High frequency of the haemochromatosis C282Y mutation in Hungary could argue against a Celtic origin of the mutation.
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Cohort Studies↗
Biomedical subjects
B Sarkadi
Publications and source records attributed to B Sarkadi.
Phosphorylation site mutations in the human multidrug transporter modulate its drug-stimulated ATPase activity.
In the human multidrug transporter (MDR1), three serine residues located in the "linker" region of the protein are targets of in vivo phosphorylation. These three serines, or all eight serines and threonines in the linker, were substituted by alanines (mutants 3A and 8A) or with glutamic acids (mutants 3E and 8E). The wild-type and mutant proteins were expressed in baculovirus-infected Spodoptera frugiperda (Sf9) ovarian insect cells, and the vanadate-sensitive, drug-stimulated ATPase activity was measured in isolated membrane preparations. The maximum drug-stimulated MDR1-ATPase activity was similar for the wild-type and the mutant proteins. However, wild-type MDR1, which is known to be phosphorylated in Sf9 membranes, and the 3E and 8E mutants, which mimic the charge of phosphorylation, achieved half-maximum activation of MDR1-ATPase activity at lower verapamil, vinblastine, or rhodamine 123 concentrations than the nonphosphorylatable 3A and 8A variants. For some other drugs (e.g. valinomycin or calcein acetoxymethylester) activation of the MDR1-ATPase for any of the mutants was indistinguishable from that of the wild-type protein. Kinetic analysis of the data obtained for the 3A and 8A MDR1 variants indicated the presence of more than one drug interaction site, exhibiting an apparent negative cooperativity. This phenomenon was not observed for the wild-type or the 3E and 8E MDR1 proteins. The dependence of the MDR1-ATPase activity on ATP concentration was identical in the wild-type and the mutant proteins, and Hill plots indicated the presence of more than one functional ATP-binding site. These results suggest that phosphorylation of the linker region modulates the interaction of certain drugs with MDR1, especially at low concentrations, although phosphorylation does not alter the maximum level of MDR1-ATPase activity or its dependence on ATP concentration.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region.
A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters.
A group of ATP-binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance-associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP-related proteins are also characterized by a special, common membrane topology pattern. The most studied ABC transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance (MDR) proteins, were shown to contain a tandem repeat of six transmembrane helices, each set followed by an ATP-binding domain. According to the present study, in contrast to various membrane topology predictions proposed for the different MRP-related proteins, they all seem to have a CFTR/MDR-like core structure, and an additional, large, N-terminal hydrophobic region. This latter domain is predicted to contain 4-6 (most probably 5) transmembrane helices, and is occasionally glycosylated on the cell surface. Since all the MRP-related transporters were shown to interact with anionic compounds, the N-terminal membrane-bound domain may have a key role in these interactions.
ATP-Binding Cassette Transporters↗
Search for specific inhibitors of multidrug resistance in cancer.
This paper deals with the theoretical background, methods and clinical significance of the search for specific inhibitors of multidrug resistance (MDR) in cancer. After discussing the basic features of the drug pump membrane proteins (MDR1 and MRP) responsible for this phenomenon, the possible mechanism of action of MDR inhibitors is reviewed. Modulators of drug pump expression may include inhibitors of the transcription and translation, as well as the processing and post-translational modifications of MDR1 and MRP. The function of the drug pumps can be effectively inhibited by substrate analogs, inhibitors of ATP-binding and utilization, specific monoclonal antibodies, and by various agents with still non-specified mechanisms. Basic methods are presented for the in vitro screening of MDR inhibitors in whole cells and in pump protein preparations, as well as for the in vivo assessment of the efficiency and safety of such inhibitors. The review also discusses some current problems and perspectives of MDR modulation in clinical tumor therapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Prevalence of factor V Leiden (Arg506Gln) in Hungary.
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Factor V↗
Membrane topology and glycosylation of the human multidrug resistance-associated protein.
The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells.
