{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T02:50:55Z","timestamp":1787021455529,"version":"build-2736575974"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.17964.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,10,19]],"date-time":"2022-10-19T00:00:00Z","timestamp":1666137600000}}],"reference-count":44,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2022,10,19]],"date-time":"2022-10-19T00:00:00Z","timestamp":1666137600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"crossref","award":["210622"],"award-info":[{"award-number":["210622"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns4:p>\n                    Targeted protein degradation is an invaluable tool in studying the function of proteins. Such a tool was not available in\n                    <ns4:italic>Trypanosoma brucei<\/ns4:italic>\n                    , an evolutionarily divergent eukaryote that causes human African trypanosomiasis. Here, we have adapted deGradFP (degrade green fluorescent protein [GFP]), a protein degradation system based on the SCF E3 ubiquitin ligase complex and anti-GFP nanobody, in\n                    <ns4:italic>T. brucei<\/ns4:italic>\n                    . As a proof of principle, we targeted a kinetoplastid kinetochore protein (KKT3) that constitutively localizes at kinetochores in the nucleus. Induction of deGradFP in a cell line that had both alleles of KKT3 tagged with yellow fluorescent protein (YFP) caused a more severe growth defect than RNAi in procyclic (insect form) cells. deGradFP also worked on a cytoplasmic protein (COPII subunit, SEC31). Given the ease in making GFP fusion cell lines in\n                    <ns4:italic>T. brucei<\/ns4:italic>\n                    , deGradFP can serve as a powerful tool to rapidly deplete proteins of interest, especially those with low turnover rates.\n                  <\/ns4:p>","DOI":"10.12688\/wellcomeopenres.17964.2","type":"journal-article","created":{"date-parts":[[2022,10,19]],"date-time":"2022-10-19T08:30:09Z","timestamp":1666168209000},"page":"175","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":5,"title":["Targeted protein degradation using deGradFP in Trypanosoma brucei"],"prefix":"10.12688","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9597-9458","authenticated-orcid":false,"given":"Midori","family":"Ishii","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6010-394X","authenticated-orcid":false,"given":"Bungo","family":"Akiyoshi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2022,10,19]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"e356","DOI":"10.1002\/wdev.356","article-title":"Reflections on the use of protein binders to study protein function in developmental biology.","volume":"8","author":"G Aguilar","year":"2019","journal-title":"Wiley Interdiscip Rev Dev Biol."},{"key":"ref-2","doi-asserted-by":"publisher","first-page":"1247-1258","DOI":"10.1016\/j.cell.2014.01.049","article-title":"Discovery of unconventional kinetochores in kinetoplastids.","volume":"156","author":"B Akiyoshi","year":"2014","journal-title":"Cell."},{"key":"ref-3","doi-asserted-by":"publisher","first-page":"915-924","DOI":"10.1101\/gr.115089.110","article-title":"High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.","volume":"21","author":"S Alsford","year":"2011","journal-title":"Genome Res."},{"key":"ref-4","doi-asserted-by":"publisher","first-page":"jcs242271","DOI":"10.1242\/jcs.242271","article-title":"Control of assembly of extra-axonemal structures: the paraflagellar rod of trypanosomes.","volume":"133","author":"A Alves","year":"2020","journal-title":"J Cell Sci."},{"key":"ref-5","doi-asserted-by":"publisher","first-page":"170095","DOI":"10.1098\/rsos.170095","article-title":"A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids.","volume":"4","author":"T Beneke","year":"2017","journal-title":"R Soc Open Sci."},{"key":"ref-6","doi-asserted-by":"publisher","first-page":"217-224","DOI":"10.1016\/j.molbiopara.2007.08.005","article-title":"The F-box protein CFB2 is required for cytokinesis of bloodstream-form Trypanosoma brucei.","volume":"156","author":"C Benz","year":"2007","journal-title":"Mol Biochem Parasitol."},{"key":"ref-7","doi-asserted-by":"publisher","first-page":"289-292","DOI":"10.5169\/seals-312533","article-title":"Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.19908.r51223, Cynthia He Y, Department of Biological Sciences, Centre for BioImaging Sciences, National University of Singapore, Singapore, Singapore, 27 Jun 2022, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-175\/v1#referee-response-51223","order":0,"name":"referee-response-51223","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19908.r51226, Christine Clayton, Heidelberg University Centre for Molecular Biology (ZMBH), Heidelberg, Germany, 29 Jun 2022, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-175\/v1#referee-response-51226","order":1,"name":"referee-response-51226","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19908.r51224, Christopher L. de Graffenried, Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, USA, 11 Jul 2022, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-175\/v1#referee-response-51224","order":2,"name":"referee-response-51224","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19908.r51225, David Horn, The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, UK, 11 Jul 2022, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/7-175\/v1#referee-response-51225","order":3,"name":"referee-response-51225","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome (210622\/Z\/18\/Z ; a Senior Research Fellowship to BA); the European Molecular Biology Organization Young Investigator Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}