title: Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro.
abstract: 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.
authors.affiliation: Department of Chemistry, Chemical Biological Center, Umeå University, 901 87 Umeå, Sweden.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Palm
authors.forename: Christoph F
authors.initials: CF
authors.lastname: Weise
authors.forename: Christina
authors.initials: C
authors.lastname: Lundin
authors.forename: Gunnar
authors.initials: G
authors.lastname: Wingsle
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Nygren
authors.forename: Erik
authors.initials: E
authors.lastname: Björn
authors.forename: Peter
authors.initials: P
authors.lastname: Naredi
authors.forename: Magnus
authors.initials: M
authors.lastname: Wolf-Watz
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry* / Q000493:pharmacokinetics; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002945:Cisplatin / Q000737:chemistry* / Q000493:pharmacokinetics; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D006801:Humans; D056511:Metallochaperones; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: C474500:ATOX1 protein, human; D000970:Antineoplastic Agents; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D003300:Copper; D004247:DNA; D002945:Cisplatin