dc.contributor.author | Bakos, G | |
dc.contributor.author | Yu, L | |
dc.contributor.author | Gak, IA | |
dc.contributor.author | Roumeliotis, TI | |
dc.contributor.author | Liakopoulos, D | |
dc.contributor.author | Choudhary, JS | |
dc.contributor.author | Mansfeld, J | |
dc.date.accessioned | 2019-02-20T07:48:43Z | |
dc.date.issued | 2018-11-14 | |
dc.identifier.citation | Nature communications, 2018, 9 (1), pp. 4776 - ? | |
dc.identifier.issn | 2041-1723 | |
dc.identifier.uri | https://repository.icr.ac.uk/handle/internal/3062 | |
dc.identifier.eissn | 2041-1723 | |
dc.identifier.doi | 10.1038/s41467-018-07251-5 | |
dc.description.abstract | Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models. | |
dc.format | Electronic | |
dc.format.extent | 4776 - ? | |
dc.language | eng | |
dc.language.iso | eng | |
dc.publisher | NATURE PUBLISHING GROUP | |
dc.rights.uri | https://creativecommons.org/licenses/by/4.0 | |
dc.subject | Cell Line | |
dc.subject | Hela Cells | |
dc.subject | Humans | |
dc.subject | Saccharomyces cerevisiae | |
dc.subject | Ubiquitin-Activating Enzymes | |
dc.subject | Ubiquitin-Conjugating Enzymes | |
dc.subject | Ubiquitin-Protein Ligases | |
dc.subject | Saccharomyces cerevisiae Proteins | |
dc.subject | Ubiquitins | |
dc.subject | SUMO-1 Protein | |
dc.subject | Ubiquitin | |
dc.subject | Anaphase-Promoting Complex-Cyclosome | |
dc.title | An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules. | |
dc.type | Journal Article | |
dcterms.dateAccepted | 2018-10-10 | |
rioxxterms.versionofrecord | 10.1038/s41467-018-07251-5 | |
rioxxterms.licenseref.uri | https://creativecommons.org/licenses/by/4.0 | |
rioxxterms.licenseref.startdate | 2018-11-14 | |
rioxxterms.type | Journal Article/Review | |
dc.relation.isPartOf | Nature communications | |
pubs.issue | 1 | |
pubs.notes | Not known | |
pubs.organisational-group | /ICR | |
pubs.organisational-group | /ICR/Primary Group | |
pubs.organisational-group | /ICR/Primary Group/ICR Divisions | |
pubs.organisational-group | /ICR/Primary Group/ICR Divisions/Cancer Biology | |
pubs.organisational-group | /ICR/Primary Group/ICR Divisions/Cancer Biology/Post-translational modifications and cell proliferation | |
pubs.organisational-group | /ICR | |
pubs.organisational-group | /ICR/Primary Group | |
pubs.organisational-group | /ICR/Primary Group/ICR Divisions | |
pubs.organisational-group | /ICR/Primary Group/ICR Divisions/Cancer Biology | |
pubs.organisational-group | /ICR/Primary Group/ICR Divisions/Cancer Biology/Post-translational modifications and cell proliferation | |
pubs.publication-status | Published | |
pubs.volume | 9 | |
pubs.embargo.terms | Not known | |
icr.researchteam | Post-translational modifications and cell proliferation | |
dc.contributor.icrauthor | Roumeliotis, Theodoros | |
dc.contributor.icrauthor | Choudhary, Jyoti | |
dc.contributor.icrauthor | Mansfeld, Joerg | |