Department of Physics, State Key Laboratory of Surface Physics, and Key Laboratory for Computational Physical Sciences (Ministry of Education), Fudan University, Shanghai 200438, P. R. China.
MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, School of Physics, Xi'an Jiaotong University, Xi'an 710049, P. R. China.
Phys Chem Chem Phys. 2022 Jul 6;24(26):16263-16273. doi: 10.1039/d2cp01625g.
Amyotrophic lateral sclerosis (ALS) is intensively associated with insoluble aggregates formed by transactivation response element DNA-binding protein 43 (TDP-43) in the cytoplasm of neuron cells. A recent experimental study reported that two ALS-linked familial variants, A315E and A315pT (pT, phosphorylated threonine), can induce irreversible aggregation of the TDP-43 NFGAFS segment (TDP-43). However, the underlying molecular mechanism remains largely elusive. Here, we investigated the early aggregation process of the wild type (WT) NFGAFS segment and its A315E and A315pT variants by performing multiple microsecond all-atom molecular dynamics simulations. Our simulations show that the two variants display lower fluidity than WT, consistent with their decreased labilities observed in previous denaturation assay experiments. Despite each of the two variants carrying one negative charge, unexpectedly, we find that both A315E mutation and A315pT phosphorylation enhance intermolecular interactions and result in the formation of more compact oligomers. Compared to WT, A315E oligomers possess low β-sheet content but a compact hydrophobic core, while A315pT oligomers have high β-sheet content and large β-sheets. Side chain hydrogen-bonding and hydrophobic interactions as well as N312-E315 salt bridges contribute most to the increased aggregation propensity of the A315E mutant. By contrast, main chain and side chain hydrogen-bonding interactions, side chain hydrophobic and aromatic interactions, are crucial to the enhanced aggregation capability of the A315pT variant. These results indicate that glutamate mutation and phosphorylation at position 315 induce the irreversible aggregation of TDP-43 peptides through differential mechanisms, which remind us that we should be careful in the investigation of the phosphorylation effect on protein aggregation by using phosphomimetic substitutions. This study provides mechanistic insights into the A315E/A315pT-induced irreversible aggregation of TDP-43, which may be helpful for the in-depth understanding of ALS-mutation/phosphorylation-associated liquid-to-solid phase transition of TDP-43 protein aggregates.
肌萎缩侧索硬化症（ALS）与神经元细胞质中转录激活反应元件结合蛋白 43（TDP-43）形成的不溶性聚集体密切相关。最近的一项实验研究报告称，两种与 ALS 相关的家族变体 A315E 和 A315pT（pT，磷酸化苏氨酸）可以诱导 TDP-43 NFGAFS 片段（TDP-43）的不可逆聚集。然而，其潜在的分子机制在很大程度上仍未被揭示。在这里，我们通过进行多次微秒全原子分子动力学模拟，研究了野生型（WT）NFGAFS 片段及其 A315E 和 A315pT 变体的早期聚集过程。我们的模拟表明，这两个变体的流动性低于 WT，这与之前变性实验中观察到的它们的稳定性降低一致。尽管这两个变体中的每一个都带有一个负电荷，但出乎意料的是，我们发现 A315E 突变和 A315pT 磷酸化都增强了分子间相互作用，导致形成更紧凑的寡聚物。与 WT 相比，A315E 寡聚物具有低的β-折叠含量但具有紧凑的疏水性核心，而 A315pT 寡聚物具有高的β-折叠含量和大的β-折叠。侧链氢键和疏水相互作用以及 N312-E315 盐桥对 A315E 突变体增加的聚集倾向贡献最大。相比之下，主链和侧链氢键相互作用、侧链疏水性和芳香族相互作用对于 A315pT 变体增强的聚集能力至关重要。这些结果表明，谷氨酸突变和 315 位磷酸化通过不同的机制诱导 TDP-43 肽的不可逆聚集，这提醒我们，在用磷酸模拟取代物研究磷酸化对蛋白质聚集的影响时应小心谨慎。这项研究为 A315E/A315pT 诱导的 TDP-43 不可逆聚集提供了机制上的见解，这可能有助于深入了解 ALS 突变/磷酸化相关的 TDP-43 蛋白聚集体从液相到固相的转变。
