Activity-Based Probes for HECT E3 Ubiquitin Ligases.
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.
Mesh Terms:
Amino Acid Sequence, Humans, Molecular Probes, Sequence Alignment, Ubiquitin-Protein Ligases
Amino Acid Sequence, Humans, Molecular Probes, Sequence Alignment, Ubiquitin-Protein Ligases
Chembiochem
Date: Dec. 18, 2016
PubMed ID: 28425671
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