Mechanisms for phase separation between TDP-43 and ubiquitin in vitro
10.7644/j.issn.1674-9960.2024.02.001
- VernacularTitle:TAR DNA结合蛋白43与泛素体外共相分离机制初步研究
- Author:
Lijuan HE
1
;
Lijie ZHOU
;
Yingwei GE
;
Lingqiang ZHANG
Author Information
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
- Keywords:
TAR DNA binding protein-43;
ubiquitin;
phase separation;
aggregates;
amyotrophic lateral sclerosis
- From:
Military Medical Sciences
2024;48(2):81-87
- CountryChina
- Language:Chinese
-
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.
