Citation: Shuai Peng, Shaowen Wang, Xiaotong Liu, Hongrui Xu, Guoqiang Xu, Jia-Bin Li. E. coli-mediated expedient biosynthesis of biotin-labeled ubiquitin probe enables activity-based profiling of deubiquitinases in cytoplasmic and nuclear compartments[J]. Chinese Chemical Letters, ;2026, 37(8): 111760. doi: 10.1016/j.cclet.2025.111760 shu

E. coli-mediated expedient biosynthesis of biotin-labeled ubiquitin probe enables activity-based profiling of deubiquitinases in cytoplasmic and nuclear compartments

Shuai Peng ^a ,
Shaowen Wang ^a ,
Xiaotong Liu ^a ,
Hongrui Xu ^{b, *,} ,
Guoqiang Xu ^{a, c, d, e, *,} ,
Jia-Bin Li ^{a, *,}

a.
Jiangsu Key Laboratory of Drug Discovery and Translational Research for Brain Diseases, College of Pharmaceutical Sciences, The Fourth Affiliated Hospital of Soochow University, Jiangsu Province Engineering Research Center of Precision Diagnostics and Therapeutics Development, Jiangsu Key Laboratory of Preventive and Translational Medicine for Major Chronic Non-communicable Diseases, Suzhou Key Laboratory of Drug Research for Prevention and Treatment of Hyperlipidemic Diseases, Soochow University, Suzhou 215123, China
b.
Suzhou Municipal Center for Disease Control and Prevention, Suzhou 215004, China
c.
Suzhou International Joint Laboratory for Diagnosis and Treatment of Brain Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China
d.
MOE Key Laboratory of Geriatric Diseases and Immunology, Suzhou Medical College of Soochow University, Suzhou 215123, China
e.
Suzhou Key Laboratory of Geriatric Neurological Disorders, the First People’s Hospital of Taicang, Taicang Affiliated Hospital of Soochow University, Suzhou 215400, China

christinerania@163.com

gux2002@suda.edu.cn

lijiabin@suda.edu.cn

Received Date: 6 June 2025
Revised Date: 25 August 2025
Accepted Date: 27 August 2025
Available Online: 28 August 2025

Figures(4)

Deubiquitinases (DUBs) that remove conjugated ubiquitin from substrates are critical regulators of protein homeostasis and cellular signaling. Understanding the spatial distribution of DUBs in subcellular compartments is essential for uncovering their roles in stress responses and disease pathogenesis. Herein, we developed an Escherichia coli (E. coli)-based biosynthetic strategy for the expedient generation of biotin-labeled ubiquitin probe (Biotin-Ub-PA) through co-expression of Avi-Ub and BirA in E. coli, thus bypassing tedious chemical synthesis. Combined with affinity purification and proteomics, we utilized the biosynthetic probe to covalently capture active DUBs in subcellular compartments and revealed the distribution of DUBs in the cytoplasm and nucleus. As a proof of concept, hydrogen peroxide-induced activity changes of DUBs in the cytoplasm and nucleus were analyzed by the same probe. The results suggest that some DUBs, such as ubiquitin-specific protease 36 (USP36) and USP16, may undergo subcellular translocation and activity alterations under oxidative stress. Our study presents a reliable workflow for the spatial distribution analysis of DUBs under different stimuli or disease conditions.
- Activity-based ubiquitin probes,
- Chemical protein synthesis,
- Biotin-labeling,
- Deubiquitinase,
- Proteomic profiling
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