{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:48:33Z","timestamp":1787028513182,"version":"build-2736575974"},"reference-count":42,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2023,6,26]],"date-time":"2023-06-26T00:00:00Z","timestamp":1687737600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS KAKENHI","award":["JP25291024"],"award-info":[{"award-number":["JP25291024"]}]},{"name":"JSPS KAKENHI","award":["JP23K05779"],"award-info":[{"award-number":["JP23K05779"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The extracellular ubiquitin\u2013proteasome system is involved in sperm binding to and\/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the \u201csperm reaction\u201d. However, the de novo function of ubiquitin-activating enzyme (UBA\/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.<\/jats:p>","DOI":"10.3390\/ijms241310662","type":"journal-article","created":{"date-parts":[[2023,6,27]],"date-time":"2023-06-27T01:42:02Z","timestamp":1687830122000},"page":"10662","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4363-7696","authenticated-orcid":false,"given":"Hitoshi","family":"Sawada","sequence":"first","affiliation":[{"name":"Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, Japan"},{"name":"Department of Food and Nutritional Environment, College of Human Life and Environment, Kinjo Gakuin University, Omori 2-1723, Moriyama-ku, Nagoya 463-8521, Japan"},{"name":"Department of Applied Life Sciences, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shukumi","family":"Inoue","sequence":"additional","affiliation":[{"name":"Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3969-9192","authenticated-orcid":false,"given":"Takako","family":"Saito","sequence":"additional","affiliation":[{"name":"Department of Applied Life Sciences, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan"},{"name":"Shizuoka Institute for the Study of Marine Biology and Chemistry, Shizuoka University, Shizuoka 422-8529, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kei","family":"Otsuka","sequence":"additional","affiliation":[{"name":"Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, Japan"},{"name":"Department of Life Science, Faculty of Life Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maki","family":"Shirae-Kurabayashi","sequence":"additional","affiliation":[{"name":"Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1146\/annurev.biochem.67.1.425","article-title":"The ubiquitin system","volume":"67","author":"Hershko","year":"1998","journal-title":"Annu. Rev. Biochem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/0955-0674(95)80031-X","article-title":"Ubiquitin, proteasomes, and the regulation of intracellular protein degradation","volume":"2","author":"Hochstrasser","year":"1995","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1093\/oxfordjournals.jbchem.a021922","article-title":"Proteasomes: Structure and biology","volume":"123","author":"Tanaka","year":"1998","journal-title":"J. Biochem."},{"key":"ref_4","first-page":"57","article-title":"The ubiquitin-proteasome pathway and pathogenesis of human diseases","volume":"50","author":"Schwartz","year":"1999","journal-title":"Annu. Rev. Biochem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1104\/pp.112.200667","article-title":"The expanding universe of ubiquitin and ubiquitin-like modifiers","volume":"160","author":"Viestra","year":"2012","journal-title":"Plant Physiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1146\/annurev.biochem.70.1.503","article-title":"Mechanisms underlying ubiquitination","volume":"70","author":"Pickart","year":"2001","journal-title":"Annu. Rev. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1146\/annurev.cellbio.22.010605.093503","article-title":"Modification of proteins by ubiquitin and ubiquitin-like proteins","volume":"22","author":"Kerscher","year":"2006","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1021\/acs.chemrev.6b00737","article-title":"Ubiquitin-like conjugation: Structures, chemistry, and mechanism","volume":"118","author":"Cappadocia","year":"2018","journal-title":"Chem. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.tibs.2008.01.005","article-title":"Activating the ubiquitin family: UBA6 challenges the field","volume":"33","author":"Groettrup","year":"2008","journal-title":"Trends Biochem. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"23010","DOI":"10.1074\/jbc.C700111200","article-title":"UBE1L2, a novel E1 enzyme specific for ubiquitin","volume":"282","author":"Pelzer","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1135","DOI":"10.1038\/nature05902","article-title":"Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging","volume":"447","author":"Jin","year":"2007","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1014","DOI":"10.1016\/j.molcel.2007.08.020","article-title":"E1-L2 activates both ubiquitin and FAT10","volume":"27","author":"Chiu","year":"2007","journal-title":"Mol. Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3483","DOI":"10.1128\/MCB.25.9.3483-3491.2005","article-title":"FAT10, a ubiquitin-independent signal for proteasomal degradation","volume":"25","author":"Hipp","year":"2008","journal-title":"Mol. Cell. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1038\/nrm2673","article-title":"Ubiquitin-like protein activation by E1 enzymes: The apex for downstream signaling pathway","volume":"10","author":"Schulman","year":"2009","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Wang, F., and Zhao, B. (2019). UBA6 and its biospecific pathway for ubiquitin and FAT10. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20092250"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1002\/mrd.1120050309","article-title":"Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian, Halocynthia roretzi","volume":"5","author":"Sawada","year":"1982","journal-title":"Gamete Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1007\/BF01963132","article-title":"Ascidian sperm chymotrypsin-like enzyme; participation in fertilization","volume":"39","author":"Sawada","year":"1983","journal-title":"Experimentia"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2900","DOI":"10.1016\/S0021-9258(17)43233-9","article-title":"Purification and characterization of two types of trypsin-like enzymes from sperm of the ascidian (Prochordata), Halocynthia roretzi: Evidence for the presence of spermosin, a novel acrosin-like enzyme","volume":"259","author":"Sawada","year":"1984","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1016\/0012-1606(84)90281-1","article-title":"Evidence for the participation of two sperm proteases, spermosin and acrosin, in fertilization of the ascidian, Halocynthia roretzi: Inhibitory effects of leupeptin analogs on enzyme activities and fertilization","volume":"105","author":"Sawada","year":"1984","journal-title":"Dev. