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Updated: Jul 18, 2025

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta
Published on: December 5, 2019
Artificial RING finger reveals unique auto-ubiquitination with E2 specificity
Kazuhide Miyamoto1, Atsushi Matsumoto1
1Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, Yamaguchi, Japan.
Abstract:
Ubiquitin (Ub)-conjugating enzymes (E2s) transfer activated Ub from Ub-activating enzymes (E1s) to substrates and are associated with various cancers and neurological disorders. In this study, the unique properties of E2-binding and auto-ubiquitination of artificial RING fingers (ARFs) were demonstrated in ubiquitination assays. Circular dichroism spectra indicated the characteristic structures of ARFs. Point mutations of 31 PKLTC35 in ARF by tryptophan (Trp) resulted in dramatic changes in E2 specificity and the type of Ub chain elongation of mono- and polyubiquitination. The Trp residue was a cue that changed the ubiquitination activity of ARF via E2-binding. Furthermore, the ARF mutants interacted with all 11 E2s and then promoted auto-ubiquitination. Thus, the use of the ARF mutants allowed specific detection of E2 activities during ubiquitination. The present study opens up a new avenue for researching E2 activities related to the fatal diseases.
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