Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multidrug resistant (MDR) cells overexpress a 170-180 kDa membrane glycoprotein, the P-glycoprotein, which is believed to export drugs in an ATP-dependent manner. Plasma membrane vesicles from the MDR CHRC5 cell line, but not the AuxB1 drug-sensitive parent, showed uptake of [3H]colchicine and [3H]vinblastine that was stimulated by the presence of ATP and an ATP-regenerating system. Steady-state uptake of drugs was achieved by 10 min and was stable for greater than 30 min. Non-hydrolysable ATP analogues were unable to support drug uptake, indicating that ATP hydrolysis is essential for transport. ATP-stimulated drug uptake appeared to result from drug transport into inside-out vesicles, since uptake was osmotically sensitive and could be prevented by detergent permeabilization. Steady-state uptake was half-maximal at 100 microM colchicine and 200 nM vinblastine and was inhibited by a 10-100-fold excess of MDR drugs and chemosensitizers, in the order vinblastine greater than verapamil greater than daunomycin greater than colchicine. In addition to being vanadate-sensitive, drug uptake was inhibited by 10-200 microM concentrations of several sulfhydryl-modifying reagents, suggesting that cysteine residues play an important role in drug transport. Vesicular colchicine was rapidly exchanged by an excess of unlabelled drug, demonstrating that drug association is the net result of opposing colchicine fluxes across the membrane.
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PMID:Transport properties of P-glycoprotein in plasma membrane vesicles from multidrug-resistant Chinese hamster ovary cells. 135 67

The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E. (1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide-binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 microM for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains may need to bind ATP to couple drug binding to ATPase activity.
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PMID:Covalent modification of human P-glycoprotein mutants containing a single cysteine in either nucleotide-binding fold abolishes drug-stimulated ATPase activity. 755 32

A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect to its ability to confer resistance to various drugs. The cysteine-less P-glycoprotein-A52 retained the ability to confer resistance to vinblastine, colchicine, doxorubicin, and actinomycin D with only a small decrease in efficiency relative to wild-type enzyme. Cysteine residues were then reintroduced into predicted extracellular or cytoplasmic loops of the cysteine-less P-glycoprotein-A52, and the topology of the protein was determined using membrane-permeant and impermeant thiol-specific reagents. It was found that 8 of 15 cysteine residues introduced into P-glycoprotein-A52 could be biotinylated, when cells expressing the mutant P-glycoprotein were incubated with membrane-permeant biotin maleimide. Biotinylation of a cysteine residue placed in predicted extracellular loops between transmembrane segment (TM) 5 and TM6, TM7 and TM8, or TM11 and TM12 was blocked by pretreatment of the cells with a membrane-impermeant maleimide, suggesting that these residues have an extracellular location. By contrast, biotinylation of cysteine residues located in the predicted cytoplasmic loops between TM2 and TM3, TM4 and TM5, TM8 and TM9, or TM10 and TM11 were not blocked by pretreatment with membrane impermeant maleimide, suggesting that these residues were in the cytoplasm. These results are consistent with the model of P-glycoprotein, which predicts six transmembrane segments in each of the two homologous halves of the molecule.
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PMID:Membrane topology of a cysteine-less mutant of human P-glycoprotein. 782 20

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.
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PMID:Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells. 793 53

