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