Phosphomimetic substitutions in TDP-43's transiently α-helical region suppress phase separation.
Phosphomimetic substitutions in TDP-43's transiently α-helical region suppress phase separation.
复制标题
TDP-43 的瞬时α 螺旋区域中的拟磷取代可抑制相分离。
DOI：
10.1016/j.bpj.2024.01.001
复制
发表时间：
2024
影响因子：
3.4
通讯作者：
Surewicz,WitoldK
中科院分区：
文献类型：
--
作者：
Haider,Raza;Penumutchu,Srinivasa;Boyko,Solomiia;Surewicz,WitoldK
43 kDa磷酸化的TAR dna结合蛋白（TDP-43）存在于几种与年龄相关的神经退行性疾病的聚集物中，如肌萎缩性侧索硬化症、额颞叶变性和阿尔茨海默病，以至于磷酸化的TDP-43的存在被认为是其中一些疾病的标志。在肌萎缩性侧索硬化症和额颞叶变性患者中检测到的大多数已知TDP-43磷酸化位点位于低复杂性结构域（LCD），该结构域已被证明对TDP-43液-液相分离（LLPS）至关重要。然而，这些LCD磷酸化位点对TDP-43 LLPS的影响在很大程度上尚未被探索，并且所做的任何工作主要集中在LCD的c端附近的位点。在这里，我们使用了一种拟磷方法来探索残基S332和S333的磷酸化对蛋白LLPS的影响，这些位点位于TDP-43的瞬时α-螺旋区，在疾病中被观察到被磷酸化。我们的浊度测定和荧光显微镜数据表明，这些拟磷取代极大地抑制了LLPS，溶液核磁共振数据强烈表明，这种影响至少部分是由于拟磷蛋白变体的α-螺旋倾向的丧失。我们还发现，S332D和S333D的替代减缓了TDP-43液晶液滴的老化和蛋白质的纤颤。总的来说，这些发现为了解TDP-43 LCD的瞬时α-螺旋区磷酸化对蛋白LLPS和纤颤的影响提供了生物物理基础，表明残基332和333的磷酸化不一定与致病过程直接相关。
Phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) is present within the aggregates of several age-related neurodegenerative disorders, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease, to the point that the presence of phosphorylated TDP-43 is considered a hallmark of some of these diseases. The majority of known TDP-43 phosphorylation sites detected in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients is located in the low-complexity domain (LCD), the same domain that has been shown to be critical for TDP-43 liquid-liquid phase separation (LLPS). However, the effect of these LCD phosphorylation sites on TDP-43 LLPS has been largely unexplored, and any work that has been done has mainly focused on sites near the C-terminal end of the LCD. Here, we used a phosphomimetic approach to explore the impact of phosphorylation at residues S332 and S333, sites located within the transientlyα-helical region of TDP-43 that have been observed to be phosphorylated in disease, on protein LLPS. Our turbidimetry and fluorescence microscopy data demonstrate that these phosphomimetic substitutions greatly suppress LLPS, and solution NMR data strongly suggest that this effect is at least in part due to the loss ofα-helical propensity of the phosphomimetic protein variant. We also show that the S332D and S333D substitutions slow TDP-43 LCD droplet aging and fibrillation of the protein. Overall, these findings provide a biophysical basis for understanding the effect of phosphorylation within the transientlyα-helical region of TDP-43 LCD on protein LLPS and fibrillation, suggesting that phosphorylation at residues 332 and 333 is not necessarily directly related to the pathogenic process.