Abstract 833: The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin

Abstract

Abstract ATP7A and ATP7B are copper (Cu) efflux transporters that are up-regulated in many types of cancer that have acquired resistance to platinum (Pt)-containing drugs. Over-expression of ATP7A or ATP7B in cultured cells also leads to Pt drug resistance, but the mechanisms by which these proteins mediate resistance are unknown. The N-terminal domain of both ATP7A and ATP7B contains 6 metal binding domains (MBD) whose βαββαβ fold is similar to that of ATOX1, a protein with which cisplatin (cDDP) is known to bind. Each MBD contains a CXXC motif similar to that known to be the cDDP binding site in ATOX1. This study explored the role of the MBDs of ATP7B in mediating cDDP resistance. Gel filtration, gel electrophoresis and UV spectrometry was employed to determine whether the sixth MBD of ATP7B (MBD6), which is essential for Cu efflux, interacts with cDDP. Recombinant MBD6 was produced as a maltose binding protein (MBP) fusion protein in E. coli. Purified recombinant MBD6 interacted with cDDP very rapidly and became multimerized producing dimers, trimers, tetramers and pentamers. Purified wild type MBP-MBD6 bound 1.75 ± 0.19 mol Pt/mol whereas the MBP tag alone bound only 0.57 ± 0.012 mol Pt/mol (p = 0.0057). MBD6 accumulated cDDP at a rate of 0.08 mol Pt/min/mol. Blocking the CXXC motif in MBD6 with a thiol reactive agent, or converting the motif to SXXS caused the loss of cDDP binding and multimerization. MBD6 in which the CXXC motif was converted to SXXS bound only 0.42 ± 0.013 mol Pt/mol of protein. The reaction of cDDP with MBD6 was rapid as dimers and trimers were detected with just a 10 min exposure to a 20-fold molar excess of cDDP. cDDP reacted with MBS6 even after pre-loading of the protein with Cu, presumably dislodging Cu from the protein. The interaction of cDDP with MBD6 was strong and only very slowly reversible under extreme reducing conditions. Using the lentiviral vector pLVX-mCherry-C1, we constructed vectors capable of expressing mCherry-tagged wild type ATP7B, variants in which either the first 5 (mC-ATP7B α1-5) or all 6 (mC-ATP7B α1-6) of the MBD were deleted, and a variant in which the CXXC motifs of all 6 MBDs were converted to SXXS (mC-ATP7B SXXS). These constructs were expressed in 2008 ovarian carcinoma and HEK293Tcell lines and their effects on the intracellular accumulation and cytotoxicity of cDDP as well as their traffic pattern following cDDP administration was investigated. The wild type ATP7B conferred resistance to 2008 cells, significantly reduced cDDP accumulation and exhibited cDDP- and Cu-induced trafficking from the TGN to superficially located vesicles; however, the other variants did not. We conclude that cDDP binds to ATP7B and that, in the case of MBD6, the site of interaction is the CXXC motif. Conversion of the CXXC motif in all 6 MBD rendered ATP7B unable to mediate cDDP resistance of traffic from the TGN indicating that cDDP binding to at least one of the MBD is essential to ATP7B-mediated detoxification of cDDP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 833. doi:1538-7445.AM2012-833