{"doi":"10.1074/jbc.ra120.016858","title":"A new FRET-based platform to track substrate ubiquitination by fluorescence","abstract":"Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences. Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences. Post-translational modification of protein by ubiquitin (Ub) alters the abundance, subcellular location, or function of the target protein in a profound fashion that impacts numerous biological processes and directly contributes to myriad cellular defects or disease states, such as cancer (1Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6463) Google Scholar, 2Rajalingam K. Dikic I. SnapShot: expanding the ubiquitin code.Cell. 2016; 164: 1074-1074.e1Abstract Full Text PDF PubMed Scopus (24) Google Scholar). The biochemical process of covalently attaching Ub to a protein substrate, termed ubiquitination, is driven by three enzymes: the E1-activating enzyme, E2-conjugating enzyme, an","journal":"Journal of Biological Chemistry","year":2020,"id":74663,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9509,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":390510,"name":"Kevin Ching","orcid":"0000-0002-9008-5612","position":1,"is_corresponding":false},{"id":391593,"name":"Robert Chong","orcid":null,"position":2,"is_corresponding":false},{"id":383458,"name":"Zhen‐Qiang Pan","orcid":"0000-0001-9504-8768","position":3,"is_corresponding":false},{"id":383454,"name":"Kenneth Wu","orcid":"0000-0003-3513-5143","position":0,"is_corresponding":true}],"reference_count":31,"raw_metadata":null,"created_at":"2026-07-18T21:46:11.663457Z","pmid":"33361156","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}