Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na+/K+-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.

b Department of Protein Biochemistry and Proteomics Faculty of Science Centre of Region Haná for Biotechnological and Agricultural Research Palacký University Olomouc Czech Republic

Department of Biophysics Faculty of Science Centre of Region Haná for Biotechnological and Agricultural Research Palacký University Olomouc Czech Republic

Zobrazit více v PubMed

Cepeda V, Fuertes M, Castilla J, et al. . Biochemical mechanisms of cisplatin cytotoxicity. Anticancer Agents Med Chem 2007;7:3–18. PubMed

Barabas K, Milner R, Lurie D, Adin C.. Cisplatin: a review of toxicities and therapeutic applications. Vet Comp Oncol 2008;6:1–18. PubMed

Dzagnidze A, Katsarava Z, Makhalova J, et al. . Repair capacity for platinum-DNA adducts determines the severity of cisplatin-induced peripheral neuropathy. J Neurosci 2007;27:9451–7. PubMed PMC

Pabla N, Dong Z.. Cisplatin nephrotoxicity: mechanisms and renoprotective trategies. Kidney Int 2008;73:994–1007. PubMed

Miller RP, Tadagavadi RK, Ramesh G, Reeves WB.. Mechanisms of cisplatin nephrotoxicity. Toxins (Basel) 2010;2:2490–518. PubMed PMC

Huličiak M, Vacek J, Šebela M, et al. . Covalent binding of cisplatin impairs the function of Na+/K+-ATPase by binding to its cytoplasmic part. Biochem Pharmacol 2012;83:1507–13. PubMed

Kubala M, Geleticova J, Huliciak M, et al. . Na+/K+-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity. Biomed Pap 2014;158:194–200. PubMed

Kaplan JH.Biochemistry of Na,K-ATPase. Annu Rev Biochem 2002;71:511–35. PubMed

Geering K. The functional role of beta subunits in oligomeric P-type ATPases. J Bioenerg Biomembr 2001;33:425–38. PubMed

Huliciak M, Reinhard L, Laursen M, et al. Crystals of Na+/K+-ATPase with bound cisplatin. Biochem Pharmacol 2014;92:494–8. PubMed

Jamieson ER, Lippard SJ.. Structure, recognition, and processing of cisplatin-DNA adducts. Chem Rev 1999;99:2467–98. PubMed

Tran CM, Farley RA.. Catalytic activity of an isolated domain of Na, K-ATPase expressed in Escherichia coli. Biophys J 1999;77:258–66. PubMed PMC

Gatto C, Wang AX, Kaplan JH.. The M4M5 cytoplasmic loop of the Na, K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein*. J Biol Chem 1998;273:10578–85. PubMed

Grycova L, Sklenovsky P, Lansky Z, et al. . ATP and magnesium drive conformational changes of the Na+/K+-ATPase cytoplasmic headpiece. Biochim Biophys Acta Biomembr 2009;1788:1081–91. PubMed

Kubala M, Obsil T, Obsilová V, et al. . Protein modeling combined with spectroscopic techniques: an attractive quick alternative to obtain structural information. Physiol Res 2004;53:S187–S97. PubMed

Kubala M, Plášek J, Amler E.. Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+,K+-ATPase. Physiol Res 2004;53:109–13. PubMed

Kubala M, Plášek J, Amler E.. Limitations in linearized analyses of binding equilibria: binding of TNP-ATP to the H 4-H 5 loop of Na/K-ATPase. Eur Biophys J 2003;32:363–9. PubMed

Lánský Z, Kubala M, Ettrich R, et al. . The hydrogen bonds between Arg423 and Glu472 and other key residues, Asp443, Ser477, and Pro489, are responsible for the formation and a different positioning of TNP-ATP and ATP within the nucleotide-binding site of Na+/K+-ATPase. Biochemistry 2004;43:8303–11. PubMed

Kubala M, Teisinger J, Ettrich R, et al. . Eight amino acids form the ATP recognition site of Na(+)/K(+)-ATPase. Biochemistry 2003;42:6446–52. PubMed

