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Conference Paper: Expression, purification and characterization of the N-terminal domain of the human copper transporter (hCtr1)

TitleExpression, purification and characterization of the N-terminal domain of the human copper transporter (hCtr1)
Authors
Issue Date2009
PublisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00775/index.htm
Citation
The 14th International Conference on Biological Inorganic Chemistry, Nagoya, Japan, 25-30 July 2009. In Journal of Biological Inorganic Chemistry, 2009, v. 14 n. Suppl1, p. S196 Abstract no.P534 How to Cite?
AbstractCopper is an essential element required for many important cellular proteins and reactions, however, excess copper is toxic. In cells, copper homeostasis is tightly regulated, resulting in very low free cellular metal concentration. It has been shown previously that the high affinity copper (Cu (I)) uptake at the plasma membrane in humans is mediated by the hCtr1 protein, a three-putative transmembrane protein (190 aa) [1, 2]. The methionine-rich region, which is regarded as potential metal-binding sites, is located in the extracellular domain in the N-terminus (hCtr1_N). Surprisingly, the widely used anticancer drug, cisplatin [cis-diammedichloroplatinum (II)], which bears a different coordination from Cu+, was also found to be transported by the protein both in yeast and mammalian [3]. In this work, we overexpressed, purified and characterized the N-terminal extracellular domain of hCtr1 (hCtr1_N, residues 1-55). Two independent methods, i.e. dialysis equilibrium and the protein’s prevention effect on the copper-catalyzed ascorbic acid oxidation, revealed that the protein could bind 3 Cu+ ions per monomer. An average dissociation constant was determined to beKD = (K1K2K3)1/3 = 10-14 M for binding of Cu+ to hCtr1_N protein via competition with the ligand bicinchoninic acid (BCA). The binding of Cu+ to hCtr1_N is reversible as the bound Cu+ can be released in the presence of chelating ligand, e.g. bathocuproine disulfonate bcs or BCA. Replacement of Met within the two Met-rich motifs with Ala greatly reduced the Cu+ binding affinity of the protein, also with a change of stoichiometry( n) value. Interestingly, both the apo- and Cu+-bound hCtr1_N existed as monomer in solution based on gel filtration chromatography and native electrophoresis. The implication for copper transport will be discussed. Acknowledgement: This work was supported by Research Grants Council of Hong Kong (HKU7512/05 M, HKU7043/06P, HKU2/06C, HKU1/07C, HKU7038/08P) and the University of Hong Kong! References 1. Ishida S, Lee J, Thiele DJ, Herskowitz I (2002) Proc Natl Acad Sci USA 99:14298–14302 2. Lin X, Okuda T, Holzer A, Howell SB (2002) Mol Pharmacol 62:1154–1159 3. Kim BE, Nevitt T, Thiele DJ (2008) Nat Chem Biol 4:176– 185
Persistent Identifierhttp://hdl.handle.net/10722/96883
ISSN
2015 Impact Factor: 2.495
2015 SCImago Journal Rankings: 0.882

 

DC FieldValueLanguage
dc.contributor.authorDu, X-
dc.contributor.authorWang, X-
dc.contributor.authorChan, DSB-
dc.contributor.authorSun, H-
dc.date.accessioned2010-09-25T16:48:30Z-
dc.date.available2010-09-25T16:48:30Z-
dc.date.issued2009-
dc.identifier.citationThe 14th International Conference on Biological Inorganic Chemistry, Nagoya, Japan, 25-30 July 2009. In Journal of Biological Inorganic Chemistry, 2009, v. 14 n. Suppl1, p. S196 Abstract no.P534-
dc.identifier.issn0949-8257-
dc.identifier.urihttp://hdl.handle.net/10722/96883-
dc.description.abstractCopper is an essential element required for many important cellular proteins and reactions, however, excess copper is toxic. In cells, copper homeostasis is tightly regulated, resulting in very low free cellular metal concentration. It has been shown previously that the high affinity copper (Cu (I)) uptake at the plasma membrane in humans is mediated by the hCtr1 protein, a three-putative transmembrane protein (190 aa) [1, 2]. The methionine-rich region, which is regarded as potential metal-binding sites, is located in the extracellular domain in the N-terminus (hCtr1_N). Surprisingly, the widely used anticancer drug, cisplatin [cis-diammedichloroplatinum (II)], which bears a different coordination from Cu+, was also found to be transported by the protein both in yeast and mammalian [3]. In this work, we overexpressed, purified and characterized the N-terminal extracellular domain of hCtr1 (hCtr1_N, residues 1-55). Two independent methods, i.e. dialysis equilibrium and the protein’s prevention effect on the copper-catalyzed ascorbic acid oxidation, revealed that the protein could bind 3 Cu+ ions per monomer. An average dissociation constant was determined to beKD = (K1K2K3)1/3 = 10-14 M for binding of Cu+ to hCtr1_N protein via competition with the ligand bicinchoninic acid (BCA). The binding of Cu+ to hCtr1_N is reversible as the bound Cu+ can be released in the presence of chelating ligand, e.g. bathocuproine disulfonate bcs or BCA. Replacement of Met within the two Met-rich motifs with Ala greatly reduced the Cu+ binding affinity of the protein, also with a change of stoichiometry( n) value. Interestingly, both the apo- and Cu+-bound hCtr1_N existed as monomer in solution based on gel filtration chromatography and native electrophoresis. The implication for copper transport will be discussed. Acknowledgement: This work was supported by Research Grants Council of Hong Kong (HKU7512/05 M, HKU7043/06P, HKU2/06C, HKU1/07C, HKU7038/08P) and the University of Hong Kong! References 1. Ishida S, Lee J, Thiele DJ, Herskowitz I (2002) Proc Natl Acad Sci USA 99:14298–14302 2. Lin X, Okuda T, Holzer A, Howell SB (2002) Mol Pharmacol 62:1154–1159 3. Kim BE, Nevitt T, Thiele DJ (2008) Nat Chem Biol 4:176– 185-
dc.languageeng-
dc.publisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00775/index.htm-
dc.relation.ispartofJournal of Biological Inorganic Chemistry-
dc.rightsThe final publication is available at Springer via http://dx.doi.org/[insert DOI]-
dc.titleExpression, purification and characterization of the N-terminal domain of the human copper transporter (hCtr1)-
dc.typeConference_Paper-
dc.identifier.emailDu, X: xbdu@HKUCC-COM.hku.hk-
dc.identifier.emailSun, H: hsun@hku.hk-
dc.identifier.authoritySun, H=rp00777-
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1007/s00775-009-0515-7-
dc.identifier.hkuros164173-
dc.identifier.volume14-
dc.identifier.issueSuppl1-
dc.identifier.spageS196 Abstract no.P534-
dc.identifier.epageS196 Abstract no.P534-
dc.publisher.placeGermany-

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