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.

Fig. 1

Biochemical functions of deubiquitinating enzymes. Ubiquitin (Ub) is synthesized as a fusion protein and must be processed by ubiquitin-specific proteases (USPs). Removal of conjugated Ub by USPs serves an editing function. At the proteasome, the poly-Ub chain must be removed from the docked conjugates and disassembled to replenish the Ub pool and prevent accumulation of free poly-Ub chains. “Salvage” is the removal of cellular thiols, amines, and other small adducts that inactivate free Ub (catalyzed by the ubiquitin terminal hydrolase [UCH] enzymes).

Fig. 2

Schematic map of the pET24-6H-SUMO–3D^pol expression vector. The vector contains the T7 promoter driving the expression of the His-tagged SUMO–3D^pol fusion protein. DNA and protein sequence at the junction between the two proteins is shown on the right, with the SUMO protease cleavage site indicated.

Fig. 3

Expression of SUMO–3D^pol. Coomassie-stained SDS–polyacrylamide (10%) gel of bacterial cell lysates before IPTG induction and after 4 h of induction at 37 °C. The induced SUMO–3D^pol migrates at the 66-kDa marker. Lane 1, molecular weight markers; lane 2, uninduced; lane 3, induced.

Fig. 4

Purification of SUMO–3D^pol. Coomassie-stained SDS–polyacrylamide (10%) gel of the samples from each purification step. Lane 1, molecular weight markers; lane 2, uninduced; lane 3, induced; lane 4, lysate; lane 5, clarified lysate; lane 6, pellet; lane 7, Ni–NTA elution; lane 8, phospho-cellulose elution; lane 9, Q Sepharose elution. Approximately, 1.0–2.5 μg of total protein was loaded in lanes 7–9.

Fig. 5

Cleavage of SUMO–3D^pol by SUMO protease: Coomassie-stained SDS–polyacrylamide (10%) gel of purified SUMO–3D^pol before and after proteolytic cleavage by SUMO protease. SUMO–3D^pol (5 μg) was incubated with 2 U of SUMO protease on ice for 30 min prior to loading samples on the gel.

Fig. 6

Poly(rU) polymerase activity assay of SUMO–3D^pol. Reactions contained 0.05 μM SUMO–3D^pol and were performed as described in Materials and methods. The figure shows kinetics of nucleotide incorporation by 3D^pol where SUMO–3D^pol was added to the reaction in the absence of SUMO protease (filled circles), where SUMO–3D^pol was added to a reaction containing SUMO protease (filled squares), and where SUMO–3D^pol was incubated with SUMO protease on ice for 30 min prior to initiating the reaction (open circles). Conventionally purified 3D^pol is shown as a control (open squares).

Fig. 7

Primer extension. Reactions contained 1 μM sym/sub (0.5 μM duplex), 1 μM enzyme, and 500 μM ATP. Reactions were initiated by the addition of enzyme and were quenched at the indicated times by EDTA. SUMO–3D^pol was preincubated with SUMO protease on ice for 30 min prior to initiating the reaction.