Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases

Abstract

As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo™, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo™. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo™. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z′ =0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases.

Deubiquitylating enzymes and drug discovery: emerging opportunities

“…Novel technologies based on chemically synthesized DUB substrates containing isopeptide linkages, ubiquitin chains and/or assay technologies that are less prone to false positives -such as luminescence, time-resolved fluorescence or mass spectrometry -are advancing screening campaigns and are therefore now being exploited 76,[226][227][228] . For example, a ubiquitin-aminoluciferin substrate was used with a variety of DUBs to demonstrate a suitable assay window for high-throughput screening 207,229 .…”

A novel reporter for real-time, quantitative imaging of AKT-directed K63-poly-ubiquitination in living cells

“…Most studies to measure ubiquitination in live cells by fluorescence [ 46 ] or bioluminescence [ 47 , 48 ] have focused on the ubiquitin-proteosome system (UPS). Reporters for imaging non-proteosomal ubiquitination events have been described; however, they either work in a cell free system where pure components need to be supplied [ 49 , 50 ] or they are based on fusing the ubiquitin binding domain (UBD) to fluorescent proteins [ 39 , 51 , 52 ]. Therefore, they monitor total pools of the K63-ubiquitin chains in cells, and not target-specific E3-ubiquitin ligase activity.…”

How to Target Viral and Bacterial Effector Proteins Interfering with Ubiquitin Signaling

“…Fluorogenic reporters used in such type of reagents are aminomethylcoumarin (AMC) (Dang et al 1998) or substituted rhodamine-110 (RHO) (Hassiepen et al 2007) scaffolds that show favorable fluorescent properties. In a similar setup, DUB mediated aminoluciferin release can be assayed in a bioluminescence approach using a luciferase assay (Orcutt et al 2012). All of these reporters are conjugated to a mono-Ub recognition element, and hence, the preference of a DUB to specific poly-Ub topologies cannot be assessed.…”