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1006\/dbio.1993.1182","article-title":"High-molecular-weight protease complexes (proteasomes) of sperm of the ascidian, Halocynthia roretzi","volume":"158","author":"Saitoh","year":"1993","journal-title":"Dev. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.1073\/pnas.032389499","article-title":"Extracellular ubiquitination and proteasome-mediated degradation of the ascidian sperm receptor","volume":"99","author":"Sawada","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1002\/mrd.10089","article-title":"Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi","volume":"62","author":"Sawada","year":"2002","journal-title":"Mol. Reprod. Dev."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1002\/jez.1402670112","article-title":"Effects of protease inhibitors on binding of sperm to the vitelline coat of ascidian eggs: Implications for participation of a proteasome (multicatalytic proteinase complex)","volume":"267","author":"Takizawa","year":"1993","journal-title":"J. Exp. Zool."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.ydbio.2003.07.016","article-title":"Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: isolation and characterization","volume":"264","author":"Sakai","year":"2003","journal-title":"Dev. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1016\/0012-1606(79)90293-8","article-title":"Calcium-mediated mitochondrial movement in ascidian sperm during fertilization","volume":"69","author":"Lambert","year":"1979","journal-title":"Dev. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"15615","DOI":"10.1073\/pnas.0401928101","article-title":"Self\/nonself recognition in ascidian fertilization: Vitelline coat protein HrVC70 is a candidate allorecognition molecule","volume":"101","author":"Sawada","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/0012-1606(81)90322-5","article-title":"Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzi: Effects of protease inhibitors","volume":"86","author":"Hoshi","year":"1981","journal-title":"Dev. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4789","DOI":"10.1038\/s41467-022-32040-6","article-title":"Structures of UBA6 explain its dual specificity for ubiquitin and FAT10","volume":"13","author":"Truongvan","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"18337","DOI":"10.1074\/jbc.RA118.003975","article-title":"Crystal structure of a human ubiquitin E1-ubiquitin complex reveals conserved functional elements essential for activity","volume":"29","author":"Lv","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1111\/j.1440-169X.1988.00325.x","article-title":"Sperm binding and penetration during ascidian fertilization","volume":"30","author":"Lambert","year":"1988","journal-title":"Dev. Growth Differ."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1111\/j.1365-2605.2011.01217.x","article-title":"Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization","volume":"35","author":"Yi","year":"2012","journal-title":"Int. J. Androl."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.jri.2009.07.006","article-title":"The sperm proteasome during sperm capacitation and fertilization","volume":"83","author":"Zimmerman","year":"2009","journal-title":"Reprod. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2731","DOI":"10.1021\/acs.jmedchem.9b01138","article-title":"Advances in discovering deubiquitinating enzyme (DUB) inhibitors","volume":"63","author":"Schauer","year":"2020","journal-title":"J. Med. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Korenev, G., Yakukhnov, S., Druk, A., Golovina, A., Chasov, V., Mirgayazova, R., Ivanov, R., and Bulatov, E. (2022). USP7 inhibitors in cancer immunotherapy: Current status and perspective. Cancers, 14.","DOI":"10.3390\/cancers14225539"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"9472","DOI":"10.1158\/0008-5472.CAN-07-0568","article-title":"Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics","volume":"67","author":"Yang","year":"2007","journal-title":"Cancer Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"640","DOI":"10.1002\/mrd.21198","article-title":"Identification of testis-specific ubiquitin-conjugating enzyme in the ascidian Ciona intestinalis","volume":"77","author":"Yokota","year":"2010","journal-title":"Mol. Reprod. Dev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1101\/gr.108175.110","article-title":"The ANISEED database: Digital representation, formalization, and elucidation of a chordate developmental program","volume":"20","author":"Tassy","year":"2010","journal-title":"Genome Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"D808","DOI":"10.1093\/nar\/gkv966","article-title":"ANISEED 2015: A digital framework for the comparative developmental biology of ascidians","volume":"44","author":"Brozovic","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: Molecular evolutionary genetics analysis across computing platforms","volume":"35","author":"Kumar","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_41","first-page":"406","article-title":"The neighbor-joining method: A new method for reconstructing phylogenetic trees","volume":"4","author":"Saitou","year":"1987","journal-title":"Mol. Biol. Evol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript server","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/13\/10662\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:01:04Z","timestamp":1760126464000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/13\/10662"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,6,26]]},"references-count":42,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["ijms241310662"],"URL":"https:\/\/doi.org\/10.3390\/ijms241310662","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,6,26]]}}}