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)

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F1-ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.
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PMID:Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues. 1532 76

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mm), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[alpha-32P]ATP binds to MRP4 (concentration for half-maximal binding approximately 3 microm) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 microm). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4.8-azido[alpha-32P]ADP.Vi and MRP4.8-azido[alpha-32P]ADP.beryllium fluoride can be generated, nucleotide trapping is approximately 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co2+ increases 8-azido[alpha-32P]ATP binding and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mm, respectively) and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping, whereas Pi has no effect up to 20 mm. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.
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PMID:Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state. 1536 14

Expression of multiple drug resistant (MDR) phenotype and over-expression of P-glycoprotein (P-gp) in the human hepatocellular carcinoma (HCC) cell clone P1(0.5), derived from the PLC/PRF/5 cell line (P5), are associated with strong resistance to oxidative stress and a significant (p < 0.01) increase in intracellular vitamin E content as compared with the parental cell line. This study evaluates the role of vitamin E in conferring resistance to drugs and oxidative stress in P1(0.5) cells. Parental drug-sensitive cells, P5, were incubated in alpha-tocopherol succinate (alpha-TS, 5 microM for 24 h) enriched medium to increase intracellular vitamin E content to levels comparable to those observed in P1(0.5) cells at basal conditions. Susceptibility to lipid peroxidation and oxidative DNA damage were assessed by measuring the concentration of thiobarbituric-reactive substances (TBARS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) at basal and after experimental conditions. Cell capacity to form colonies and resistance to doxorubicin were also studied. P5 cells, treated with alpha-TS, became resistant to ADP-Fe3+ and to ionizing radiation-induced lipid peroxidation as P1(0.5) cells. Exposure to ADP-Fe3+ or ionizing radiation increased TBARS and the 8-OHdG content in the P5 cells, while vitamin E enrichment abolished these effects. Irradiation doses at 5 cGy increased TBARS and 8-OHdG. They also inhibited cell capacity to form colonies in the untreated P5 cells. Incubation with alpha-TS fully reverted this effect and significantly (p < 0.01) reduced the inhibitory effect of cell proliferation induced by irradiation doses at >500 cGy. Resistance to doxorubicin was not affected by alpha-TS. These observations demonstrate the role of vitamin E in conferring protection from lipid peroxidation, ionizing radiation and oxidative DNA damage on the human HCC cell line. They also rule out any role of P-gp over-expression as being responsible for these observations in cells with MDR phenotype expression.
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PMID:Vitamin E protects DNA from oxidative damage in human hepatocellular carcinoma cell lines. 1545 40

P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.
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PMID:ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein. 1604 2

Flavonoids and their in vivo metabolites are neuroprotective, cardioprotective and chemopreventive agents acting as hydrogen-donating antioxidants or modulators functioning at protein kinase and lipid signaling pathways. In presented study treatments of human leukemia cells HL60 and their MDR-1 resistant subline HL60/VCR by flavonoids apigenin (API), luteolin (LUT), quercetin (QU) and anticancer drug doxorubicin (DOX) are reported. Of all flavonoids used only QU treatments led in both cell lines to DNA fragmentation, cleavage of poly (ADP- ribose) polymerase (PARP), up-regulation of proapoptotic Bax and posttranslational modification (phosphorylation) of antiapoptotic Bcl-2. Cytochrome c and p21WAF1/CIP1 levels remained unchanged in these cells. Furthermore, treatments of both cell lines by QU and in its combined application with DOX increased phosphorylation of ERK, while Akt-1 and phosphorylated Akt-1 levels were not changed. All these events resulted in effective induction of apoptosis associated with down-regulation of P-glycoprotein in resistant cells. Presented results suggest that in human leukemia cells QU is a potent regulator of the cell apoptotic program associated with the modulation of several signaling molecules.
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PMID:Flavonoid quercetin, but not apigenin or luteolin, induced apoptosis in human myeloid leukemia cells and their resistant variants. 1605 41

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.
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PMID:Modulator-induced interference in functional cross talk between the substrate and the ATP sites of human P-glycoprotein. 1648 67

ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis.
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PMID:Allosteric modulation bypasses the requirement for ATP hydrolysis in regenerating low affinity transition state conformation of human P-glycoprotein. 1650 85

The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.
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PMID:Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates. 1662 45

The multidrug resistance efflux pump P-glycoprotein (Pgp) couples drug export to ATP binding and hydrolysis. Details regarding drug trajectory, as well as the molecular basis for coupling, remain unknown. Nearly all drugs exported by Pgp have been assayed for competitive behavior with rhodamine123 transport at a canonical "R" drug binding site. Tetramethylrosamine (TMR) displays a relatively high affinity for Pgp when compared to other rhodamines. Here, we present the construction and characterization of a library of compounds based upon the TMR scaffold and use this set to assess the determinants of drug binding to the "R" site of Pgp. This set contained modifications in (1) the number, location, and conformational mobility of hydrogen-bond acceptors; (2) the heteroatom in the xanthylium core; and (3) the size of the substituent in the 9-position of the xanthylium core. Relative specificity for coupling to the distal ATP catalytic site was assessed by ATPase stimulation. We found marked ( approximately 1000-fold) variation in the ATPase specificity constant within the library of TMR analogues. Using established methods involving ADP-Vi trapping by wild-type Pgp and ATP binding by catalytic carboxylate mutant Pgp, these effects can be extended to ATP hydrolysis transition-state stabilization and ATP occlusion at a single site. These data support the idea that drugs trigger the engagement of ATP catalytic site residues necessary for hydrolysis. Further, the nature of the drug binding site and coupling mechanism may be dissected by variation of a drug-like scaffold. These studies may facilitate development of novel competitive inhibitors at the "R" drug site.
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PMID:Stimulation of P-glycoprotein ATPase by analogues of tetramethylrosamine: coupling of drug binding at the "R" site to the ATP hydrolysis transition state. 1680 Jun 28

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.
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PMID:Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter. 1680 57


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