Chemically labeled ThUBD permits rapid and super-sensitive imaging of polyubiquitination signals

作者全名："Xiao, Weidi; Huang, Shuai; Gao, Yuan; Liu, Tong; Li, Naikang; Sui, Xinying; Wang, Yonghong; Yang, Guan; Chang, Lei; Kang, Xianjiang; Duan, Changzhu; Qin, Weijie; Xu, Ping; Li, Yanchang"

作者地址："[Xiao, Weidi; Huang, Shuai; Liu, Tong; Yang, Guan; Chang, Lei; Qin, Weijie; Xu, Ping; Li, Yanchang] Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, Inst Life, State Key Lab Prote, Beijing 102206, Peoples R China; [Gao, Yuan] Nanjing Agr Univ, Cent Lab Coll Hort, Nanjing 210095, Peoples R China; [Li, Naikang; Kang, Xianjiang; Xu, Ping; Li, Yanchang] Hebei Univ, Coll Life Sci, Hebei Prov Key Lab Res & Applicat Microbial Diver, Baoding 071002, Hebei, Peoples R China; [Sui, Xinying; Duan, Changzhu] Chongqing Med Univ, Mol Med & Canc Res Ctr, Dept Cell Biol & Genet, Chongqing 400016, Peoples R China; [Wang, Yonghong; Xu, Ping] Guizhou Univ, Sch Med, Guiyang 550025, Peoples R China; [Xu, Ping] Guangzhou Univ Chinese Med, State Key Lab Dampness Syndrome Chinese Med, Affiliated Hosp 2, Guangzhou 510120, Peoples R China; [Xu, Ping] China Med Univ, Sch Publ Hlth, Shenyang 110122, Peoples R China"

通信作者："Qin, WJ; Xu, P; Li, YC (通讯作者)，Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, Inst Life, State Key Lab Prote, Beijing 102206, Peoples R China.; Xu, P; Li, YC (通讯作者)，Hebei Univ, Coll Life Sci, Hebei Prov Key Lab Res & Applicat Microbial Diver, Baoding 071002, Hebei, Peoples R China.; Xu, P (通讯作者)，Guizhou Univ, Sch Med, Guiyang 550025, Peoples R China.; Xu, P (通讯作者)，Guangzhou Univ Chinese Med, State Key Lab Dampness Syndrome Chinese Med, Affiliated Hosp 2, Guangzhou 510120, Peoples R China.; Xu, P (通讯作者)，China Med Univ, Sch Publ Hlth, Shenyang 110122, Peoples R China."

来源：ANALYST

ESI学科分类：CHEMISTRY

WOS号：WOS:000821638000001

JCR分区：Q2

影响因子：4.2

年份：2022

卷号：147

期号：15

开始页：3434

结束页：3443

文献类型：Article

关键词：　

摘要："Polyubiquitination signal deliver diverse cellular signal, which have been recognized as a sophisticated ubiquitin code. The perception and transduction of ubiquitination signal depend on the specificity and sensitivity of the ubiquitin-binding domain. Accurate and sensitive detection of polyubiquitination signal is crucial for revealing the dynamic cellular ubiquitin-regulated events. Western blotting (WB) and immunohistochemistry (IHC) are the most widely used biochemical strategies to detect ubiquitination signal on substrates under diverse physiological and pathological conditions. However, anti-ubiquitin antibodies fail to reflect polyubiquitination signal unbiasedly because of their strong preference for K63-linked ubiquitin chains. Herein, we demonstrated that our previously developed tandem hybrid ubiquitin-binding domain (ThUBD) chemically labeled with a reporter group such as horseradish peroxidase (ThUBD-HRP) could significantly improve the robustness and sensitivity of polyubiquitination signal detection. This advanced method was named TUF-WB Plus (TUF-WB+). The TUF-WB+ method significantly increases the sensitivity and accuracy of ubiquitin detection and requires a shorter experimental operation time. Furthermore, it enables the ThUBD-HRP probe to function as a powerful tool for spatial in situ polyubiquitination detection in cells by immunohistochemistry. Our newly developed ThUBD-HRP probe and TUF-WB+ method provide a robust and powerful tool for ubiquitination signal detection with hypersensitivity in an unbiased manner."

基金机构："National Key R&D Program of China [2020YFE0202200, 2017YFA0505002, 2017YFA0505100, 2017YFA0505700]; National Natural Science Foundation of China [32071431, 31901037, 31870824, 91839302, 32070668]; Innovation Foundation of Medicine [19SWAQ17, 20SWAQX34, AWS17J008]; CAMS Innovation Fund for Medical Sciences [2019-I2M-5-017]; Beijing-Tianjin-Hebei Basic Research Cooperation Project [J200001]; Foundation of State Key Lab of Proteomics [SKLP-K201704, SKLP-K201901]"

基金资助正文："This study was supported by the National Key R&D Program of China (2020YFE0202200, 2017YFA0505002, 2017YFA0505100 and 2017YFA0505700), the National Natural Science Foundation of China (32071431, 31901037, 31870824, 91839302 and 32070668), the Innovation Foundation of Medicine (19SWAQ17, 20SWAQX34, and AWS17J008), the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-017), the Beijing-Tianjin-Hebei Basic Research Cooperation Project (J200001), and the Foundation of State Key Lab of Proteomics (SKLP-K201704 and SKLP-K201901)."