A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity

Anal Biochem. 2009 Jul 1;390(1):85-7. doi: 10.1016/j.ab.2009.03.035. Epub 2009 Mar 27.


Neural precursor cell expressed, developmentally down-regulated gene 8 (NEDD8) is a recently discovered ubiquitin-like posttranslational modifier. NEDD8 acts predominantly as a regulator of ubiquitin-protein ligases and as a decoy for proteins targeted for proteasomal degradation. It thereby controls key events in cell cycle progression and embryogenesis. Deneddylase-1 (DEN1/NEDP1/SENP8) features a selective peptidase activity converting the proNEDD8 precursor to its mature form and an isopeptidase activity deconjugating NEDD8 from substrates such as cullins and p53. In this study, we describe a high-throughput screening (HTS)-compatible time-resolved fluorescent resonance energy transfer (TR-FRET) assay measuring the peptidase activity of DEN1.

MeSH terms

  • Endopeptidases / metabolism*
  • Enzyme Inhibitors / analysis
  • Enzyme Inhibitors / chemistry
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • NEDD8 Protein
  • Time Factors
  • Ubiquitins / metabolism


  • Enzyme Inhibitors
  • NEDD8 Protein
  • NEDD8 protein, human
  • Ubiquitins
  • Endopeptidases
  • SENP8 protein, human