ArticleProceedings of the National Academy of Sciences of the United States of America2015
A comprehensive method for detecting ubiquitinated substrates using TR-TUBE.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 64 papers.
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64 citing papers in PubMed, 117 citations in OpenAlex.
- A guide to mapping ubiquitin and ubiquitin-like E3 ligases to their substrates.The FEBS journal · 2026Review
- EID1 overexpression in brown adipose tissue enhances cold-induced thermogenesis in male mice.iScience · 2026Article
- Optineurin calibrates STING-mediated type I interferon response.The FEBS journal · 2026Article
- Protein tyrosine kinase-6 is post-translationally regulated by arginine methylation and ubiquitination.Cell death & disease · 2026Article
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- Identification of E3 ligase substrates and PROTAC-induced ubiquitylation sites using proximity-based identification of ubiquitin sites (PrIUS).Communications biology · 2026Article
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- Small Molecule-Induced Alterations of Protein Polyubiquitination Revealed by Mass-Spectrometric Ubiquitome Analysis.Angewandte Chemie (International ed. in English) · 2025Article
- TanGIBLE: A selective probe for evaluating hydrophobicity-exposed defective proteins in live cells.The Journal of cell biology · 2025Article
- Ubiquitylomics: An Emerging Approach for Profiling Protein Ubiquitylation in Skeletal Muscle.Journal of cachexia, sarcopenia and muscle · 2024Review
- Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates.Nature chemical biology · 2024Article
- Ubiquitin E3 Ligase FBXO9 Regulates Pluripotency by Targeting DPPA5 for Ubiquitylation and Degradation.Stem cells (Dayton, Ohio) · 2024Article
- Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD.Nature communications · 2024Article
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- FBXO21 mediated degradation of p85α regulates proliferation and survival of acute myeloid leukemia.Leukemia · 2023Article
- Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells.JACS Au · 2023Article
- Strigolactones promote plant freezing tolerance by releasing the WRKY41-mediated inhibition of CBF/DREB1 expression.The EMBO journal · 2023Article
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- FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.The EMBO journal · 2023Article
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4 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.
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