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)

The permeation characteristics of a model opioid peptide, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs [acyloxyalkoxy-based cyclic prodrug of DADLE (AOA-DADLE), coumarinic acid-based cyclic prodrug of DADLE (CA-DALE), and oxymethyl-modified coumarinic acid-based cyclic prodrug of DADLE (OMCA-DADLE)] across the blood-brain barrier (BBB) were determined using an in situ perfused rat brain model. The rat brains were perfused with Krebs-bicarbonate buffer containing test compounds in the absence or presence of a specific P-glycoprotein inhibitor (GF-120918). Brain samples were collected after perfusion and processed by a capillary depletion method. After liquid phase extraction with acetonitrile, samples were analyzed using high-performance liquid chromatography with tandem mass spectrometric detection. Linear uptake kinetics of DADLE and its cyclic prodrugs was observed within the range of 60 to 240 s of perfusion. The apparent permeability coefficient (P(app)) of DADLE across the BBB was very low (<10(-7) cm/s), probably due to its unfavorable physicochemical properties (e.g., charge, hydrophilicity, and high hydrogen-bonding potential). All three cyclic prodrugs, however, also exhibited low membrane permeation (P(app) <10(-7) cm/s) in spite of their more favorable physicochemical properties (e.g., no charge, high hydrophobicity, and low hydrogen-bonding potential). Inclusion of GF-120918 (10 microM) in the perfusates fully inhibited the P-gp activity in the BBB and dramatically increased the P(app) values of AOA-DADLE, CA-DADLE, and OMCA-DADLE by approximately 50-, 460-, and 170-fold, respectively. In contrast, GF-120918 had no effect on the P(app) value of DADLE. In addition, the observed bioconversions of the prodrugs to DADLE in the rat brains after 240-s perfusion were very low (5.1% from AOA-DADLE, 0.6% from CA-DADLE, and 0.2% from OMCA-DADLE), which was consistent with the in vitro bioconversion rates determined previously in rat brain homogenates.
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PMID:Evaluation of the permeation characteristics of a model opioid peptide, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs across the blood-brain barrier using an in situ perfused rat brain model. 1238 72

Bidirectional transport studies were conducted to determine whether substrates of five intestinal transporters showed carrier-mediated asymmetric transport across MDCK (Madin-Darby canine kidney) cell monolayers grown under standard conditions. Drug concentrations were quantitated using liquid scintillation counting, liquid chromatography/mass spectrometry/mass spectrometry, or liquid chromatography/mass spectrometry. In the presence of a pH gradient, benzoic acid exhibited net apical-to-basolateral transport, with apparent permeability ratios (apical-to-basolateral permeability/basolateral-to-apical permeability) ranging from 14 to 25. The addition of valproic acid reduced the permeability ratio by 70-90%. Cephalexin transport also exhibited net absorption in the presence of a pH gradient, with apparent permeability ratios ranging from 14 to 71, depending on growth conditions. Radiolabeled phenylalanine exhibited a low level of carrier-mediated absorption with an apparent permeability ratio of 1.8 that was reduced to 1.0 in the presence of unlabeled L-phenylalanine. Taurocholic acid did not exhibit carrier-mediated absorption. Cyclosporine and fexofenadine exhibited P-glycoprotein-mediated efflux from both MDCK and Caco-2 cells, which was more sensitive to inhibition in MDCK cells. These results suggest that although MDCK cell monolayers may be a useful model for evaluating transport by the absorptive monocarboxylic acid and peptide transporters and the efflux transporter, P-glycoprotein, they are not useful for predicting large neutral amino acid or bile acid transport in the intestine.
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PMID:Functional characterization of monocarboxylic acid, large neutral amino acid, bile acid and peptide transporters, and P-glycoprotein in MDCK and Caco-2 cells. 1243 7

Oligopeptides are generally thought to have poor permeability across biological membranes. Recent studies, however, suggest significant distribution of [Dmt1]DALDA (Dmt-D-Arg-Phe-Lys-NH2; Dmt is 2',6'-dimethyltyrosine), a 3+ net charge opioid peptide, to the brain and spinal cord after subcutaneous administration. Peptide transporters (PEPT1 and PEPT2) play a major role in the uptake of di- and tripeptides across cell membranes, but their ability to transport tetrapeptides is not clear. The purpose of this study was to determine whether [Dmt1]DALDA can translocate across Caco-2 cell monolayers and whether PEPT1 plays a role in the uptake process. Our results show that [3H][Dmt1]DALDA can readily translocate across Caco-2 cells, with a permeability coefficient estimated to be 1.24 x 10(-5) cm/s. When incubated with Caco-2 cells, [3H][Dmt1]DALDA was detected in cell lysates by 5 min. The internalization of [Dmt1]DALDA was confirmed visually with a fluorescent [Dmt1]DALDA analog (H-Dmt-D-Arg-Phe-dnsDap-NH2; dnsDap is beta-dansyl-L-alpha,beta-diaminopropionic acid). The uptake of [3H][Dmt1]DALDA was concentration-dependent but temperature- and pH-independent. Treatment with diethylpyrocarbonate (DEPC) inhibited [14C]glycine-sarcosine uptake but increased [3H][Dmt1]DALDA uptake 34-fold. These findings suggest that PEPT1 is not involved in [Dmt1]DALDA internalization. [Dmt1]DALDA uptake was also observed in SH-SY5Y, human embryonic kidney 293, and CRFK cells, and was independent of whether the cells expressed opioid receptors. The efflux of [3H][Dmt1]DALDA from Caco-2 cells was temperature-dependent and was inhibited by DEPC, but was not affected by verapamil, an inhibitor of P-glycoprotein. These data show transcellular translocation of a highly polar 3+ charge tetrapeptide and suggest that [Dmt1]DALDA may not only distribute across the blood-brain barrier but also it may even have reasonable oral absorption.
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PMID:Transcellular transport of a highly polar 3+ net charge opioid tetrapeptide. 1249 Jun 19

