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Published online before print April 18, 2008, 10.1110/ps.083450408
Protein Science (2008), 17:1035-1043. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 The Protein Society
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Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities

Benjamin Nicholson1, Craig A. Leach1, Seth J. Goldenberg1, Dana M. Francis1, Matthew P. Kodrasov1, Xufan Tian1, John Shanks2, David E. Sterner1, Alejandro Bernal1, Michael R. Mattern1, Keith D. Wilkinson2, and Tauseef R. Butt1

1 Division of Research and Development, Progenra, Inc., Malvern, Pennsylvania 19355, USA
2 Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA

(RECEIVED January 14, 2008; FINAL REVISION March 8, 2008; ACCEPTED March 10, 2008)

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A2 (PLA2). Isopeptidase activity releases PLA2, which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA2 assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA2 assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.

Keywords: ubiquitin; deubiquitylase; deSUMOylase; deISGylase; deNEDDylase


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R. J. Peroutka, N. Elshourbagy, T. Piech, and T. R. Butt
Enhanced protein expression in mammalian cells using engineered SUMO fusions: Secreted phospholipase A2
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[Abstract] [Full Text] [PDF]


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