A toolbox approach to high-throughput TR-FRET-based SUMOylation and DeSUMOylation assays.
The posttranslational modification of target substrates by the ubiquitin-like proteins, specifically the small ubiquitin-like modifier (SUMO), has emerged as an essential mechanism to regulate protein function and control intracellular trafficking. Traditional methods for monitoring either the attachment or removal of SUMO, such as gel electrophoresis or western blot, are effective but typically suffer from a lack of throughput. Here, we report the development and application of time-resolved Förster resonance energy transfer (TR-FRET)-based assays capable of detecting SUMOylation or deSUMOylation in a high-throughput screening (HTS) format. Using Ran GTPase-activating protein (RanGAP1) as a model target substrate, we have demonstrated that the SUMOylation of this protein can be detected using LanthaScreen (Invitrogen, Carlsbad, CA) TR-FRET technology. Additionally, we have generated reagents useful for assessing the deSUMOylation activity of a sentrin-specific protease. All assays are performed in 384-well format and display excellent statistical data (Z' > 0.7) with high signal-to-background levels. Together, this collection of tools can be utilized in a modular approach to develop HTS assays for inhibitors of SUMOylation or deSUMOylation.
Carlson
Coby B
CB
Invitrogen Discovery Assays and Services, Madison, Wisconsin 53719, USA. coby.carlson@invitrogen.com
Horton
Robert A
RA
Vogel
Kurt W
KW
D055808:Drug Discovery / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004798:Enzymes / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D020690:GTPase-Activating Proteins / Q000737:chemistry; D007202:Indicators and Reagents; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D004798:Enzymes; D020690:GTPase-Activating Proteins; D007202:Indicators and Reagents; D025841:Small Ubiquitin-Related Modifier Proteins