Ubiquitin Chloromethylketone Probe Enables Activity-Based Protein Profiling of E2 Ubiquitin Conjugating Enzymes.
6 affiliations
- 1Department of Biochemistry and Biophysics, College of Agriculture and Life Sciences, Texas A&M University, College Station, Texas77843, United States.
- 2Texas A&M Drug Discovery Center and Department of Chemistry, College of Arts and Sciences, Texas A&M University, College Station, Texas77843, United States.
- 3Texas A&M High Performance Research Computing, Texas A&M University, College Station, Texas77843, United States.
- 4Institute of Biosciences and Technology and Department of Translational Medical Sciences, School of Medicine, Texas A&M University, Houston, Texas77030, United States.
- 5Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, Texas77843, United States.
- 6Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University, College Station, Texas77843, United States.
Abstract
Ubiquitin (Ub) conjugating enzymes (E2s) are central to Ub signaling, yet their systematic activity-based profiling remains challenging because of the weak nucleophilicity and elevated pKa of their catalytic cysteines. Existing Ub probes primarily target deubiquitinases (DUBs), and the only reported E2-targeting probe requires E1-dependent activation to capture limited E2s. To profile E2s broadly, here, Ub chloromethylketone (UbCMK) is reported as a standalone activity-based probe. Density functional theory calculations identified CMK as a highly electrophilic warhead with a low activation barrier for reaction with weakly nucleophilic thiolates. UbCMK was synthesized via activated cysteine-based protein ligation and irreversibly labeled multiple E2s and cysteine DUBs. Activity-based protein profiling and quantitative proteomics in HEK293T cell lysates revealed broad enrichment of E2 enzymes, including many previously inaccessible to other probes. UbCMK furthermore enables activity-dependent quantification of endogenous E2 mobilization across oxidative, proteotoxic, inflammatory, metabolic, lipid oxidative, and genotoxic stress conditions. In addition, UbCMK engages both E1s and DUBs, as well, indicating its broad utility as a probe. Collectively, these results establish UbCMK as a powerful chemical tool that expands activity-based protein profiling coverage across the Ub-proteasome system and enables functional interrogation of E2 enzymes under physiological and pathological conditions.
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BibTeX
@article{chanda2026ubiquitin,
title = {{Ubiquitin Chloromethylketone Probe Enables Activity-Based Protein Profiling of E2 Ubiquitin Conjugating Enzymes.}},
author = {Saibal Chanda and Alan Pham and Yugendar R Alugubelli and Nathan Axtell and SreeNidhi Karnati and Yaswanth Aravapalli and James X Mao and Wenyue Cao and Wenshe Ray Liu},
journal = {Journal of the American Chemical Society},
year = {2026},
month = sep,
doi = {10.1021/jacs.6c08586},
url = {https://europepmc.org/article/MED/42937821},
note = {CC BY},
}
RIS
TY - JOUR
TI - Ubiquitin Chloromethylketone Probe Enables Activity-Based Protein Profiling of E2 Ubiquitin Conjugating Enzymes.
AU - Saibal Chanda
AU - Alan Pham
AU - Yugendar R Alugubelli
AU - Nathan Axtell
AU - SreeNidhi Karnati
AU - Yaswanth Aravapalli
AU - James X Mao
AU - Wenyue Cao
AU - Wenshe Ray Liu
PY - 2026
DA - 2026/09/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c08586
UR - https://europepmc.org/article/MED/42937821
AB - Ubiquitin (Ub) conjugating enzymes (E2s) are central to Ub signaling, yet their systematic activity-based profiling remains challenging because of the weak nucleophilicity and elevated pKa of their catalytic cysteines. Existing Ub probes primarily target deubiquitinases (DUBs), and the only reported E2-targeting probe requires E1-dependent activation to capture limited E2s. To profile E2s broadly, here, Ub chloromethylketone (UbCMK) is reported as a standalone activity-based probe. Density functional theory calculations identified CMK as a highly electrophilic warhead with a low activation barrier for reaction with weakly nucleophilic thiolates. UbCMK was synthesized via activated cysteine-based protein ligation and irreversibly labeled multiple E2s and cysteine DUBs. Activity-based protein profiling and quantitative proteomics in HEK293T cell lysates revealed broad enrichment of E2 enzymes, including many previously inaccessible to other probes. UbCMK furthermore enables activity-dependent quantification of endogenous E2 mobilization across oxidative, proteotoxic, inflammatory, metabolic, lipid oxidative, and genotoxic stress conditions. In addition, UbCMK engages both E1s and DUBs, as well, indicating its broad utility as a probe. Collectively, these results establish UbCMK as a powerful chemical tool that expands activity-based protein profiling coverage across the Ub-proteasome system and enables functional interrogation of E2 enzymes under physiological and pathological conditions.
SN - 0002-7863
SN - 1520-5126
ER -