The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1(-/-) cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu(263) was comparable to that of TAP1(-/-) cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe(265) of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu(263) of the TAP1 protein, but not the Phe(265), is critical for proper TAP function.
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PMID:Impaired transporter associated with antigen processing (TAP) function attributable to a single amino acid alteration in the peptide TAP subunit TAP1. 1251 60

Well-characterised cell lines represent important tools for the study of endogenous solute or xenobiotic transport. A brain microvascular cell line, b.End3, isolated from mice transformed with the Polyoma virus middle T-antigen is available commercially. Here we report the characterisation of some features of b.End3 of relevance to its use in blood-brain barrier transport investigations. The b.End3 cells displayed a distinctive spindle-like squamous morphology in culture. Clathrin coated pits and numerous uncoated intracellular vesicles were evident within the cells, as was the expression of the vesicle-associated proteins, clathrin, caveolin-1, flotillin and dynamin II. In the presence of C6 astroglial co-culture b.End3 monolayers achieved a maximal transendothelial electrical resistance of 130 Omega cm2, but lacked real discrimination with respect to the permeation of transcellular and paracellular probes, e.g. permeability coefficients (x 10(-6) cm s(-1)) for propranolol of approximately 23 vs. 16 for sucrose. RT-PCR analysis confirmed the presence within the b.End3 cells of mRNA transcripts for the following transporters: GLUT-1; MCT 1 and 2; OAT1; Oatp1; mdr 1a and 1b; MRP 1 and 5; beta-alanine, system L and system y+L amino acid carriers; the nucleoside transporters cNT1 and 2, eNT1 and 2, and the tight junctional elements, ZO-1, JAM, occludin, claudin-1 and -5. The b.End3 cells actively accumulated D-glucose in a sodium-independent manner with characteristics consistant with that of GLUT-1. Functionality for P-glycoprotein efflux was evident as assessed by a rhodamine-123 accumulation and retention assay. The system L LAT1/4F2hc amino acid transporter was examined through uptake of L-leucine and L-phenylalanine and provided Km and Vmax values of approximately 16 microM and 350-480 pmol/mg protein/10 min, respectively; the affinity of transport for these substrates being weaker, approximately threefold, when the b.End3 cells were grown in the presence of C6 astroglial factors. Although the b.End3 cells appear unsuitable for transendothelial permeability assessments they display characteristics that would allow their worthwhile use in studies addressing blood-brain barrier transport mechanisms.
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PMID:Evaluation of the immortalised mouse brain capillary endothelial cell line, b.End3, as an in vitro blood-brain barrier model for drug uptake and transport studies. 1456 34

Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca(2+) ATPase (SERCA-1). Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1.2Ca(2+) transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase.
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PMID:Specific structural requirements for the inhibitory effect of thapsigargin on the Ca2+ ATPase SERCA. 1497 Feb 6

The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (DeltaF508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585-27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.
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PMID:Processing mutations located throughout the human multidrug resistance P-glycoprotein disrupt interactions between the nucleotide binding domains. 1524 15

The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.
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PMID:The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator. 1527 10

The effects on the drug efflux of $3,3',4,4',5$ -pentachlorobiphenyl (PCB-126), the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs), were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine ( ${\text{KB3 - Phe}};{61} $ ). In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In ${\text{KB3 - Phe}};{61} $, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 $\mu$ M PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in ${\text{KB3 - Phe}};{61} $. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.
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PMID:$3,3',4,4',5$ -Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein. 1529 79

P-glycoprotein (P-gp, ABCB1) actively transports a broad range of cytotoxic compounds out of the cell. The COOH terminus of P-gp contains a dileucine motif (Leu(1260)-Leu(1261)) and a conserved phenylalanine (Phe(1268)). Similar residues in SUR1 (ABCC8) were reported to be important plasma membrane-targeting signals (Sharma, N., Crane, A., Clement, J. P. t., Gonzalez, G., Babenko, A. P., Bryan, J., and Aguilar-Bryan, L. (1999) J. Biol. Chem. 274, 20628-20632). Here, we used alanine-scanning mutagenesis to test whether these residues were essential for trafficking of P-gp to the cell surface. Mutant L1260A expressed a 150-kDa immature protein that did not reach the cell surface and was sensitive to digestion by Endo H(f). By contrast, mutants L1261A, F1268A, and wild-type P-gps expressed the 170-kDa mature proteins at the cell surface. Mutation of Leu(1260) to Gly, Ile, Trp, Lys, or Glu also resulted in the expression of the 150-kDa immature protein. All of the mutants, however, expressed the 170-kDa protein in the presence of the drug substrate/specific chemical chaperone cyclosporin A. Mutant L1260A P-gp exhibited drug-stimulated ATPase activities similar to that of wild-type enzyme after rescue with cyclosporin A. Deletion of the last 22 amino acids (Q(1259)-Q(1280)) also caused misprocessing. The mutant, however, was rescued by expression in the presence of cyclosporin A and conferred resistance to colchicine in transfected cells. These results show that the dileucine motif is not a plasma membrane targeting signal. The COOH terminus is required for proper folding of P-gp but not for activity.
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PMID:The dileucine motif at the COOH terminus of human multidrug resistance P-glycoprotein is important for folding but not activity. 1554 93


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