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:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a 'flippase' for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD-GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD-GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD-GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD-lactosylceramide drastically reduced the rate of flipping compared with NBD-PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.
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PMID:The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids. 1579 13

Valspodar (Amdray, SDZ PSC 833) is derived from cyclosporin, but lacks the immunosuppressive and most of the collateral activities of cyclosporin A (CsA, Sandimmune, Neoral); it exhibits an enhanced capacity to chemosensitise tumour cells showing the classical type multiple drug-resistance (MDR) associated with MDR1 P-glycoprotein (Pgp) overexpression. This valspodar-mediated chemosensitisation of MDR tumour cells is reviewed with regard to its mechanism of inhibition on Pgp flippase function, and its potential inhibition of anticancer drug (ACD) metabolisation by CYP3A enzymes is discussed. Potent inhibition of the membranous and cytoplasmic detoxification mechanisms expressed by cells at the absorption and clearance borders in the body by valspodar results in the many pharmacokinetic interactions with other drugs that are substrates of either, or both, Pgp and CYP classes of detoxifying enzyme. In view of the present ability to restrict oral bioavailability of valspodar within a narrow range, and to adapt adequately the chemotherapeutic dosages to achieve their equivalent exposure in the presence or absence of valspodar, current clinical data on its efficacy and safety permit optimism for ongoing Phase III trials. The potential of valspodar to increase exposure or to modulate the biodistribution of other chemotherapeutics, such as HIV protease inhibitors to the brain, is further evoked, as this might become another application of the new drug. This evaluation of valspodar compared to CsA attempts to interpret its mechanisms of action, rather than to serve as a complete and comparative repertoire of all published preclinical and clinical data.
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PMID:Valspodar: current status and perspectives. 1599 33

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.
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PMID:P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains. 1617 66

Active extrusion of drugs from the cell interior by primary and secondary efflux pumps is an essential mechanism underlying the phenomenon of multidrug resistance. The first discovered and best characterized primary efflux pump found in humans is the ABC transporter P-glycoprotein (PGP), which shows very broad substrate specificity. Many of these molecules are lipophilic, and binding most likely takes place within the membrane. PGP could either translocate them from the inner to the outer leaflet (flippase) or extrude them from the membrane into the extracellular environment (hydrophobic vacuum cleaner). Recognition and binding of such a diverse set of substrates must be associated with a preferred membrane location, determined by molecular properties and lipid interactions. Therefore, a systematic study of the interaction among seven PGP substrates (phenazine, doxorubicin, cephalexin, ampicillin, chloramphenicol, penicillin G, and quercetin) and two modulators (quinidine and nicardipine) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model membranes is reported here. The location profile of these molecules across the membrane was determined by (1)H NOESY MAS NMR based on (1)H-(1)H cross-peaks between their aromatic fingerprint region and lipid resonances. Although structurally rather diverse, all tested substances are found to have their highest concentration between the phosphate of the lipid headgroup and the upper segments of the lipid hydrocarbon chains. Our findings are consistent with PGP substrate and modulator binding from the membrane interface region.
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PMID:Localization of multidrug transporter substrates within model membranes. 1668 93

The MDR1 P-glycoprotein, an ATP-binding cassette (ABC) superfamily member that functions as an ATP-driven drug efflux pump, has been linked to resistance of human tumors to multiple chemotherapeutic agents. P-glycoprotein binds and actively transports a large variety of hydrophobic drugs and peptides. P-glycoprotein in reconstituted proteoliposomes is also an outwardly directed flippase for membrane phospholipids and simple glycosphinglipids. This review focuses on recent advances in our understanding of P-glycoprotein structure and function, particularly through the use of fluorescence spectroscopic approaches. Progress is being made towards understanding the structure of the transporter, especially the spatial relationship between the two nucleotide-binding domains. Exploration of the P-glycoprotein catalytic cycle using vanadate-trapped complexes has revealed that drug transport likely takes place by concerted conformational changes linked to relaxation of a high energy intermediate. Low resolution mapping of the protein using fluorescence resonance energy transfer showed that both the H and R drug-binding sites are located within the cytoplasmic leaflet. Two drugs can bind to the R-site simultaneously, suggesting that the protein contains a large flexible binding region.
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PMID:New insights into the drug binding, transport and lipid flippase activities of the p-glycoprotein multidrug transporter. 1669 87

P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell, driven by ATP hydrolysis. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers, leading to multidrug resistance (MDR). The protein also plays an important physiological role in limiting drug uptake in the gut and entry into the brain. Substrates partition into the lipid bilayer before interacting with Pgp, which has been proposed to function as a hydrophobic vacuum cleaner. Low- and medium-resolution structural models of Pgp suggest that the 2 nucleotide-binding domains are closely associated to form a nucleotide sandwich dimer. Pgp is an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The ATPase catalytic cycle of the protein is thought to proceed via an alternating site mechanism, although the details are not understood. The lipid bilayer plays an important role in Pgp function, and may regulate both the binding and transport of drugs. This review focuses on the structure and function of Pgp, and highlights the importance of fluorescence spectroscopic techniques in exploring the molecular details of this enigmatic transporter.
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PMID:Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1). 1721 84

