Lin Pin-Han, Wu Guan-Wei, Lin Yu-Hao, Huang Jing-Rou, Jeng U-Ser, Liu Wei-Min, Huang Jie-Rong
Institute of Biochemistry and Molecular Biology, National Yang Ming Chiao Tung University, No. 155 Section 2, Li-nong Street, Taipei 11221, Taiwan.
National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan.
ACS Chem Neurosci. 2024 Oct 2;15(20):3767-75. doi: 10.1021/acschemneuro.4c00503.
Intrinsically disordered regions (IDRs) in proteins can undergo liquid-liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than β-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to β-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.
蛋白质中的内在无序区域（IDR）可经历液-液相分离（LLPS）以进行功能组装，但这增加了形成与疾病相关的淀粉样原纤维的可能性。然而，并非所有淀粉样原纤维都是通过LLPS形成的，LLPS相对于原纤维形成中其他途径的重要性仍不清楚。我们使用硫黄素T（ThT）荧光、核磁共振（NMR）、透射电子显微镜（TEM）和广角X射线散射（WAXS）实验，在TDP-43（一种导致运动神经元疾病和痴呆的蛋白质，可发生LLPS）中研究了这个问题。使用从ThT修饰而来的荧光探针，该探针经过精心设计以靶向蛋白质组装而非β-折叠，并得到TEM图像的支持，我们提出在有利于LLPS的条件下观察到的双相ThT信号是由于无定形聚集体的存在。这些聚集体代表了一种中间状态，它偏离了直接形成以β-折叠为主的原纤维的途径。相比之下，在非LLPS条件下（低pH或在去除关键LLPS残基的构建体中的生理条件下），该蛋白质形成水凝胶。实时WAXS数据、ThT信号和TEM图像共同表明，凝胶化过程绕过了LLPS，但仍导致形成纤维状结构网络。我们认为，无论形成途径如何，TDP-43的IDR都会形成致病的淀粉样原纤维。我们的发现揭示了为什么在与TDP-43相关的疾病中会同时发现促进LLPS和抑制LLPS的突变体。