In this paper we demonstrate that the expression of the multidrug resistance-associated protein (MRP) in a variety of intact human tumour cells results in the ATP-dependent, mutually exclusive extrusion of both the acetoxymethyl ester and the free anion forms of the fluorescent dye calcein, as well as that of a fluorescent pyrenemaleimide-glutathione conjugate. The MRP-dependent transport of all these three model compounds closely correlates with the expression level of MRP and is cross-inhibited by hydrophobic anticancer drugs, by reversing agents for MDR1, and also by compounds not influencing MDR1, such as hydrophobic anions, alkylating agents, and inhibitors of organic anion transporters. Cellular glutathione depletion affects neither the MRP-dependent extrusion of calcein AM or free calcein, nor its modulation by most hydrophobic or anionic compounds, although eliminating the cross-inhibitory effect of glutathione conjugates. These results suggest that the outward pumping of both hydrophobic uncharged and water-soluble anionic compounds, including glutathione conjugates, is an inherent property of MRP, and offer sensitive methods for the functional diagnostics of this transport protein as well as for the rapid screening of drug-resistance modulating agents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein.
The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system. The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells. In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied. We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied. Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity. In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either. However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine. These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells. These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
The multidrug transporters--proteins of an ancient immune system.
The multidrug resistance proteins, discovered as membrane transporters producing chemotherapy-resistance in cancer, are functioning as complex cellular defence systems through recognition and energy-dependent removal of a large variety of toxic agents. The multidrug transporters belong to the ATP-binding cassette (ABC) transporters, present both in prokaryotes and eukaryotes and built from a combination of characteristic membrane-spanning helices and cytoplasmic ATP-binding domains. In mammals the MDR1 (P-glycoprotein) extrudes large hydrophobic compounds and provides the basis of the blood-brain and the blood-testis barrier for such molecules. The multidrug resistance-associated protein (MRP) and its homologues have a major role in the cellular export of large organic anions, including e.g. conjugated bile salts and glutathione-conjugates. The substrate recognition, that is the self and non-self discrimination and the ATP-dependent foreign agent extrusion are directly coupled within the structure of these large plasma membrane proteins. Here we suggest that the multidrug transporters are essential parts of our immune-defence system, working as 'cellular antitoxic' mechanisms.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells.
A rapid, functional and quantitative diagnostic method for the estimation of the P-glycoprotein (P-gp)-dependent multidrug resistance is required in the clinical treatment of human tumours, as chemotherapy protocols and resistance-reversing agents could be applied accordingly. In the present work, by using a calcein accumulation method in combination with immunorecognition and drug-resistance studies, a new method is described for the quantitative estimation of the expression and function of the multidrug transporter. MDR1-transfected and drug-selected tumour cell lines with various levels of drug resistance were examined. The expression of P-gp and its cell-surface appearance were assessed by quantitative immunoblotting and by immunofluorescence cytometry. The transport function of the P-gp was assessed by measuring the extrusion of calcein acetoxymethyl ester (AM) with fluorometry and flow cytometry, while in parallel experiments drug resistance was directly examined in cell survival assays. The MDR1 activity factor (MAF), calculated from the calcein AM extrusion assay, is demonstrated to provide a reliable quantitative measure for MDR1 specific activity, reflecting cellular drug resistance. This relatively simple and rapid new functional P-gp assay surpasses the formerly used techniques in both sensitivity and reproducibility.
3T3 Cells↗
Drug-stimulated ATPase activity of a deletion mutant of the human multidrug-resistance protein (MDR1).
The baculovirus-insect cell system has been used for the functional expression of the human multidrug resistance protein (MDR1) and a mutant MDR1 variant lacking a twenty amino acid segment from the first extracellular loop (delta aa78-97 MDR1). Both MDR1 proteins were found to be correctly inserted into the insect cell membrane as indicated by their interaction with MRK 16 antibody. The removal of the 78-97 segment from the first extracellular loop dramatically altered drug-stimulated ATPase activity. Rhodamine 123 or vinblastine were not able to stimulate the mutant protein and Calcein AM had also little effect. In contrast, verapamil increased the ATPase activity of the mutant almost to the same maximal level as that of the wild type. However, the verapamil concentration needed for the half maximal stimulation of the ATPase activity was found to be about hundred times higher than that for the wild type MDR1. These results indicate that a partial deletion of an extracellular loop modulates the affinity of MDR1 for its transportable substrates in a variable fashion.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Calcein accumulation as a fluorometric functional assay of the multidrug transporter.
Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein-Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493-21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.
3T3 Cells↗
Pyridine nucleotide redox potential modulates cystic fibrosis transmembrane conductance regulator Cl- conductance.
Cl- conductance of the apical membrane of airway epithelial cells has properties of a passive diffusion mechanism but is decreased by inhibition of oxidative metabolism. Recent reports that cAMP-dependent Cl- conductance also requires ATP at the intracellular domains of the cystic fibrosis transmembrane conductance regulator (CFTR) suggests that ATP concentration could mediate metabolic regulation of Cl- conductance. However, metabolic inhibitors affect processes other than ATP free energy levels, including notably the metabolic pathways that set the redox potential of pyridine nucleotides within the cell. We have investigated the possibility that CFTR-mediated Cl- conductance is affected by the ratio of oxidized to reduced intracellular pyridine nucleotides. CFTR was expressed in airway and heterologous cells and studied under whole cell voltage clamp conditions, which permitted the intracellular NAD(P)+/NAD(P)H ratio to be varied independently of ATP concentration. In three cell types expressing CFTR, whole cell dialysis with reduced pyridine nucleotides inhibited activation of Cl- currents by forskolin and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), whereas dialysis with oxidized pyridines increased both basal and stimulated CFTR-mediated Cl- conductance. In cell-attached membrane patches, the open probability of 5-6-picosiemens Cl- channels that had been activated by forskolin and CPT-cAMP was further and reversibly increased by permeant oxidants. Neither swelling-induced whole cell K+ currents in CFTR-expressing cells nor swelling-induced whole cell Cl- currents in multidrug resistance protein-expressing cells were affected by NADPH. Pyridine nucleotide redox potential had little effect on phosphorylation of histone by protein kinase A. We conclude that CFTR Cl- conductance function can be modulated by pyridine nucleotide redox potential. This effect points to the existence of a mechanism or mechanisms by which cytosolic nucleotides other than ATP can affect plasma membrane Cl- conductance and may help explain how a passive ion conductance is linked to cellular energy metabolism.
3T3 Cells↗
Controlled proteolysis of Ca(2+)-ATPases in human platelet and non-muscle cell membrane vesicles. Evidence for a multi-sarco/endoplasmic reticulum Ca(2+)-ATPase system.
Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.
Blood Platelets↗
Interaction of bioactive hydrophobic peptides with the human multidrug transporter.
In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1-ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high-affinity interaction with the MDR1-ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.
3T3 Cells↗
Fluorescent cellular indicators are extruded by the multidrug resistance protein.
In this report we show that NIH-3T3 mouse fibroblasts stably expressing the human multidrug transporter (MDR1 or P-glycoprotein), in contrast to the control NIH-3T3 cells, actively extrude the hydrophobic acetoxymethyl ester (AM) derivatives used for cellular loading of various fluorescent calcium and pH indicators. This dye extrusion is blocked by competing substrates and inhibitors of the multidrug transporters, e.g. by verapamil, vincristine, sodium orthovanadate, oligomycin, and a monoclonal anti-MDR1 antibody. The hydrophilic free acid forms of the indicators are not exported by MDR1. We also demonstrate that in isolated cell membranes the MDR1-ATPase, similar to that by known substrates of the transporter, is stimulated by the AM derivatives of fluorescent dyes whereas the free acid forms of the dyes are without effect. Since (i) the AM derivatives of the fluorescent indicators rapidly permeate the cell membrane and are readily cleaved by high activity and large capacity cytoplasmic esterases and (ii) the free acid forms are not substrates for export by MDR1, the observations above suggest that dye extrusion by MDR1 may occur without a cytoplasmic appearance of the AM compounds. These data also call attention to the possible interaction of widely used hydrophobic fluorescent indicators with MDR1 and offer an efficient detection of MDR1-expressing tumor cells as well as a screening method for examining drug interactions with the multidrug transporter.
3T3 Cells↗
Effects of equisetin on rat liver mitochondria: evidence for inhibition of substrate anion carriers of the inner membrane.
The effect of equisetin, an antibiotic produced by Fusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F1-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate+malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD+ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca(2+)-uptake of mitochondria energized both by ATP or succinate without affecting the Ca(2+)-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the Pi and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.
Adenosine Diphosphate↗