Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach.

Yan Liu,Lei Xu,Yali Shen,Yang Song,Jiayu Liao,J Jefferson P P Perry

doi:10.3390/biom11050673

Abstract

Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.

Highlights

We have recently developed the highly sensitive and qFRET-based protease assay, so as to characterize Sentrin/SUMO-specific proteases (SENPs)’ endopeptidase kinetics [27,28,29,30]
The substrate digestions were monitored over 5 min with 10 s intervals (Figure 6) and the initial under ([S] = 100 nM) were derived by the above qFRET analysis (Table111).of The results indicated that SENP1 exhibited higher activity toward SUMO deconjugation than pre-SUMO maturation
In our systematical effort of developing a qFRET universal assay methodology to determine all kinetics of SUMOylation cascade and others, we have focused on developing a qFRET analysis method for various biochemical reactions

Summary

Introduction

Reversible post-translational modification of proteins by small chemical groups, sugars, lipids, and polypeptides is an important means to alter their function, activity, or localization after their synthesis have been completed [1,2]. SUMO (small ubiquitin-like modifier) covalently modifies and regulates the activities of proteins that have important roles in diverse cellular processes, including cell cycle regulation, cell survival and apoptosis, DNA damage responses, and stress responses [2,3,4,5,6]. SUMO conjugation occurs through a cascade of reactions performed by an activating enzyme (E1), a conjugating enzyme (E2) and, usually, a SUMO ligase (E3). RanGAP1 was the first identified SUMO target, and its SUMOylation cycle mediates the constant shuttling of this protein between the cytoplasm and nucleus [7]

Methods

Discussion

Conclusion