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.

• MeSH
• cisplatina chemie   farmakologie   MeSH
• cystein antagonisté a inhibitory   metabolismus   MeSH
• cytoplazma účinky léků   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• mutageneze cílená MeSH
• myši MeSH
• protinádorové látky chemie   farmakologie   MeSH
• simulace molekulární dynamiky MeSH
• sodíko-draslíková ATPasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

This study was aimed at verifying the hypothesis that acute kidney failure accompanying cisplatin administration in the cancer therapy could be due to cisplatin interaction with the cytoplasmic part of Na(+)/K(+)-ATPase. Our results demonstrated that cisplatin-binding caused inhibition of Na(+)/K(+)-ATPase, in contrast to other platinated chemotherapeutics such as carboplatin and oxaliplatin, which are known to be much less nephrotoxic. To acquire more detailed structural information, we performed a series of experiments with the isolated large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45 loop) of Na(+)/K(+)-ATPase. Electrochemistry showed that cisplatin is bound to the cysteine residues of the C45 loop, mass spectrometry revealed a modification of the C45 peptide fragment GSHMASLEAVETLGSTSTICSDK, which contains the conserved phosphorylated residue Asp369. Hence, we hypothesize that binding of cisplatin to Cys367 can cause sterical obstruction during the phosphorylation or dephosphorylation step of the Na(+)/K(+)-ATPase catalytic cycle.

• MeSH
• cisplatina metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• mozková kůra enzymologie   MeSH
• prasata MeSH
• protinádorové látky metabolismus   farmakologie   MeSH
• sodíko-draslíková ATPasa antagonisté a inhibitory   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Combination of fluorescence techniques and molecular docking was used to monitor interaction of Na,K-ATPase and its large cytoplasmic loop connecting fourth and fifth transmembrane helices (C45) with fluorone dyes (i.e. eosin Y, 5(6)-carboxyeosin, rose bengal, fluorescein, and erythrosine B). Our data suggested that there are at least two binding sites for all used fluorone dyes, except of 5(6)-carboxyeosin. The first binding site is located on C45 loop, and it is sensitive to the presence of nucleotide. The other site is located on the extracellular part of the enzyme, and it is sensitive to the presence of Na(+) or K(+) ions. The molecular docking revealed that in the open conformation of C45 loop (which is obtained in the presence of ATP) all used fluorone dyes occupy position directly inside the ATP-binding pocket, while in the closed conformation (i.e. in the absence of any ligand) they are located only near the ATP-binding site depending on their different sizes. On the extracellular part of the protein, the molecular docking predicts two possible binding sites with similar binding energy near Asp897(α) or Gln69(β). The former was identified as a part of interaction site between α- and β-subunits, the latter is in contact with conserved FXYD sequence of the γ-subunit. Our findings provide structural explanation for numerous older studies, which were performed with fluorone dyes before the high-resolution structures were known. Further, fluorone dyes seem to be good probes for monitoring of intersubunit interactions influenced by Na(+) and K(+) binding.

• MeSH
• adenosintrifosfát chemie   MeSH
• červeň bengálská chemie   MeSH
• chemické modely MeSH
• cytoplazma metabolismus   MeSH
• draslík chemie   MeSH
• eosin chemie   MeSH
• erythrosin chemie   MeSH
• Escherichia coli metabolismus   MeSH
• fluorescein chemie   MeSH
• fluoresceiny chemie   farmakologie   MeSH
• fluorescenční barviva farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární konformace MeSH
• sodík chemie   MeSH
• sodíko-draslíková ATPasa chemie   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• kinetika MeSH
• mutageneze cílená metody   MeSH
• simulace molekulového dockingu MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• tryptofan metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A set of single-tryptophan mutants of the Na(+)/K(+)-ATPase isolated, large cytoplasmic loop connecting transmembrane helices M4 and M5 (C45) was prepared to monitor effects of the natural cytoplasmic ligands (i.e., Mg(2+) and/or ATP) binding. We introduced a novel method for the monitoring of the changes in the electrostatic surface potential (ESP) induced by ligand binding, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. This approach opens a new way to understanding the interactions within the proteins. Our experiments revealed that the C45 conformation in the presence of the ATP (without magnesium) substantially differed from the conformation in the presence of Mg(2+) or MgATP or in the absence of any ligand not only in the sense of geometry but also in the sense of the ESP. Notably, the set of ESP-sensitive residues was different from the set of geometry-sensitive residues. Moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. This property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain.

• MeSH
• adenosintrifosfát farmakologie   metabolismus   MeSH
• akrylamidy farmakologie   metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• fluorescence MeSH
• hořčík farmakologie   metabolismus   MeSH
• jodidy farmakologie   metabolismus   MeSH
• konformace proteinů účinky léků   MeSH
• ligandy MeSH
• molekulární modely MeSH
• mutace MeSH
• myši MeSH
• povrchové vlastnosti MeSH
• sodíko-draslíková ATPasa genetika   chemie   metabolismus   MeSH
• statická elektřina MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• radiační ochrana MeSH

• Konspekt
• Veřejné zdraví a hygiena
• NLK Obory
• environmentální vědy