title: A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity.
abstract: 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.
authors.affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA. iengels@gnf.org
authors.forename: Ingo H
authors.initials: IH
authors.lastname: Engels
authors.forename: Catherine
authors.initials: C
authors.lastname: Daguia
authors.forename: Truc
authors.initials: T
authors.lastname: Huynh
authors.forename: Hugo
authors.initials: H
authors.lastname: Urbina
authors.forename: Jörg
authors.initials: J
authors.lastname: Buddenkotte
authors.forename: Andrew
authors.initials: A
authors.lastname: Schumacher
authors.forename: Jeremy S
authors.initials: JS
authors.lastname: Caldwell
authors.forename: Achim
authors.initials: A
authors.lastname: Brinker