Electrocatalytic assay for monitoring methylglyoxal-mediated protein glycation.

Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl3 M KCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.

Fluorone dyes have binding sites on both cytoplasmic and extracellular domains of Na,K-ATPase.

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(ß). The former was identified as a part of interaction site between α- and ß-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.

Palmatine activates AhR and upregulates CYP1A activity in HepG2 cells but not in human hepatocytes.

The protoberberine alkaloid palmatine is present in preparations from medicinal plants such as Coptis chinensis and Corydalis yanhusuo. This study examined whether palmatine affects the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells grown as monolayer or spheroids. Gene reporter assays showed that palmatine significantly activated the aryl hydrocarbon receptor (AhR) and increased the activity of CYP1A1 gene promoter in transiently transfected HepG2 cells. In HepG2 monolayer culture, palmatine also significantly increased mRNA and activity levels of CYP1A1, albeit with considerably less potency than 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical CYP1A inducer. On the other hand, CYP1A activity was not significantly elevated by palmatine in HepG2 spheroids. Moreover, palmatine induced mild or negligible changes in CYP1A1 and CYP1A2 mRNA expression without affecting CYP1A activity levels in primary human hepatocytes. It is concluded that palmatine activates the AhR-CYP1A pathway in HepG2 monolayer, while the potential for CYP1A induction is irrelevant in cell systems which are closer to the in vivo situation, i.e. in HepG2 spheroids and primary cultures of human hepatocytes. Possible induction of CYP1A enzymes by palmatine in vivo remains to be investigated.

Covalent binding of cisplatin impairs the function of Na(+)/K(+)-ATPase by binding to its cytoplasmic part.

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.