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Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities

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

Reprint requests to: Benjamin Nicholson, Progenra, Inc., 271A Great Valley Parkway, Malvern, PA 19355, USA; e-mail: nicholson{at}progenra.com; fax: (610) 647-4705.

Abbreviations: DTT, dithiothreitol; DUB, deubiquitylase; EDTA, Ethylenediaminetetraacetic acid; FRET, fluorescence resonance energy transfer; HTS, high-throughput screening; IPTG, isopropyl β-D-1-thiogalactopyranoside; MJD, Machado–Joseph domain; NBD C₆-HPC, 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine; MOI, multiplicity of infection; NEM, N-Ethylmaleimide; OTU, ovarian tumor related; PCR, polymerase chain reaction; PLA₂, phospholipase A₂; PLP2, papain-like protease 2; RFU, relative fluorescence unit; SARS-CoV PLpro, severe acute respiratory syndrome coronavirus papain-like protease; S/B, signal, background; Ub, ubiquitin; Ub-ald, ubiquitin aldehyde; Ub-AMC, Ub-7-amino-4-methylcoumarin; UBL, ubiquitin-like; UCH, ubiquitin C-terminal hydrolase; USP/UBP, ubiquitin-specific protease.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.083450408.

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