The serine residue located at position 939 and 941 in the predicted transmembrane segment 11 of P-glycoprotein (P-gp) encoded by mouse mdr3 and mdr1, respectively, appears to be important for interaction of chemotherapeutic drugs and reversal agents with P-gp. To further understand the role of this residue in this process and to identify the structural requirements involved, we have replaced this serine residue by alanine, cysteine, threonine, tyrosine, tryptophan, and aspartic acid and tested the effect of these mutations on the overall activity and substrate specificity of mdr1 and mdr3. All mutant proteins could be expressed at high levels in the membrane fractions of LR73 Chinese hamster cells transfected with the corresponding mutant cDNAs. All introduced mutations had limited effect on the capacity of mdr1 and mdr3 to confer resistance to vinblastine. The modulatory effect of mutations on resistance to colchicine, adriamycin, and actinomycin D was more dramatic. The hydroxyl group of serine did not seem essential for interaction with these drugs since mutant mdr1 and mdr3 bearing alanine or cysteine at that position behaved essentially as wild type, while threonine-bearing mutants showed significantly reduced resistance to these drugs. The insertion at that site of residues with bulkier side chains had more complex effects on P-gp function. While introducing tyrosine, tryptophan, or aspartic acid caused an almost complete loss of colchicine and adriamycin resistance in both mdr1 and mdr3, the replacement to tyrosine or tryptophan had the opposite effect on mdr1 and mdr3 for actinomycin D resistance, causing either a 3-fold increase or a 4-8-fold decrease in resistance to this drug, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulatory effects on substrate specificity of independent mutations at the serine939/941 position in predicted transmembrane domain 11 of P-glycoproteins. 810 79

Cysteine-containing amino acid sequences (CAAX, CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any amino acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl) isoprenoids to peptides and proteins by specific prenyltransferases. Although much work has centered on the enzymatic mechanisms of these enzymes, the biological consequences of the differential isoprenylation they catalyze remain to be elucidated. Farnesylation of the a-factor mating pheromone of Saccharomyces cerevisiae is a known prerequisite for its biological activity and its secretion through a pathway utilizing the yeast STE6 protein, a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein. We generated specific mutations in the a-factor gene to encode isoprenylation targets for geranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in place of the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factors containing these modified prenylation sites were successfully exported by a STE6-dependent mechanism. Furthermore, these peptides, as well as synthetic geranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons of synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate containing the geranylgeranylation target CVIL can be both farnesylated and geranylgeranylated. These results clearly demonstrate that in vivo (i) different prenyltransferases may recognize the same substrate; (ii) both farnesylated and geranylgeranylated a-factor peptides are substrates for export via STE6, a MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are both biologically active.
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PMID:Consequences of altered isoprenylation targets on a-factor export and bioactivity. 810 1

Calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cell-permeable synthetic tripeptide with an aldehyde at its C terminus specifically inhibits the activity of cysteine proteases. Since the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase in Chinese hamster ovary (CHO) cells is blocked by ALLN and ALLN has a cytotoxic effect on cells, we attempted to isolate ALLN-resistant cells that overproduce an ALLN-sensitive protease(s). However, we obtained an ALLN-resistant cell line that overproduced P-glycoprotein (Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R. T., and Simoni, R. D. (1992) J. Biol. Chem. 267, 5731-5734). To circumvent the multidrug resistance (MDR) phenotype during selection, we have stepwise selected an ALLN-resistant cell line of CHO cells in the presence of verapamil, a competitive inhibitor of P-glycoprotein. These non-MDR ALLN-resistant cells overexpress a 35-kDa protein and have increased aldo-keto reductase activity. Partial amino acid sequences of the 35-kDa protein are highly homologous to members of the aldo-keto reductase superfamily. The aldo-keto reductases are NADPH-dependent oxidoreductases and catalyze reduction of a wide range of carbonyl compounds such as aldehydes, sugars, and ketones. Our findings support the concept that a physiological function for aldo-keto reductases may be detoxification.
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PMID:Cellular detoxification of tripeptidyl aldehydes by an aldo-keto reductase. 844 54

