Related Experiment Video
Updated: May 26, 2026

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay
Published on: September 27, 2024
The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain
Alex C Faesen1, Mark P A Luna-Vargas, Paul P Geurink
1Division of Biochemistry and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Abstract:
Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k(cat)) and ubiquitin (Ub) binding (K(M)) between USPs. Further activity modulation by intramolecular domains affects both the k(cat) and K(M), whereas the intermolecular activators UAF1 and GMPS mainly increase the k(cat). Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.
Related Concept Videos
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Regulated Protein Degradation
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
Regulated Protein Degradation
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
Protein Complexes with Interchangeable Parts
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Regulation of Expression at Multiple Steps

