液 pH 值和盐调节 TDP43-LCD LLPS。pH 值逐渐降低至等电点 9.8 以下，由于单体之间的电荷-电荷排斥，导致 TDP43-LCD LLPS 单调下降，而在 pH 值 6 及以下时，未观察到 LLPS。在 pH 6 的 TDP43-LCD 溶液中添加肝素，肝素与 TDP43-LCD 摩尔比为 1:2，可促进 TDP43-LCD LLPS，而在较高浓度下，它会通过重入相变破坏 LLPS。在 pH 6 下孵育后，TDP43-LCD 会发生凝胶化而不会发生相分离。然而，在存在高肝素浓度的重入状态下，它会形成较厚的淀粉样蛋白聚集体，其比凝胶具有明显更强的 SDS 耐受性。结果表明，TDP43-LCD 组装产品的材料性质可以通过肝素进行调节，这在 TDP-43 蛋白质病中观察到的液-固相变过程中具有重要意义。我们的发现对于由于涉及其他 LCD 和 RNA 的各种多价生物分子之间的分子水平相互作用的改变而可能发生的类似转变也至关重要。