title: Probing the interaction of cisplatin with the human copper chaperone Atox1 by solution and in-cell NMR spectroscopy.
abstract: Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH(3))(2)}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in E. coli reduces the amount of DNA platination and, consequently, the degree of cell filamentation.
authors: Department Farmaco-Chimico, University of Bari A. Moro, via Edoardo Orabona 4, 70125 Bari, Italy.
authors: Fabio
authors: F
authors: Arnesano
authors: Lucia
authors: L
authors: Banci
authors: Ivano
authors: I
authors: Bertini
authors: Isabella C
authors: IC
authors: Felli
authors: Maurizio
authors: M
authors: Losacco
authors: Giovanni
authors: G
authors: Natile
mesh_terms: D027682:Cation Transport Proteins / Q000737:chemistry*; D002945:Cisplatin / Q000737:chemistry*; D000079922:Copper Transport Proteins; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D056511:Metallochaperones; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry*; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D012996:Solutions; D002945:Cisplatin