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Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro
Umeå University, Faculty of Science and Technology, Department of Chemistry. Center of Life and Food Sciences, Technische Universität München, Freising, Germany.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2015 (English)In: Biometals, ISSN 0966-0844, E-ISSN 1572-8773, Vol. 28, no 3, 577-585 p.Article in journal (Refereed) Published
Abstract [en]

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P-1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Place, publisher, year, edition, pages
Springer, 2015. Vol. 28, no 3, 577-585 p.
Keyword [en]
Human copper transport, Atox1, Copper chaperone for superoxide dismutase, (SOD), Size exclusion chromatography, Proton-NMR
National Category
Chemical Sciences
URN: urn:nbn:se:umu:diva-100334DOI: 10.1007/s10534-015-9832-1ISI: 000354273900014PubMedID: 25673218OAI: diva2:791586
Available from: 2015-03-01 Created: 2015-03-01 Last updated: 2015-07-13Bibliographically approved
In thesis
1. Human copper ion transfer: from metal chaperone to target transporter domain
Open this publication in new window or tab >>Human copper ion transfer: from metal chaperone to target transporter domain
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many processes in living systems occur through transient interactions among proteins. Those interactions are often weak and are driven by small changes in free energy. Due to the short-living nature of these interactions, our knowledge about driving forces, dynamics and structures of these types of protein-protein heterocomplexes are though limited. This is especially important for cellular copper (Cu) trafficking:

Copper ions are essential for all eukaryotes and most bacteria. As a cofactor in many enzymes, copper is especially vital in respiration or detoxification. Since the same features that make copper useful also make it toxic, it needs to be controlled tightly. Additionally, in the reducing environment of the cytosol, Cu is present as insoluble Cu(I). To circumvent both toxicity and solubility issues, a system has evolved where copper is comforted by certain copper binding proteins, so-called Cu-chaperones. They transiently interact with each other to distribute the Cu atoms in a cell. In humans, one of them is Atox1. It binds copper with a binding site containing two thiol residues and transfers it to other binding sites, mostly those of a copper pump, ATP7B (also known as Wilsons disease protein).

My work was aimed at understanding copper-mediated protein-protein interactions on a molecular and mechanistic level. Which amino acids interact with the metal? Which forces drive the transfer from one protein to the other? Using biophysical and biochemical methods such as chromatography and calorimetry on wild type and point-mutated proteins in vitro, we found that the copper is transferred via a dynamic intermediate complex that keeps the system flexible while shielding the copper against other interactions.

Although similar transfer interactions can be observed in other organisms, and many conclusions in the copper field are drawn from bacterial and yeast analogs, we believe that it is important to investigate human proteins, too. Not only is their regulation different, but also only in humans we find the diseases linked to the proteins: Copper level regulation diseases are to be named first, but atypical copper levels have also been linked to tumors and amyloid dispositions. In summary, my observations and conclusions are of basic research character and can be of importance for both general copper and human medicinal research.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2015. 96 p.
copper homeostasis, copper chaperone, Atox1, ATP7B, Wilson disease protein, metal transport, size exclusion chromatography, thermodynamics, isothermal calorimetry
National Category
Inorganic Chemistry Biophysics Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-100511 (URN)978-91-7601-203-1 (ISBN)
Public defence
2015-03-27, Lilla Hörsalen, KBC KB3A9, Umeå Universitet, Umeå, 10:00 (English)
Available from: 2015-03-06 Created: 2015-03-03 Last updated: 2015-03-27Bibliographically approved

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