Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells
Ubiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual fun...
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Bio-protocol LLC
2025-06-01
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| author | Urbi Mukhopadhyay Sophie Levantovsky Christian Behrends Sagar Bhogaraju |
| author_facet | Urbi Mukhopadhyay Sophie Levantovsky Christian Behrends Sagar Bhogaraju |
| author_sort | Urbi Mukhopadhyay |
| collection | DOAJ |
| description | Ubiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual function of 1) transferring covalently attached ubiquitin from the catalytic cysteine of E2 (E2~Ub) to the substrate and 2) providing substrate specificity. Our current knowledge of their individual substrate pools is incomplete due to the difficult capture of these transient substrate–E3 ligase interactions. Here, we present an efficient protocol that enables the selective biotinylation of substrates of a given ubiquitin ligase. In brief, the candidate E3 ligase is fused to the biotin ligase BirA and ubiquitin to a biotin acceptor peptide, an Avi-tag variant (-2) AP. Cells are co-transfected with these fusion constructs and exposed to biotin, resulting in a BirA-E3 ligase-catalyzed biotinylation of (-2) AP-Ub when in complex with E2. As the next step, the biotinylated (-2) AP-Ub is transferred covalently to the substrate lysine, which enables an enrichment via denaturing streptavidin pulldown. Substrate candidates can then be identified via mass spectrometry (MS). Our ubiquitin-specific proximity-dependent labeling (Ub-POD) method allows robust biotinylation of the ubiquitylation substrates of a candidate E3 ligase thanks to the wild-type BirA and biotin acceptor peptide fused to the E3 and Ub, respectively. Because of the highly Ub-specific labeling, Ub-POD is more appropriate for identifying ubiquitination substrates compared to other conventional proximity labeling or immunoprecipitation (IP) approaches. |
| format | Article |
| id | doaj-art-12e0a23ae04a449b90f6a3e1e9341b45 |
| institution | OA Journals |
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| language | English |
| publishDate | 2025-06-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-12e0a23ae04a449b90f6a3e1e9341b452025-08-20T02:20:52ZengBio-protocol LLCBio-Protocol2331-83252025-06-01151210.21769/BioProtoc.5351Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human CellsUrbi Mukhopadhyay0Sophie Levantovsky1Christian Behrends2Sagar Bhogaraju3Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Frankfurt am Main, GermanyEuropean Molecular Biology Laboratory, 71 avenue des Martyrs, 38042 Grenoble, FranceMunich Cluster for Systems Neurology, Medical Faculty, Ludwig-Maximilians-Universität München, Munich, GermanyMunich Cluster for Systems Neurology, Medical Faculty, Ludwig-Maximilians-Universität München, Munich, GermanyEuropean Molecular Biology Laboratory, 71 avenue des Martyrs, 38042 Grenoble, FranceUbiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual function of 1) transferring covalently attached ubiquitin from the catalytic cysteine of E2 (E2~Ub) to the substrate and 2) providing substrate specificity. Our current knowledge of their individual substrate pools is incomplete due to the difficult capture of these transient substrate–E3 ligase interactions. Here, we present an efficient protocol that enables the selective biotinylation of substrates of a given ubiquitin ligase. In brief, the candidate E3 ligase is fused to the biotin ligase BirA and ubiquitin to a biotin acceptor peptide, an Avi-tag variant (-2) AP. Cells are co-transfected with these fusion constructs and exposed to biotin, resulting in a BirA-E3 ligase-catalyzed biotinylation of (-2) AP-Ub when in complex with E2. As the next step, the biotinylated (-2) AP-Ub is transferred covalently to the substrate lysine, which enables an enrichment via denaturing streptavidin pulldown. Substrate candidates can then be identified via mass spectrometry (MS). Our ubiquitin-specific proximity-dependent labeling (Ub-POD) method allows robust biotinylation of the ubiquitylation substrates of a candidate E3 ligase thanks to the wild-type BirA and biotin acceptor peptide fused to the E3 and Ub, respectively. Because of the highly Ub-specific labeling, Ub-POD is more appropriate for identifying ubiquitination substrates compared to other conventional proximity labeling or immunoprecipitation (IP) approaches.https://bio-protocol.org/en/bpdetail?id=5351&type=0 |
| spellingShingle | Urbi Mukhopadhyay Sophie Levantovsky Christian Behrends Sagar Bhogaraju Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells Bio-Protocol |
| title | Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells |
| title_full | Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells |
| title_fullStr | Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells |
| title_full_unstemmed | Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells |
| title_short | Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells |
| title_sort | ub pod a ubiquitin specific proximity dependent labeling technique to identify e3 ubiquitin ligase substrates in human cells |
| url | https://bio-protocol.org/en/bpdetail?id=5351&type=0 |
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