{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T10:54:58Z","timestamp":1788173698868,"version":"build-2803163510"},"reference-count":43,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,1,3]],"date-time":"2020-01-03T00:00:00Z","timestamp":1578009600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000009","name":"Foundation for the National Institutes of Health","doi-asserted-by":"publisher","award":["ZIA EY000476-10"],"award-info":[{"award-number":["ZIA EY000476-10"]}],"id":[{"id":"10.13039\/100000009","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (&lt;25 \u00b0C). At higher temperatures, &gt;31 \u00b0C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.<\/jats:p>","DOI":"10.3390\/ijms21010331","type":"journal-article","created":{"date-parts":[[2020,1,3]],"date-time":"2020-01-03T11:55:07Z","timestamp":1578052507000},"page":"331","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Protein Stability and Functional Characterization of Intra-Melanosomal Domain of Human Recombinant Tyrosinase-Related Protein 1"],"prefix":"10.3390","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2427-1118","authenticated-orcid":false,"given":"Monika B.","family":"Dolinska","sequence":"first","affiliation":[{"name":"National Eye Institute, National Institutes of Health, 31 Center Drive MSC 2510, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3950-1921","authenticated-orcid":false,"suffix":"II","given":"Kenneth L.","family":"Young","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, 31 Center Drive MSC 2510, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"Kassouf","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, 31 Center Drive MSC 2510, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emilios K.","family":"Dimitriadis","sequence":"additional","affiliation":[{"name":"National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 9000 Rockville Pike, Building 13, Room 3N18 Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9515-4752","authenticated-orcid":false,"given":"Paul T.","family":"Wingfield","sequence":"additional","affiliation":[{"name":"National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 31 Center Dr. - MSC 2350, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7204-6572","authenticated-orcid":false,"given":"Yuri V.","family":"Sergeev","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, 31 Center Drive MSC 2510, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,1,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"9812","DOI":"10.1002\/anie.201704616","article-title":"Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis","volume":"56","author":"Lai","year":"2017","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1111\/j.1600-0749.1992.tb00547.x","article-title":"Functional Properties of Cloned Melanogenic Proteins","volume":"5","author":"Hearing","year":"1992","journal-title":"Pigment Cell Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1111\/1523-1747.ep12392743","article-title":"High-Molecular-Weight Forms of Tyrosinase and the Tyrosinase-Related Proteins: Evidence for a Melanogenic Complex","volume":"103","author":"Orlow","year":"1994","journal-title":"J. Investig. Dermatol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4261","DOI":"10.1242\/jcs.017913","article-title":"Direct interaction of tyrosinase with Tyrp1 to form heterodimeric complexes in vivo","volume":"120","author":"Kobayashi","year":"2007","journal-title":"J. Cell Sci."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Dolinska, M.B., Kovaleva, E., Backlund, P., Wingfield, P.T., Brooks, B.P., and Sergeev, Y.V. (2014). Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0084494"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Kus, N.J., Dolinska, M.B., Young, K.L., Dimitriadis, E.K., Wingfield, P.T., and Sergeev, Y.V. (2018). Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0198247"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1515\/hsz-2017-0178","article-title":"The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase","volume":"399","author":"Dolinska","year":"2017","journal-title":"Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1111\/pcmr.12546","article-title":"Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity","volume":"30","author":"Dolinska","year":"2017","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1002\/humu.22315","article-title":"DNA variations in oculocutaneous albinism: An updated mutation list and current outstanding issues in molecular diagnostics","volume":"34","author":"Simeonov","year":"2013","journal-title":"Hum. Mutat."},{"key":"ref_10","first-page":"1145","article-title":"Mutation in and lack of expression of tyrosinase-related protein-1 (TRP-1) in melanocytes from an individual with brown oculocutaneous albinism: A new subtype of albinism classified as OCA3","volume":"58","author":"Boissy","year":"1996","journal-title":"Am. J. Hum. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1111\/j.1600-0749.2006.00298.x","article-title":"Oculocutaneous albinism with TYRP1 gene mutations in a Caucasian patient","volume":"19","author":"Rooryck","year":"2006","journal-title":"Pigment Cell Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1578","DOI":"10.1002\/ajmg.a.32930","article-title":"A case of Asian Indian OCA3 patient","volume":"149","author":"Chiang","year":"2009","journal-title":"Am. J. Med. Genet. Part A"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1111\/j.1755-148X.2009.00636.x","article-title":"New insights into the active site structure and catalytic mechanism of tyrosinase and its related proteins","volume":"22","author":"Olivares","year":"2009","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1111\/pcmr.12791","article-title":"The TYRP1-mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains","volume":"32","author":"Dolinska","year":"2019","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"D\u2019Mello, S.A.N., Finlay, G.J., Baguley, B.C., and Askarian-Amiri, M.E. (2016). Signaling Pathways in Melanogenesis. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17071144"},{"key":"ref_16","first-page":"2773","article-title":"Glycoproteins as differentiation markers in human malignant melanoma and melanocytes","volume":"43","author":"Tai","year":"1983","journal-title":"Cancer Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1002\/ijc.2910470221","article-title":"Purification of an autoantigenic 75-kDa human melanosomal glycoprotein","volume":"47","author":"Vijayasaradhi","year":"1991","journal-title":"Int. J. Cancer"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2013\/697051","article-title":"In Silico Screening and Molecular Dynamics Simulation of Disease-Associated nsSNP in TYRP1 Gene and Its Structural Consequences in OCA3","volume":"2013","author":"Kamaraj","year":"2013","journal-title":"BioMed Res. Int."