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

Biochim Biophys Acta. 2012 Nov;1823(11):2079-86. doi: 10.1016/j.bbamcr.2012.06.004. Epub 2012 Jun 15.


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. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Endopeptidases / metabolism*
  • High-Throughput Screening Assays*
  • Humans
  • Luminescent Measurements*
  • NEDD8 Protein
  • Proteasome Endopeptidase Complex / metabolism*
  • Sensitivity and Specificity
  • Small Ubiquitin-Related Modifier Proteins / metabolism
  • Ubiquitin / metabolism*
  • Ubiquitins / metabolism


  • NEDD8 Protein
  • NEDD8 protein, human
  • SUMO2 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • Ubiquitin
  • Ubiquitins
  • Endopeptidases
  • Proteasome Endopeptidase Complex