Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No. 155 Section 2, Li-nong Street, Taipei, Taiwan.
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, No. 155 Section 2, Li-nong Street, Taipei, Taiwan.
Biochim Biophys Acta Proteins Proteom. 2018 Feb;1866(2):214-223. doi: 10.1016/j.bbapap.2017.10.001. Epub 2017 Oct 4.
The TAR DNA-binding protein of 43kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43's intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43's functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43's physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320-340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.
TDP-43 是一种 43kDa 的 TAR DNA 结合蛋白，已被鉴定为肌萎缩性侧索硬化症（ALS）细胞质内含物的主要成分。该蛋白病变与 TDP-43 固有无序的 C 端结构域之间的联系是众所周知的，但最近也表明，该结构域参与介导 TDP-43 功能的无膜细胞器的形成。支持这些无膜细胞器液-液相分离（LLPS）的机制仍然难以捉摸。但至关重要的是，这些因素可能是理解 TDP-43 生理和病理功能之间微妙平衡的关键。在这项研究中，我们使用核磁共振波谱和光学方法证明，C 端结构域中心（残基 320-340）的α-螺旋成分与蛋白质的自我缔合和 LLPS 有关。系统地分析了不同缓冲条件和不同温度下 ALS 相关 TDP-43 突变体（G298S、M337V 和 Q331K），我们证明这种相分离是由疏水相互作用驱动的，但被静电排斥所抑制。基于这些发现，我们合理地引入了一个突变体 W334G，并证明该突变体不会破坏这种α-螺旋倾向，但会破坏 LLPS。这种色氨酸可能是该蛋白 LLPS 中的关键残基。
