Ubiquitination of an artificial RING finger without a substrate and a tag

Authors


Kazuhide Miyamoto, Department of Pharmaceutical Health Care, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Hyogo 670-8524, Japan. E-mail: miyamoto@himeji-du.ac.jp

Abstract

Alpha-helical region substitution was applied to the SIAH1 and EL5 RING fingers. The Williams–Beuren syndrome transcription factor (WSTF) PHD_SIAH1 and WSTF PHD_EL5 RING fingers were created as the artificial ubiquitin-ligating enzyme (E3). These fingers possess E3 activities of mono-ubiquitination and poly-ubiquitination, respectively, with ubiquitin-conjugating enzyme (E2)-binding capabilities. Artificial E3s bind two zinc atoms and adopt a zinc-dependent ordered structure and ubiquitinate upon themselves without a substrate and a tag. Ubiquitination experiments using biotinylated ubiquitin showed that the WSTF PHD_EL5 RING finger is poly-ubiquitinated via residue Lys63 of ubiquitin. Substitution of alpha-helical region might be applicable to various RING fingers with mono-ubiquitination or poly-ubiquitination. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

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