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 highthroughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases. Keywords Isopeptidases; Protein degradation; N terminus; 3D polymeraseThe ubiquitin-proteasomal pathway regulates the cellular content and/or compartmentalization of many proteins [1,2] and is a promising, albeit underexploited, area for drug discovery. The therapeutic value of this pathway was recently validated by the introduction and clinical success of Velcade, a proteasome inhibitor, for the treatment of refractory relapsed multiple myeloma [3]. Other ubiquitin-like proteins (UBLs) 1 have recently been shown to contribute similarly to the cellular regulation of proteins, with the most extensively characterized UBL being small ubiquitin-like modifying protein (SUMO) [4].Destruction of a targeted protein via the ubiquitin system initially involves recognition by a multienzyme system that attaches ubiquitin to the target protein. Energy is required to activate ubiquitin at its carboxy terminus. Activation is catalyzed by the enzyme E1, which links the ubiquitin C terminus to a cysteine side chain of the enzyme. This activated ubiquitin is * Corresponding author. Fax: +1 610 644 8616., E-mail address: mattern@progenra.com (M.R. Mattern).. 1 Abbreviations used: UBL, ubiquitin-like protein; SUMO, small ubiquitin-like modifying protein; UBP/USP, ubiquitin-specific protease; UCH, ubiquitin terminal hydrolase; DUB, deubiquitinating enzyme; Ub-AMC, Ub-7-amino-4-methylcoumarin; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; PMSF, phenylmethylsulfonylfluoride; GFP, green fluorescent protein; DTT, dithiothreitol. transferred to a second enzyme of the series, E2 (ubiquitin-conjugating protein), as a thioe...

Picornaviral polymerase structure, function, and fidelity modulation

“…Bacterial methionine aminopeptidases are fairly efficient at removing the Met1 residue, but they can be overloaded under very high-level protein expression. To circumvent this issue, the Cameron lab developed expression systems using N-terminal fusions with ubiquitin (Gohara et al, 1999) and SUMO (Arnold et al, 2006) that are cleaved in situ by co-expressed proteases, generating 3D pol with a native N-terminal glycine residue for direct purification from bacterial lysates.…”

Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins

“…The NcoI/HindIII-digested PCR products were ligated into the pET26-Ub-NHis vector (11). Cloning for 2005-2221 and 2212-2419 NS5A in the pET24-6H-SUMO vector was performed as previously reported (2). Briefly, the NS5A gene segments were amplified from the above-mentioned pHCVbart.rep1b/Ava-II using forward primers SUMOBsaI-HCV-2005-2221-for and SUMO-BsaI-HCV-2212-2419-for and reverse primers SUMO-HCV-2005-2221-HindIII-rev and SUMO-HCV-2212-2419-HindIIIrev, respectively ( Table 1).…”

Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases

“…It should be noted that WT and all variant RdRps also have L446D and R455D substitutions to prevent RdRp self-association. Overexpression and purification of PV RdRp followed procedures described previously (21,23,63,64).…”