Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay

Abstract

BackgroundThe ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging.ResultsUPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme.ConclusionsAs a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0554-z) contains supplementary material, which is available to authorized users.

RNA pull-down-Confocal Nanoscanning (RP-CONA) detects quercetin as pri-miR-7/HuR interaction inhibitor that decreases α-Synuclein levels

“…However, due to a lack of understanding of the interaction domains where pri-miR-7 binds HuR, it seems challenging to identify pri-miR-7/HuR inhibitors using similar approaches. To tackle this problem, we developed the RP-CONA technique that combines the RNA pull-down assay in human cell extracts with the CONA screening platform (38)(39)(40).…”

“…Here we developed a fluorescent on-bead screening assay based on RNA pull-down Confocal Nanoscanning (RP-CONA), to identify small molecules that modulate the strength of RNA-protein interactions. Our method uses an ultra-sensitive RNA-protein pull-down assay in cell extracts from human cultured cells (38) detecting RNA-protein complex modulators by confocal microscopy (39)(40)(41). By employing RP-CONA, we identified quercetin, a natural flavonoid and known inhibitor of HuR/TNF-α mRNA interaction (42), as the most potent pri-miR-7/HuR interaction inhibitor.…”

Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes

“…Indeed, another inhibitor of Ubc13, BAY 11–7082 [ 41 ], similarly relocalized dynein (data not shown). However, recent in vitro studies demonstrated that BAY 11-7082 additionally inhibits the ubiquitin E1 enzyme [ 42 ]. As BAY 11-7082 shares structural similarity with NSC697923, it is plausible that dynein relocalization induced by NSC697923 is actually also due to inhibition of the ubiquitin E1 enzyme.…”

Resolving the Complexity of Ubiquitin Networks

“…Fluorescently labeled Ub is enzymatically conjugated to the substrate and can be quantitatively detected on the bead periphery by confocal microscopy. UPS-CONA approach can be used for studying specific enzymes of the ubiquitination machinery, as well as for measuring the selectivity of putative ubiquitination inhibitors (Koszela et al, 2018).…”