{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,2,21]],"date-time":"2025-02-21T08:16:09Z","timestamp":1740125769180,"version":"3.37.3"},"reference-count":35,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2016,4,18]],"date-time":"2016-04-18T00:00:00Z","timestamp":1460937600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007428","name":"Naito Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100007428","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes to Cells"],"published-print":{"date-parts":[[2016,6]]},"abstract":"<jats:p>Many bacterial pathogens hijack the host ubiquitin system for their own benefit by delivering effectors with ubiquitin ligase (E3) into host cells via the type <jats:styled-content style=\"fixed-case\">III<\/jats:styled-content> secretion system. Therefore, screening for small compounds that selectively inhibit bacterial but not mammalian E3 ligases is a promising strategy for identifying molecules that could substitute for antibiotics. To facilitate high\u2010throughput screening for bacterial E3 ligase inhibitors, we developed a MiCy\/<jats:styled-content style=\"fixed-case\">mKO<\/jats:styled-content> (Midori\u2010ishi Cyan\/monomeric Kusabira\u2010Orange)\u2010based <jats:styled-content style=\"fixed-case\">FRET<\/jats:styled-content> (fluorescence resonance energy transfer) assay and validated it on <jats:italic>Shigella<\/jats:italic> IpaH E3 ligase effectors. We showed the feasibility of using the MiCy\/<jats:styled-content style=\"fixed-case\">mKO<\/jats:styled-content>\u2010based <jats:styled-content style=\"fixed-case\">FRET<\/jats:styled-content> assay to identify the most appropriate ubiquitin\u2010conjugating enzymes (E2s) and determine the lysine specificity of a given E3, both hallmarks of E3 activity. Furthermore, we showed the usefulness of the <jats:styled-content style=\"fixed-case\">FRET<\/jats:styled-content> assay in characterizing mammalian E3 ligases, such as <jats:styled-content style=\"fixed-case\">TNF<\/jats:styled-content> receptor\u2010associated factor 6 (<jats:styled-content style=\"fixed-case\">TRAF<\/jats:styled-content>6) and mouse double minute 2 homologue (<jats:styled-content style=\"fixed-case\">MDM<\/jats:styled-content>2). In addition, we confirmed the feasibility of determining the efficiency of inhibition of E3 ligase activity using inhibitors of E1 ubiquitin\u2010activating enzymes, such as <jats:styled-content style=\"fixed-case\">UBE<\/jats:styled-content>1\u201041, by measuring the <jats:styled-content style=\"fixed-case\">IC<\/jats:styled-content><jats:sub>50<\/jats:sub>. Based on these results, we concluded that the MiCy\/<jats:styled-content style=\"fixed-case\">mKO<\/jats:styled-content>\u2010based <jats:styled-content style=\"fixed-case\">FRET<\/jats:styled-content> assay is useful for characterizing E3 enzyme activity, as well as for high\u2010throughput E3 inhibitor screening.<\/jats:p>","DOI":"10.1111\/gtc.12369","type":"journal-article","created":{"date-parts":[[2016,4,19]],"date-time":"2016-04-19T03:12:44Z","timestamp":1461035564000},"page":"608-623","source":"Crossref","is-referenced-by-count":6,"title":["Midori\u2010ishi Cyan\/monomeric Kusabira\u2010Orange\u2010based fluorescence resonance energy transfer assay for characterization of various E3 ligases"],"prefix":"10.1111","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1152-4198","authenticated-orcid":false,"given":"Ryota","family":"Otsubo","sequence":"first","affiliation":[{"name":"Division of Bacterial Infection Biology Institute of Medical Science University of Tokyo 4\u20106\u20101, Shirokanedai, Minato\u2010ku Tokyo Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Minsoo","family":"Kim","sequence":"additional","affiliation":[{"name":"Division of Bacterial Infection Biology Institute of Medical Science University of Tokyo 4\u20106\u20101, Shirokanedai, Minato\u2010ku Tokyo Japan"},{"name":"The Hakubi Center for Advanced Research Kyoto University Yoshida\u2010honmachi, Sakyo\u2010ku, Kyoto\u2010shi Kyoto 606\u20108501 Japan"},{"name":"Department of Molecular and Cellular Physiology Graduate School of Medicine Kyoto University Yoshida\u2010konoe\u2010cho, Sakyo\u2010ku, Kyoto\u2010shi Kyoto 606\u20108501 Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jihye","family":"Lee","sequence":"additional","affiliation":[{"name":"Division of Bacterial Infection Biology Institute of Medical Science University of Tokyo 4\u20106\u20101, Shirokanedai, Minato\u2010ku Tokyo Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chihiro","family":"Sasakawa","sequence":"additional","affiliation":[{"name":"Division of Bacterial Infection Biology Institute of Medical Science University of Tokyo 4\u20106\u20101, Shirokanedai, Minato\u2010ku Tokyo Japan"},{"name":"Nippon Institute for Biological Science 9\u20102221\u20101 Shinmachi, Ome Tokyo Japan"},{"name":"Medical Mycology Research Center Chiba University 1\u20108\u20101 Inohana, Chuo\u2010ku Chiba 260\u20108673 Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2016,4,18]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro3259"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb2006"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003409"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.2780"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.medici.2015.01.007"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20141450"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1111\/imm.12255"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncb0805-758"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M114.609164"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(05)99044-7"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2011.06.003"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mib.2009.11.008"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.01.054"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1089\/154065803321537890"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.22.12436"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20040321"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(05)99045-9"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.01360-13"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.3390\/cells3030848"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.onc.1207298"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1042\/BST0370937"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060310-170328"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0043398"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0914821107"},{"key":"e_1_2_7_26_1","doi-asserted-by":"publisher","DOI":"10.1177\/1087057111423417"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1042\/BST20130128"},{"key":"e_1_2_7_28_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109745200"},{"key":"e_1_2_7_29_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0811058106"},{"key":"e_1_2_7_30_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature10894"},{"key":"e_1_2_7_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.febslet.2010.09.006"},{"key":"e_1_2_7_32_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1324021111"},{"key":"e_1_2_7_33_1","doi-asserted-by":"publisher","DOI":"10.1111\/cmi.12052"},{"key":"e_1_2_7_34_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-07-0568"},{"key":"e_1_2_7_35_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.1605"},{"key":"e_1_2_7_36_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb.1517"}],"container-title":["Genes to Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1111%2Fgtc.12369","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/gtc.12369","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,3]],"date-time":"2023-10-03T16:35:48Z","timestamp":1696350948000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/gtc.12369"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2016,4,18]]},"references-count":35,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2016,6]]}},"alternative-id":["10.1111\/gtc.12369"],"URL":"https:\/\/doi.org\/10.1111\/gtc.12369","archive":["Portico"],"relation":{},"ISSN":["1356-9597","1365-2443"],"issn-type":[{"type":"print","value":"1356-9597"},{"type":"electronic","value":"1365-2443"}],"subject":[],"published":{"date-parts":[[2016,4,18]]}}}