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A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.


ABSTRACT: The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.

SUBMITTER: Watanabe M 

PROVIDER: S-EPMC7576197 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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A substrate-trapping strategy to find E3 ubiquitin ligase substrates identifies Parkin and TRIM28 targets.

Watanabe Masashi M   Saeki Yasushi Y   Takahashi Hidehisa H   Ohtake Fumiaki F   Yoshida Yukiko Y   Kasuga Yusuke Y   Kondo Takeshi T   Yaguchi Hiroaki H   Suzuki Masanobu M   Ishida Hiroki H   Tanaka Keiji K   Hatakeyama Shigetsugu S  

Communications biology 20201020 1


The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method t  ...[more]

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