Characterization of Ubiquitin and Ubiquitin-Like-Protein Isopeptidase Activities

Benjamin Nicholson, Craig A Leach, Seth J Goldenberg, Dana M Francis, Matthew P Kodrasov, Xufan Tian, John Shanks, David E Sterner, Alejandro Bernal, Michael R Mattern, Keith D Wilkinson, Tauseef R Butt

Protein Sci. 2008 Jun;17(6):1035-43.

PMID: 18424514

Abstract:

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 A(2) (PLA(2)). Isopeptidase activity releases PLA(2), 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-PLA(2) 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-PLA(2) 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP157654676	NSC 632839		157654-67-6	Price