Recently, many potent inhibitors of protein serine/threonine phosphatases (PPs) have been found. Some of them have proven to be tumor promoters in mouse skin two-step carcinogenesis and rat liver medium-term tests. Among these inhibitors, okadaic acid (OA) selectively inhibits PP2A, and its use has therefore been proposed to facilitate analysis of biological roles of this phosphatase. OA shows bimodal effects on in vitro transformation and, in addition to such epigenetic changes, also induces marked genetic changes. OA treatment for more than 1 week flattened NIH 3T3 transformants irreversibly, with loss of the transfected genes. It is also known to induce diphtheria toxin-resistant mutations in Chinese hamster lung cells and sister chromatid exchanges (SCEs) in Chinese hamster ovary cells and human lymphocytes. To analyze roles of protein phosphatases in gene stability, we isolated OA-resistant mutants. They were proven to have a mutation in the PP2A alpha catalytic subunit, in which cysteine 269 had been substituted for glycine; and it was demonstrated that this region interacts with OA. The recombinant mutant protein was 4 approximately 9-fold more resistant to OA than the wild type. Although the OA resistant mutants of CHO cells expressed high levels of P-glycoprotein, inhibition of PP2A itself was suggested to lead to SCE induction. However, the number of molecular species of PP which are known to be sensitive to OA continues to increase, and we have isolated cDNA for a novel type of OA sensitive PP. Our studies indicate that the fact that the roles of PP2A cannot be elucidated using only OA is of crucial importance.
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PMID:Protein serine/threonine phosphatases as binding proteins for okadaic acid. 853 25

Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glyco- protein were prepared on the basis of structure predictions. Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility. Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography. NBD1 efficiently interacted with nucleotides. Fluorescence methods showed that ATP bound at millimolar concentrations and its 2',3'-O-(2,4,6-trinitrophenyl) derivative at micromolar concentrations, while the 2'(3')-N-methylanthraniloyl derivative had intermediate affinity. Photoaffinity labeling was achieved upon irradiation with 8-azido-ATP. The domain exhibited ATPase activity with a Km for MgATP in the millimolar range, and ATP hydrolysis was competitively inhibited by micromolar 2',3'-O-(2,4,6-trinitrophenyl)-ATP. NBD1 contained a single cysteine residue, at position 430, that was derivatized with radiolabeled N-ethylmaleimide. Cysteine modification increased 6-fold the Kd for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling. ATPase activity was inhibited with a 5-fold increase in the Km for MgATP. The results suggest that chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by altering substrate interaction.
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PMID:Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein. Overexpression, purification, and role of cysteine 430. 866 20

We have established a quantitative flow cytometry system to elucidate the causal role of P-glycoprotein in the phenomenon of multidrug resistance. We have used this method to analyze the accumulation and release of adriamycin (ADM) in intact L5178Y and L5178Y/VMDR/C.06 (L5178Y/R) cells, by determining the effect of sodium orthovanadate (Na3VO4), verapamil, bovine serum albumin (BSA) and physiologically operative materials on the cells. Based on the experiments, we prepared a standard solution that contained NaCl, D-glucose, L-cysteine, HCO3- and BSA, which was sufficient to perform transport experiments. In particular, BSA caused a decrease in ADM accumulation and a facilitation of the rate of ADM release in both L5178Y and L5178Y/R cells, probably due to its relatively high affinity for ADM as compared to the cell membrane. In multidrug-resistant L5178Y/R cells, sodium orthovanadate, a strong ATP-binding inhibitor, caused a marked increase in the accumulation of ADM, whereas vanadate-treated drug-sensitive L5178Y cells showed little increase in ADM accumulation. In a release (0-trans exit) experiment, vanadate-treated L5178Y/R cells exhibited an apparent decrease in ADM release (increase in ADM retention), to a level which was almost the same as L5178Y cells. We thus confirmed that the P-glycoprotein-mediated efflux system is coupled with P-glycoprotein-associated ATP-hydrolysis. Further, verapamil, a potent inhibitor of P-glycoprotein-mediated transport, facilitated the ADM accumulation in L5178Y/R cells up to the level of L5178Y and vanadate-treated L5178Y/R cells. A more important finding is that, in the release experiment, verapamil-treated L5178Y/R cells exhibited a much greater ADM retention than drug-sensitive L5178Y and vanadate-treated L5178Y/R cells. These findings, in particular the potent effect of verapamil on drug-resistant cells, may afford new insight into the pathophysiology of the phenomenon of multidrug resistance and the mechanism of action of the multidrug transporter.
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PMID:Quantitative characterization of P-glycoprotein-mediated transport in mdr1-gene-transfected lymphoma cells. 874 16


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