The Differential Modulation of USP Activity by Internal Regulatory Domains, Interactors and Eight Ubiquitin Chain Types

SummaryUbiquitin-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 (kcat) and ubiquitin (Ub) binding (KM) between USPs. Further activity modulation by intramolecular domains affects both the kcat and KM, whereas the intermolecular activators UAF1 and GMPS mainly increase the kcat. 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.

► Developed synthetic Ub-isopeptide linked fluorescence polarization assay
► Kinetic analysis of twelve USPs
► Comprehensive activity analysis of twelve USPs against all eight di-Ub topoisomers
► Analysis of inter- and intramolecular activity modulation