Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

Jamie Jon Arnold, Alejandro Bernal, Uzo Uche, David E. Sterner, Tauseef R. Butt, Craig Eugene Cameron, Michael R. Mattern

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.

Original languageEnglish (US)
Pages (from-to)214-221
Number of pages8
JournalAnalytical Biochemistry
Volume350
Issue number2
DOIs
StatePublished - Mar 15 2006

Fingerprint

Ubiquitins
Poliovirus
DNA-Directed RNA Polymerases
Ubiquitin
Assays
Proteins
Ubiquitination
Enzymes
RNA Replicase
Eukaryotic Cells
Proteasome Endopeptidase Complex
Ligases
Throughput
isopeptidase
Peptide Hydrolases
Fusion reactions

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Arnold, J. J., Bernal, A., Uche, U., Sterner, D. E., Butt, T. R., Cameron, C. E., & Mattern, M. R. (2006). Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity. Analytical Biochemistry, 350(2), 214-221. https://doi.org/10.1016/j.ab.2005.11.001
Arnold, Jamie Jon ; Bernal, Alejandro ; Uche, Uzo ; Sterner, David E. ; Butt, Tauseef R. ; Cameron, Craig Eugene ; Mattern, Michael R. / Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity. In: Analytical Biochemistry. 2006 ; Vol. 350, No. 2. pp. 214-221.
@article{fdd7853e61164349a91672301cd51b26,
title = "Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity",
abstract = "The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.",
author = "Arnold, {Jamie Jon} and Alejandro Bernal and Uzo Uche and Sterner, {David E.} and Butt, {Tauseef R.} and Cameron, {Craig Eugene} and Mattern, {Michael R.}",
year = "2006",
month = "3",
day = "15",
doi = "10.1016/j.ab.2005.11.001",
language = "English (US)",
volume = "350",
pages = "214--221",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

Arnold, JJ, Bernal, A, Uche, U, Sterner, DE, Butt, TR, Cameron, CE & Mattern, MR 2006, 'Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity', Analytical Biochemistry, vol. 350, no. 2, pp. 214-221. https://doi.org/10.1016/j.ab.2005.11.001

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity. / Arnold, Jamie Jon; Bernal, Alejandro; Uche, Uzo; Sterner, David E.; Butt, Tauseef R.; Cameron, Craig Eugene; Mattern, Michael R.

In: Analytical Biochemistry, Vol. 350, No. 2, 15.03.2006, p. 214-221.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

AU - Arnold, Jamie Jon

AU - Bernal, Alejandro

AU - Uche, Uzo

AU - Sterner, David E.

AU - Butt, Tauseef R.

AU - Cameron, Craig Eugene

AU - Mattern, Michael R.

PY - 2006/3/15

Y1 - 2006/3/15

N2 - The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.

AB - The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.

UR - http://www.scopus.com/inward/record.url?scp=33644674424&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33644674424&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2005.11.001

DO - 10.1016/j.ab.2005.11.001

M3 - Article

C2 - 16356462

AN - SCOPUS:33644674424

VL - 350

SP - 214

EP - 221

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -