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.
Highlights
The identification of true substrates of an E3 ligase is biologically important but biochemically difficult
We developed a method by combining the ligasetrapping method and the TR-tandem ubiquitin-binding entity (TUBE) method and we applied the method to Parkin and Tripartite motif-containing 28 (TRIM28) as E3 ligases
The ubiquitinated peptides identified by the FLAG–TUBE probe not fused with an E3 ligase or the FLAG–TUBE-fused Parkin probe, which is considered to be almost inactivated under unstimulated conditions (Supplementary Data 1), and ubiquitinated peptides identified with probes fused with E3 ligase with deletion of enzyme activity were compared as negative controls to determine ubiquitinated peptides identified for each E3 ligase
Summary
The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. 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. In the TR-TUBE method, substrate detection becomes difficult when activity of the introduced E3 is not strong enough to overcome the amount of endogenous ubiquitinated proteins already present in the cell because the method identifies ubiquitinated proteins that increase in cells as substrate candidates by overexpression of E3. Since it includes the influence of other E3 and deubiquitinating enzymes that vary depending on the introduced E3, the possibility of indirect ubiquitination cannot be excluded. To overcome their weaknesses, we considered the possibility of combining the advantages of the two methods
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