军事医学2024，Vol.48Issue(2)：81-87,7.DOI:10.7644/j.issn.1674-9960.2024.02.001
TAR DNA结合蛋白43与泛素体外共相分离机制初步研究
Mechanisms for phase separation between TDP-43 and ubiquitin in vitro
何立娟 1周丽洁 1葛英为 1张令强1
作者信息
- 1. 军事科学院军事医学研究院生命组学研究所,医学蛋白质组全国重点实验室,北京 100850
- 折叠
摘要
Abstract
Objective To explore the characteristics and mechanism of phase separation between TAR DNA binding protein-43(TDP-43)and ubiquitin.Methods The TARDBP gene and its truncated genes were inserted into vectors to construct recombinant plasmids for expression and protein purification.The phase separation system of ubiquitin and TDP-43 was constructed in vitro.The characteristics of the droplets formed via liquid-liquid phase separation were observed by fluorescence microscopy.The plasmids of ubiquitin and TDP-43 were co-transfected into HEK293T cells to observe aggregates containing TDP-43 and ubiquitin and find out whether TDP-43 could be ubiquitinated.Results The GFP-8Ub,TDP-43 full-length(FL)and truncated proteins were purified.TDP-43 FL and C-terminal domain(CTD)proteins were able to form droplets via phase separation with ubiquitin.The droplets changed into solid-like aggregates after prolonged incubation.Insolvable aggregates containing TDP-43 and ubiquitin were formed.TDP-43 was ubiquitinated under stress conditions in HEK293T cells after being co-transfected with ubiquitin and TDP-43 recombinant plasmids.Conclusion TDP-43 undergoes co-phase separation with ubiquitin,mainly driven by the multivalent interaction between TDP-43′s CTD structural domain and ubiquitin.The droplets finally form aggregates with solid-like properties.Under stress conditions,especially when the protein homeostasis is disrupted,TDP-43 and ubiquitin form aggregates while TDP-43 is ubiquitinated.This study reveals the basic mechanism of TDP-43 co-phase separation with ubiquitin and liquid-solid transformation.关键词
TAR DNA结合蛋白43/泛素/相分离/聚集体/肌萎缩侧索硬化症Key words
TAR DNA binding protein-43/ubiquitin/phase separation/aggregates/amyotrophic lateral sclerosis分类
生物科学引用本文复制引用
何立娟,周丽洁,葛英为,张令强..TAR DNA结合蛋白43与泛素体外共相分离机制初步研究[J].军事医学,2024,48(2):81-87,7.
