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Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Pernilla Wittung-Stafshede)
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Anna Linusson)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 7, e70473Article in journal (Refereed) Published
Abstract [en]

Cisplatin (CisPt) is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu) ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15) are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4), indicative of a possible drug detoxification mechanism.

Place, publisher, year, edition, pages
Public Library Science , 2013. Vol. 8, no 7, e70473
Keyword [en]
Atox1, Cisplatin, copper-transport
National Category
Other Chemistry Topics
Research subject
biological chemistry
Identifiers
URN: urn:nbn:se:umu:diva-80713DOI: 10.1371/journal.pone.0070473ISI: 000323114200087PubMedID: 23936210OAI: oai:DiVA.org:umu-80713DiVA: diva2:651188
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Copper-transporting proteins and their interactions with platinum-based anticancer substances
Open this publication in new window or tab >>Copper-transporting proteins and their interactions with platinum-based anticancer substances
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

  Cisplatin (CisPt) is an important drug that is used against various cancers, including testicular, ovarian, lung, head, and neck cancer. However, its effects are limited by cellular resistance. The resistance is believed to be multifactorial, and may be mediated to varying degree by multiple systems in cells, one of the proposed systems being the copper (Cu) transporting system. The Cu-importer Ctr1 has proven importance for cellular sensitivity to CisPt by regulating its influx, while the Golgi-localized Cu-ATP:ases ATP7A/B can putatively mediate CisPt efflux and/or drug sequestration. Atox1 is a small Cu-chaperone that normally transfers Cu between Ctr1 and ATP7A/B, prior to delivery of Cu to the proteins in the secretory pathway. Since Ctr1 and ATP7A/B are reportedly involved in CisPt-resistance, CisPt interaction with Atox1 was the focus of the project this thesis is based upon.

  Using a variety of techniques, Atox1 was found to bind CisPt, also simultaneously with Cu. The Atox1-CisPt complexes were further probed using selected mutants in studies demonstrating that only the two cysteines (Cys12 and Cys15) in the Cu-binding site of Atox1 are essential for CisPt interactions. A proposed Atox1 di-metal complex containing both Cu and CisPt was found to be monomeric, and no loss of Cu was observed. In vitro experiments demonstrated that CisPt could also bind to metal-binding domain 4 of ATP7B (WD4), and that the drug could be transferred from Atox1 to the domain. These findings indicated that Atox1 may transfer CisPt to ATP7A/B in vivo, utilizing the same transport pathway as Cu. However, the CisPt-bound Atox1 complexes were not stable over time; upon incubation, protein unfolding and aggregation were observed. Thus, in vivo, Atox1 might alternatively be a dead-end sink for CisPt.

  The effects of the ligands around the Pt-center of Pt-based anticancer drugs and drug derivatives on Atox1 binding and unfolding were also investigated. The ligands’ chemistry and geometry were shown to dictate the extent and rate of the Pt-based substances interactions with Atox1. Finally, the occurrence of Atox1-CisPt interactions in a biological environment was demonstrated by developing and applying an antibody-based method allowing analysis of metals associated with Atox1 extracted from CisPt-treated cells.

  The findings presented in this thesis show that CisPt binds to Atox1 and WD4, also simultaneously with Cu, in vitro. The results support the hypothesis that Cu-transporting proteins can mediate cellular resistance to CisPt in vivo, and provide a deeper chemical understanding of the interactions between the proteins and the drug.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2013. 96 p.
Keyword
Cisplatin, Atox1, copper transport, anticancer drug, resistance, platinum, spectroscopy.
National Category
Other Chemistry Topics
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-80717 (URN)978-91-7459-705-9 (ISBN)
Public defence
2013-10-18, KBC-huset, KB3B1, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-09-27 Created: 2013-09-24 Last updated: 2013-09-27Bibliographically approved

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