Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, 119260, Singapore.
Department of Biological Sciences, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, 119260, Singapore.
Biochem Biophys Res Commun. 2023 Jul 5;664:35-42. doi: 10.1016/j.bbrc.2023.04.101. Epub 2023 Apr 28.
hPFN1 mutations including C71G cause ALS by gain of toxicity but the mechanism still remains unknown. Stress granules (SGs) are formed by phase separation of the prion-like domain (PLD) of RNA-binding proteins including FUS, whose inclusion was also associated with ALS. C71G-hPFN1 triggers seed-dependent co-aggregation with FUS/TDP-43 to manifest the prion-like propagandation but its biophysical basis remains unexplored. Here by DIC imaging we first showed that three hPFN1 mutants have differential capacity in disrupting the dynamics of liquid droplets formed by phase separation of FUS prion-like domain (PLD). C71G-hPFN1 co-exists with the folded and unfolded states, thus allowing to simultaneously characterize conformations, hydrodynamics and dynamics of the interactions of both states with the phase separated FUS PLD by NMR. The results reveal that the folded state is not significantly affected while by contrast, the unfolded state has extensive interactions with FUS PLD. As a consequence, the dynamics of FUS liquid droplets become significantly reduced. Such interactions might act to recruit C71G-hPFN1 into the droplets, thus leading to the increase of the local concentrations and subsequent co-aggregation of C71G-hPFN1 with FUS. Our study sheds the first light on the biophysical basis by which hPFN1 mutants gain toxicity to cause ALS. As other aggregation-prone proteins have no fundamental difference from hPFN1 mutants, aggregation-prone proteins might share a common capacity in disrupting phase separation responsible for organizing various membrane-less organelles. As such, the mechanism for C71G-hPFN1 might also be utilized by other aggregation-prone proteins for gain of toxicity to trigger diseases and aging.
hPFN1 突变包括 C71G 通过获得毒性引起 ALS，但机制仍不清楚。应激颗粒 (SGs) 是由 RNA 结合蛋白的类朊病毒结构域 (PLD) 相分离形成的，包括 FUS，其包含物也与 ALS 有关。C71G-hPFN1 触发与 FUS/TDP-43 的种子依赖性共聚集，以表现出类朊病毒的传播，但其实质基础仍未被探索。在这里，我们通过 DIC 成像首次表明，三种 hPFN1 突变体在破坏 FUS 类朊病毒结构域 (PLD) 相分离形成的液滴动力学方面具有不同的能力。C71G-hPFN1 与折叠和未折叠状态共存，从而可以同时通过 NMR 对两种状态的构象、流体动力学和与相分离的 FUS PLD 的相互作用的动力学进行表征。结果表明，折叠状态没有受到显著影响，而相反，未折叠状态与 FUS PLD 有广泛的相互作用。因此，FUS 液滴的动力学显著降低。这种相互作用可能会将 C71G-hPFN1 招募到液滴中，从而导致 C71G-hPFN1 与 FUS 的局部浓度增加和随后的共聚集。我们的研究首次揭示了 hPFN1 突变体获得毒性引起 ALS 的生物物理基础。由于其他易于聚集的蛋白质与 hPFN1 突变体没有根本区别，因此易于聚集的蛋白质可能具有破坏相分离的共同能力，而相分离负责组织各种无膜细胞器。因此，C71G-hPFN1 的机制也可能被其他易于聚集的蛋白质用于获得毒性以引发疾病和衰老。
