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.

Functional Roles of Bromodomain Proteins in Cancer

“…As with other TIF1 family proteins, TRIM28 functions as an E3 ubiquitin ligase, and in 2020, Wantanabee et al developed a new E3 ligase substrate-trapping strategy in order to identify new ubiquitin substrates. They found that TRIM28 was associated with “Krüppel-associated box” proteins as expected, but also regulated cyclin A2 and transcription factor II beta (TFIIB) by mediating their degradation via ubiquitination [ 223 ]. Interestingly, the PHD finger and bromodomain region of TRIM28 do not appear to contribute to chromatin recognition.…”

Ubiquitination in Plant Meiosis: Recent Advances and High Throughput Methods

“…An approach which combines ligase-substate trapping with TUBE and di-gly was recently developed by Watanabe et al (2020) . This approach replaces the UBA-FLAG tagged E3 ligase proposed by Mark et al (2016) with a TUBE-FLAG tag, further increasing the affinity of the ligase for its substrate and protecting the substrate from degradation ( Figure 7A ) ( Watanabe et al, 2020 ). The addition of di-gly enrichment following anti-FLAG immunoprecipitation and trypsin digest of lysates lead to a dramatic increase in efficiency of putative substrate capture ( Watanabe et al, 2020 ).…”

“…This approach replaces the UBA-FLAG tagged E3 ligase proposed by Mark et al (2016) with a TUBE-FLAG tag, further increasing the affinity of the ligase for its substrate and protecting the substrate from degradation ( Figure 7A ) ( Watanabe et al, 2020 ). The addition of di-gly enrichment following anti-FLAG immunoprecipitation and trypsin digest of lysates lead to a dramatic increase in efficiency of putative substrate capture ( Watanabe et al, 2020 ). Watanabe et al (2020) also highlighted that attachment of the TUBE bait tag to the N or C terminal of the ligase affected the efficiency of capture in a ligase dependant manner.…”

“…The addition of di-gly enrichment following anti-FLAG immunoprecipitation and trypsin digest of lysates lead to a dramatic increase in efficiency of putative substrate capture ( Watanabe et al, 2020 ). Watanabe et al (2020) also highlighted that attachment of the TUBE bait tag to the N or C terminal of the ligase affected the efficiency of capture in a ligase dependant manner. In Arabidopsis , Durand et al (2016) and Lee et al (2018) expressed affinity tagged RING and F-box proteins respectively which retained their ability to interact with substrates but lacked the ability to ligate ubiquitin.…”

Identification and Functional Characterization of a Novel Androgen Receptor Coregulator, EAP1

“…As a control, LNCaP cells were infected with retrovirus carrying Dox-inducible FLAG-TR-TUBE. Established cells (1.0 × 10 6 cells) were lysed and ubiquitinated peptides were purified as previously described [ 36 ]. Briefly, the cell lysates were incubated with anti-DYKDDDDK tag antibody agarose (Fujifilm Wako Pure Chemical) for 2 hours at 4 ºC.…”