ChemBioChem
ChemBioChem
5 years ago

Activity-Based Probes for HECT E3 Ubiquitin Ligases

Activity-Based Probes for HECT E3 Ubiquitin Ligases
Julien D. F. Licchesi, Thomas Mund, Robert Byrne
Activity-based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine-based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin-ABPs (Ub-VME and Ub-PA) and a novel set of E2–Ub-ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin-ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2–Ub-ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub-PA and His-UBE2D2–Ub-ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases. Probing HECT ubiquitin ligases: Activity-based probes have provided structural, biochemical and cellular insights for deubiquitinases. Here, we show that existing Ub-ABPs and novel E2–Ub-ABPs can efficiently label HECT E3 ubiquitin ligases in vitro and also in cell lysates, in the case of the proteasomal E3 ligase UBE3C.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700006

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