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Copper-transporting proteins and their interactions with platinum-based anticancer substances
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Pernilla Wittung-Stafshede)
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 [en]
Cisplatin, Atox1, copper transport, anticancer drug, resistance, platinum, spectroscopy.
National Category
Other Chemistry Topics
Research subject
biological chemistry
Identifiers
URN: urn:nbn:se:umu:diva-80717ISBN: 978-91-7459-705-9 (print)OAI: oai:DiVA.org:umu-80717DiVA: diva2:651191
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
List of papers
1. Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro
Open this publication in new window or tab >>Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro
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2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 17, 6951-6956 p.Article in journal (Refereed) Published
Abstract [en]

Cisplatin (cisPt), Pt(NH(3))(2)Cl(2), is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1's metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-43722 (URN)10.1073/pnas.1012899108 (DOI)21482801 (PubMedID)
Available from: 2011-05-09 Created: 2011-05-09 Last updated: 2017-12-11Bibliographically approved
2. Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox1
Open this publication in new window or tab >>Reaction of platinum anticancer drugs and drug derivatives with a copper transporting protein, Atox1
2012 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 83, no 7, 874-881 p.Article in journal (Refereed) Published
Abstract [en]

Platinum (Pt) containing anticancer drugs have been used in cancer treatment for several decades as they trigger cell death upon DNA binding. Pt-containing anticancer drugs and drug derivates with a variety of ligands around the Pt center (with Cisplatin being most well known) exist today in clinics and in clinical trials. However, a major drawback with these drugs is limited efficacy due to side reactions resulting in cell resistance. The cellular copper (Cu) transport pathway is proposed to be responsible for part of these side reactions through interactions with the Pt-containing drugs and possibly cellular export of Pt. The cytoplasmic Cu chaperone, Atox1, was recently found to bind Cisplatin in vitro and, when over-expressed in Escherichia coli, in vivo. Here we investigate how the chemical properties of six Pt-substances differentially affect binding, unfolding, and aggregation of Atox1 in vitro using near- and far-UV circular dichroism (CD) spectroscopy and SDS-PAGE. The results show that both ligand type and orientation dictate the interactions with Atox1. Only substances with two good leaving groups in cis-configuration result in near-UV CD changes that report on Cu–Pt interactions. The different substances promote Atox1 unfolding in a pattern that can be explained by ligand chemistry and geometry. Our work emphasize that ligands around the Pt-center have decisive roles in tuning protein interactions (prior to DNA binding) and therefore they also dictate the level of drug side effects and cellular resistance.

Place, publisher, year, edition, pages
Elsevier, 2012
Keyword
Atox1, Cisplatin, Anticancer, Protein unfolding, Spectroscopy
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-52617 (URN)10.1016/j.bcp.2012.01.018 (DOI)
Available from: 2012-02-28 Created: 2012-02-28 Last updated: 2017-12-07Bibliographically approved
3. Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking
Open this publication in new window or tab >>Determinants for simultaneous binding of copper and platinum to human chaperone Atox1: hitchhiking not hijacking
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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
Keyword
Atox1, Cisplatin, copper-transport
National Category
Other Chemistry Topics
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-80713 (URN)10.1371/journal.pone.0070473 (DOI)000323114200087 ()23936210 (PubMedID)
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved
4. Interaction between anticancer drug Cisplatin and copper chaperone Atox1 in human melanoma cells
Open this publication in new window or tab >>Interaction between anticancer drug Cisplatin and copper chaperone Atox1 in human melanoma cells
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2014 (English)In: Protein peptide letters, ISSN 0929-8665, E-ISSN 1875-5305, Vol. 21, no 1, 63-68 p.Article in journal (Refereed) Published
Abstract [en]

Cisplatin (CisPt) is one of the most common anticancer drugs used against many severe forms of cancers. However, treatment with this drug causes many side effects and often, it results in the development of cell resistance. A majority of side effects as well as cell resistance are thought to develop due to CisPt interactions with proteins prior to reaching the nucleus and the DNA target. The copper (Cu) transport proteins Ctr1 and ATP7A/B have been implicated in cellular resistance of CisPt, possibly exporting the drug out of the cell. Recent in vitro work demonstrated that CisPt also interacts with the cytoplasmic Cu-chaperone Atox1, binding in or near the Cu-binding site, without expulsion of bound Cu. Whereas Ctr1 and ATP7B interactions with CisPt have been shown in vivo or ex vivo, there is no such information for Atox1-CisPt interactions. To address this, we developed a method to probe if CisPt interacts with Atox1 in human melanoma cells. Atox1-specific antibodies were linked to magnetic beads and used to immune-precipitate Atox1 from melanoma cells that had been pre-exposed to CisPt. Analysis of extracted Atox1 with inductively coupled plasma mass spectrometry demonstrated the presence of Pt in the protein fraction. Thus, CisPt-exposed human melanoma cells contain Atox1 molecules that bind some derivative of CisPt. This study gives the first indication for the intracellular presence of Atox1-CisPt complexes ex vivo.

Keyword
Copper-chaperone, Cisplatin, Atox1, anticancer, resistance
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-80714 (URN)10.2174/09298665113209990036 (DOI)000329022400011 ()
Available from: 2013-09-24 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved

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