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1590\/S0365-05962013000100009","article-title":"Mechanisms regulating melanogenesis","volume":"88","author":"Videira","year":"2013","journal-title":"An. Bras. Dermatol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1111\/php.12335","article-title":"MC1R, eumelanin and pheomelanin: Their role in determining the susceptibility to skin cancer","volume":"91","author":"Nasti","year":"2015","journal-title":"Photochem. Photobiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4697","DOI":"10.1021\/cr990291y","article-title":"N-Glycosylation Processing and Glycoprotein Folding\u2014Lessons from the Tyrosinase-Related Proteins","volume":"100","author":"Petrescu","year":"2000","journal-title":"Chem. Rev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1034\/j.1600-0749.2001.140603.x","article-title":"Tyrp1 and oculocutaneous albinism type 3","volume":"14","author":"Sarangarajan","year":"2001","journal-title":"Pigment Cell Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5818","DOI":"10.1002\/j.1460-2075.1994.tb06925.x","article-title":"Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis","volume":"13","author":"Kobayashi","year":"1994","journal-title":"EMBO J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1111\/j.1600-0625.1998.tb00324.x","article-title":"Human tyrosinase related protein-1 (TRP-1) does not function as a DHICA oxidase activity in contrast to murine TRP-1","volume":"7","author":"Boissy","year":"1998","journal-title":"Exp. Dermatol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1111\/j.1600-0749.1993.tb00601.x","article-title":"From Gene to Protein: Determination of Melanin Synthesis","volume":"6","author":"Urabe","year":"1993","journal-title":"Pigment. Cell Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1016\/S0959-440X(00)00172-X","article-title":"Macromolecular crowding: An important but neglected aspect of the intracellular environment","volume":"11","author":"Ellis","year":"2001","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2997","DOI":"10.1073\/pnas.95.6.2997","article-title":"Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release","volume":"95","author":"Kim","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1002\/jcp.1041160114","article-title":"Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents","volume":"116","author":"Saeki","year":"1983","journal-title":"J. Cell. Physiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"17193","DOI":"10.3390\/ijms160817193","article-title":"Protein Folding and Mechanisms of Proteostasis","volume":"16","year":"2015","journal-title":"Int. J. Mol. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"17993","DOI":"10.1016\/S0021-9258(17)32408-0","article-title":"A new enzymatic function in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1 (TRP1)","volume":"269","author":"Solano","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1177\/1087057107304113","article-title":"A Simple Assay for Detection of Small-Molecule Redox Activity","volume":"12","author":"Lor","year":"2007","journal-title":"J. Biomol. Screen."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1089\/adt.2008.151","article-title":"Development of a 384-Well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents","volume":"6","author":"Johnston","year":"2008","journal-title":"ASSAY Drug Dev. Technol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.ab.2012.01.006","article-title":"Reducing agents affect inhibitory activities of compounds: Results from multiple drug targets","volume":"423","author":"Lee","year":"2012","journal-title":"Anal. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1007\/s12575-009-9008-x","article-title":"Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy","volume":"11","author":"Erickson","year":"2009","journal-title":"Biol. Proced. Online"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1007\/s12551-016-0218-6","article-title":"Dynamic light scattering: A practical guide and applications in biomedical sciences","volume":"8","author":"Stetefeld","year":"2016","journal-title":"Biophys. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"850","DOI":"10.1021\/ac950914h","article-title":"Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels","volume":"68","author":"Shevchenko","year":"1996","journal-title":"Anal. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Lin, Y., Huo, L., Liu, Z., Li, J., Liu, Y., He, Q., Wang, X., and Liang, S. (2013). Sodium Laurate, a Novel Protease- and Mass Spectrometry-Compatible Detergent for Mass Spectrometry-Based Membrane Proteomics. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0059779"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1896","DOI":"10.1038\/nprot.2007.261","article-title":"Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips","volume":"2","author":"Rappsilber","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3551","DOI":"10.1002\/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2","article-title":"Probability-based protein identification by searching sequence databases using mass spectrometry data","volume":"20","author":"Perkins","year":"1999","journal-title":"Electrophoresis"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/S0304-3991(03)00053-6","article-title":"Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials","volume":"97","author":"Shlyakhtenko","year":"2003","journal-title":"Ultramicroscopy"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1016\/j.ab.2006.04.053","article-title":"Improved methods for fitting sedimentation coefficient distributions derived by time-derivative techniques","volume":"354","author":"Philo","year":"2006","journal-title":"Anal. Biochem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.1099\/mic.0.2007\/006833-0","article-title":"Involvement of a novel copper chaperone in tyrosinase activity and melanin synthesis in Marinomonas mediterranea","volume":"153","author":"Solano","year":"2007","journal-title":"Microbiology"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1984","DOI":"10.1271\/bbb.68.1984","article-title":"Tyrosinase inhibitor isolated from the leaves of Zanthoxylum piperitum","volume":"68","author":"Jeong","year":"2004","journal-title":"Biosci. Biotechnol. Biochem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/1\/331\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T14:03:35Z","timestamp":1760364215000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/1\/331"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,1,3]]},"references-count":43,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,1]]}},"alternative-id":["ijms21010331"],"URL":"https:\/\/doi.org\/10.3390\/ijms21010331","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,1,3]]}}}