Kubala M, Krumscheid R, Schoner W.. Phe475 and Glu446 but not Ser445 participate in ATP-binding to the α-subunit of Na+/K+-ATPase. Biochem Biophys Res Commun 2002;297:154–9. PubMed

Kubala M, Grycova L, Lansky Z, et al. . Changes in electrostatic surface potential of Na+/K+-ATPase cytoplasmic headpiece induced by cytoplasmic ligand(s) binding. Biophys J 2009;97:1756–64. PubMed PMC

Biler M, Štenclová T, Trouillas P, Biedermann D.. Flavonolignans as a novel class of sodium pump inhibitors. Front Physiol 2016;7:115. PubMed PMC

Schägger H, von Jagow G.. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368–79. PubMed

Lundblad RL, Noyes CM.. Chemical reagents for protein modification. Boca Ralton (FL): CRC Press; 1984. 192 p.

Beinhauer J, Lenobel R, Loginov D, et al. . Identification of Bremia lactucae and Oidium neolycopersici proteins extracted for intact spore MALDI mass spectrometric biotyping. Electrophoresis 2016;37:2940–52. PubMed

Nyblom M, Reinhard L, Andersson M, et al. . Crystal structure of Na+,K+-ATPase in the Na+-bound state. Science 2013;342:123–7. PubMed

DeLano WL.The PyMOL molecular graphics system. New York: Schrödinger, LLC; 2013.

Abraham MJ, Murtola T, Schulz R, et al. . GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 2015;1–2:19–25.

Berendsen HJC, Postma JPM, van Gunsteren WF, et al. . Molecular dynamics with coupling to an external bath. J Chem Phys 1984;81:3684–90.

Dabrowiak JC, Metals in medicine. Weinheim, Germany: Wiley-VCH; 2009. 334 p.

Hartinger CG, Tsybin YO, Fuchser J, Dyson PJ.. Characterization of platinum anticancer drug protein-binding sites usin a top-down mass spectrometric approach. Inorg Chem 2008;47:17–19. PubMed

Hartinger CG, Ang WH, Casisni A, et al. . Mass spectroscopic analysis of ubiquitin-platinum interactions of leading anticancer drugs: MALDI versus ESI. J Anal at Spectrom 2007;22:960–7.

Ishikawa T, Aliosman F.. Glutathione-associated cis-diamminodichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia-cells - molecular characterization of glutathione-platinum complex and its biological significance. J Biol Chem 1993;268:20116–25. PubMed

Odenheimer B, Wolf W.. Reactions of cisplatin with sulfur-containing amino-acids and peptides. 1. Cysteine and glutathione. Inorganica Chim Acta 1982;66:L41–3.

Neault JF, Benkirane A, Malonga H, Tajmir-Riahi HA.. Interaction of cisplatin drug with Na,K-ATPase: drug binding mode and protein secondary structure. J Inorg Biochem 2001;86:603–9. PubMed

Ferraro G, Massai L, Messori L, Merlino A.. Cisplatin binding to human serum albumin: a structural study. Chem Commun 2015;51:9436–9. PubMed

Messori L, Merlino A.. Cisplatin binding to proteins: molecular structure of the ribonuclease a adduct. Inorg Chem 2014;53:3929–31. PubMed

Marasco D, Messori L, Marzo T, Merlino A.. Oxaliplatin vs. cisplatin: competition experiments on their binding to lysozyme. Dalt Trans 2015;44:10392–8. PubMed

Messori L, Merlino A.. Cisplatin binding to proteins: a structural perspective. Coord Chem Rev 2016;315:67–89.

Bogdanova A, Petrushanko IY, Hernansanz-Agustín P.. “Oxygen Sensing” by Na, K-ATPase: these miraculous thiols. Front Physiol 2016;7:314. PubMed PMC

Petrushanko IY, Yakushev S, Mitkevich VA, et al. . S-glutathionylation of the Na,K-ATPase catalytic α subunit is a determinant of the enzyme redox sensitivity. J Biol Chem 2012;287:32195–205. PubMed PMC