The P-glycoprotein multidrug transporter (Pgp; ABCB1) is an ATP-binding cassette (ABC) protein that has been implicated in the multidrug resistance of human cancers. Pgp couples ATP hydrolysis to active extrusion from the cell of a broad array of amphipathic compounds via an ill-defined mechanism. Substrates are believed to interact with Pgp within the membrane. Reconstituted Pgp functions as an ATP-dependent flippase for a variety of fluorescently labelled membrane lipids. The protein may also function as a drug 'flippase', moving its substrates from the inner to the outer leaflet of the bilayer. We show that lipid-based anti-cancer drugs, such as miltefosine, and signaling molecules, such as platelet-activating factors, bind saturably to Pgp with Kd values in the low micromolar range, and modulate its ATPase activity. These compounds also inhibit Pgp-mediated flipping of fluorescent lipids and transport of Hoechst 33342 and tetramethylrosamine, which occupy different subsites in the drug-binding pocket. Bacterial lipid A modulates Pgp ATPase activity, and glycolipid flipping is inhibited by unlabelled glucosylceramide, suggesting that these lipids also interact with the transporter. These results indicate that Pgp treats a variety of lipid-based molecules as substrates, and likely interacts with lipids and drugs in the same manner.
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PMID:P-glycoprotein (ABCB1) interacts directly with lipid-based anti-cancer drugs and platelet-activating factors. 1721 88

Resistance to a broad spectrum of structurally diverse chemotherapeutic drugs (multidrug resistance; MDR) is a major impediment to the treatment of cancer. One cause of MDR is the expression at the tumor cell surface of P-glycoprotein (Pgp), which functions as an ATP-powered multidrug efflux pump. Since Pgp interacts with its substrates after they partition into the lipid bilayer, changes in membrane physicochemical properties may have substantial effects on its functional activity. Various interactions between cholesterol and Pgp have been suggested, including a role for the protein in transbilayer movement of cholesterol. We have characterized several aspects of Pgp-cholesterol interactions, and found that some of the previously reported effects of cholesterol result from inhibition of Pgp ATPase activity by the cholesterol-extracting reagent, methyl-beta-cyclodextrin. The presence of cholesterol in the bilayer modulated the basal and drug-stimulated ATPase activity of reconstituted Pgp in a modest fashion. Both the ability of drugs to bind to the protein and the drug transport and phospholipid flippase functions of Pgp were also affected by cholesterol. The effects of cholesterol on drug binding affinity were unrelated to the size of the compound. Increasing cholesterol content greatly altered the partitioning of hydrophobic drug substrates into the membrane, which may account for some of the observed effects of cholesterol on Pgp-mediated drug transport. Pgp does not appear to mediate the flip-flop of a fluorescent cholesterol analogue across the bilayer. Cholesterol likely modulates Pgp function via effects on drug-membrane partitioning and changes in the local lipid environment of the protein.
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PMID:Interaction of the P-glycoprotein multidrug efflux pump with cholesterol: effects on ATPase activity, drug binding and transport. 1904 91

Pgp (P-glycoprotein) (ABCB1) is an ATP-powered efflux pump which can transport hundreds of structurally unrelated hydrophobic amphipathic compounds, including therapeutic drugs, peptides and lipid-like compounds. This 170 kDa polypeptide plays a crucial physiological role in protecting tissues from toxic xenobiotics and endogenous metabolites, and also affects the uptake and distribution of many clinically important drugs. It forms a major component of the blood-brain barrier and restricts the uptake of drugs from the intestine. The protein is also expressed in many human cancers, where it probably contributes to resistance to chemotherapy treatment. Many chemical modulators have been identified that block the action of Pgp, and may have clinical applications in improving drug delivery and treating cancer. Pgp substrates are generally lipid-soluble, and partition into the membrane before the transporter expels them into the aqueous phase, much like a 'hydrophobic vacuum cleaner'. The transporter may also act as a 'flippase', moving its substrates from the inner to the outer membrane leaflet. An X-ray crystal structure shows that drugs interact with Pgp within the transmembrane regions by fitting into a large flexible binding pocket, which can accommodate several substrate molecules simultaneously. The nucleotide-binding domains of Pgp appear to hydrolyse ATP in an alternating manner; however, it is still not clear whether transport is driven by ATP hydrolysis or ATP binding. Details of the steps involved in the drug-transport process, and how it is coupled to ATP hydrolysis, remain the object of intensive study.
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PMID:The P-glycoprotein multidrug transporter. 2196 57

Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1), which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter's transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behavior of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding, and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp-lipid and substrate-lipid interactions, and how these affect drug-binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains, which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however, it clearly affects several aspects of its function in addition to substrate-membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp's catalytic cycle may lead to new strategies to combat clinical drug resistance.
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PMID:Complex Interplay between the P-Glycoprotein Multidrug Efflux Pump and the Membrane: Its Role in Modulating Protein Function. 2462 64


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