Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity.
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.
Arnold
Jamie J
JJ
201 Althouse Laboratory, Pennsylvania State University, University Park, PA 16802, USA.
Bernal
Alejandro
A
Uche
Uzo
U
Sterner
David E
DE
Butt
Tauseef R
TR
Cameron
Craig E
CE
Mattern
Michael R
MR
D019759:Carbon-Nitrogen Lyases / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D017955:Poliovirus / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000032:analysis*; D014452:Ubiquitins / Q000032:analysis*
D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D014452:Ubiquitins; D012321:DNA-Directed RNA Polymerases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase