Related Experiment Video
Updated: Aug 6, 2026

Protein Target Prediction and Validation of Small Molecule Compound
Published on: February 23, 2024
Mechanism of High-Affinity KEAP1 Recruitment by USP25 via a Specialized ETGQ Motif
Xuan Ning1, Fangning Hu2, Zihui Su3
1Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, China; College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, China.
Abstract:
The KEAP1-NRF2 axis is the main regulator of the cellular antioxidant response. Recent studies identified the deubiquitinase USP25 as a key stabilizer of KEAP1, but the molecular basis of their interaction is unclear. In this study, the result of gel filtration and isothermal titration calorimetry (ITC) show that the USP25 catalytic domain binds directly to the KEAP1 Kelch domain with high affinity. Using mutational analysis of the canonical Kelch binding pocket and simple sequence searching, we identify a non-canonical ETGQ motif in USP25. AlphaFold 3 structural modeling and structure-guided mutagenesis show that this ETGQ motif is required and sufficient for binding to the Kelch β-propeller. Because of this interaction, the closely related homolog USP28 does not regulate the KEAP1-NRF2 axis. This study defines the molecular architecture of the USP25-KEAP1 interface. It also provides a clear structural framework for the rational design of protein-protein interaction inhibitors aimed at treating oxidative stress-related diseases.
Related Concept Videos
The Proteasome Structure
The proteasome is an...
The Unfolded Protein Response
ER Retrieval Pathway
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms...
Regulation of the Unfolded Protein Response
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Tail-anchoring of Proteins in the ER Membrane

