State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, 320 Yueyang Road, Shanghai, 200031, P.R. China.
Department of Chemistry, Fudan University, 220 Handan Road, Shanghai, 200433, P.R. China.
Sci Rep. 2017 Jul 21;7(1):6196. doi: 10.1038/s41598-017-06263-3.
TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the β7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.
TDP-43 是一种核因子，在促进前体 mRNA 剪接中发挥作用。缺失 N 端结构域（NTD）和核定位信号（NLS）（即 TDP-35）导致其错误定位到细胞质并形成包含体。然而，NTD 在 TDP-43 活性和蛋白病中的作用仍知之甚少。本研究通过生化和生物物理方法，研究了 TDP-43 包含体形成和前体 mRNA 剪接中 NTD 的结构和功能。我们发现 TDP-43 NTD 在溶液中以浓度依赖的方式形成同源二聚体，并且形成分子间二硫键导致进一步的四聚化。基于 TDP-43 NTD 的 NMR 结构，通过突变和分子筛层析，确定了以β7 链周围的 Leu71 和 Val72 为中心的二聚化界面。细胞实验表明，N 端二聚化在保护 TDP-43 免受细胞质包含体形成和增强 TDP-43 在核内前体 mRNA 剪接活性方面发挥作用。这项研究可能为 TDP-43 的生理功能及其相关蛋白病提供机制见解。
