Modulation of MRP1 protein transport by plant, and synthetically modified flavonoids

The influence of novel synthetic and plant origin flavonoids on activity of multidrug resistance-associated protein (MRP1) was investigated in human erythrocytes used as a cell model expressing MRP1 in plasma membrane. The fluorescent probe, BCPCF (2', 7'-bis-(3-carboxy-propyl)-5-(and-6)-carboxyfluorescein), was applied as a substrate for MRP1 multidrug resistance transporter. The effect of compounds belonging to different classes of natural flavonoids: flavone, flavonol, isoflavones and flavanolignan was compared with action of new synthetic derivatives of genistein. Most of the flavonoids showed strong or moderate ability to inhibit transport carried out by MRP1. Inhibitory properties of flavonoids were compared to the effects of indomethacin, probenecid and MK-571 known as MRP1 inhibitors. Studying the influence of new synthetic genistein derivatives on BCPCF transport we have found that the presence of hydrophobic groups substituting hydrogen of hydroxyl group at the position 4' in ring B of isoflavone is more important for inhibitory properties than hydrophobic substitution at the position 7 in ring A. In case of naturally occurring isoflavones the replacement of hydrogen at position 4' by hydrophobic ring structure seems also to be favourable for inhibition potency.     

Phosphorylation by Sky1p promotes Npl3p shuttling and mRNA dissociation

Mammalian SR proteins are currently thought to function in mRNA export as well as splicing. They contain multiple phosphorylated serine/arginine (RS/SR) dipeptides. Although SR domains can be phosphorylated by many kinases in vitro, the physiologically relevant kinase(s), and the role(s) of these modifications in vivo have remained unclear. Npl3 is a shuttling protein in budding yeast that we showed previously to be a substrate for the mammalian SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1. Here we demonstrate that Sky1p phosphorylates only one of Npl3p's eight SR/RS dipeptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results in the cytoplasmic accumulation of Npl3p. The redistribution of Npl3p is accompanied by its increased association with poly(A)+ RNA and decreased association with its import receptor, Mtr10p, in vivo. We propose that phosphorylation of Npl3p by the cytoplasmically localized Sky1p is required for efficient release of mRNA upon termination of export.     

Phenothiazine maleates stimulate MRP1 transport activity in human erythrocytes

The expression of multidrug resistance-associated protein (MRP1) results in ATP-dependent reduction of drugs' concentration in cancer cells, i.e., multidrug resistance (MDR). Since the majority of projects are concentrated on the search of the new MDR modulators, there are very few reports on drug-induced stimulation of MDR transporters activity. In the present work, by means of functional fluorescence assay we have shown that MRP1-mediated efflux of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by phenothiazine maleates that have been already identified as P-glycoprotein inhibitors. Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2',7'-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes has been also demonstrated. Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. To our best knowledge, this report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. We conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site.     

Human MDR1 and MRP1 recognize berberine as their transport substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim-/- fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.     

Microtubule acetylation promotes kinesin-1 binding and transport

Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.     

Ion and solute transport by Prestin in Drosophila and Anopheles

The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO(3)(-) transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (Prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl(-)/nHCO(3)(-), Cl(-)/SO(4)(2-) and Cl(-)/oxalate(2-)) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.     

Functional reconstitution of substrate transport by purified multidrug resistance protein MRP1 (ABCC1) in phospholipid vesicles

The 190-kDa multidrug resistance protein MRP1 (ABCC1) is a polytopic transmembrane protein belonging to the ATP-binding cassette transporter superfamily. In addition to conferring resistance to various antineoplastic agents, MRP1 is a transporter of conjugated organic anions, including the cysteinyl leukotriene C(4) (LTC(4)). We previously characterized the ATPase activity of reconstituted immunoaffinity-purified native MRP1 and showed it could be stimulated by its organic anion substrates (Mao, Q., Leslie, E. M., Deeley, R. G., and Cole, S. P. C. (1999) Biochim. Biophys. Acta 1461, 69-82). Here we show that purified reconstituted MRP1 is also capable of active transport of its substrates. Thus LTC(4) uptake by MRP1 proteoliposomes was osmotically sensitive and could be inhibited by two MRP1-specific monoclonal antibodies. LTC(4) uptake was also markedly reduced by the competitive inhibitor, S-decyl-glutathione, as well as by the MRP1 substrates 17 beta-estradiol 17-beta-(d-glucuronide), oxidized glutathione, and vincristine in the presence of reduced glutathione. The K(m) for ATP and LTC(4) were 357 +/- 184 microm and 366 +/- 38 nm, respectively, and 2.14 +/- 0.75 microm for 17 beta-estradiol 17-beta-(d-glucuronide). Transport of vincristine required the presence of both ATP and GSH. Conversely, GSH transport was stimulated by vincristine and verapamil. Our data represent the first reconstitution of transport competent purified native MRP1 and confirm that MRP1 is an efflux pump, which can transport conjugated organic anions and co-transport vincristine together with GSH.     

The copper transport protein Atox1 promotes neuronal survival

Atox1, a copper transport protein, was recently identified as a copper-dependent suppressor of oxidative damage in yeast lacking superoxide dismutase. We have previously reported that Atox1 in the rat brain is primarily expressed in neurons, with the highest levels in distinct neuronal subtypes that are characterized by their high levels of metal, like copper, iron, and zinc. In this report, we have transfected the Atox1 gene into several neuronal cell lines to increase the endogenous level of Atox1 expression and have demonstrated that, under conditions of serum starvation and oxidative injury, the transfected neurons are significantly protected against this stress. This level of protection is comparable with the level of protection seen with copper/zinc superoxide dismutase and the anti-apoptotic gene bcl-2 that had been similarly transfected. Furthermore, neuronal cell lines transfected with a mutant Atox1 gene, where the copper binding domain has been modified to prevent metal binding, do not afford protection against serum starvation resulting in apoptosis. Therefore, Atox1 is a component of the cellular pathways used for protection against oxidative stress.     

Inhibition of Na transport by 2-chloroadenosine: dissociation from production of cyclic nucleotides

The adenosine analogue 2-chloroadenosine (2-CA) is often used to determine the biologic effects of adenosine because 2-CA is less susceptible to degradation than adenosine. We studied the effects of 2-CA on primary cultures of rat inner medullary collecting ducts because there is good evidence that adenosine can influence cell function through its effects on second messengers. 2-CA inhibited Na+ transport across the apical membrane and increased cAMP content of the cells. The major adenosine receptors in these cells appear to be the stimulatory (A2) type. Stimulation of cAMP by 2-CA was more potent when applied to the apical membrane than to the basolateral membrane, an effect opposite to that of vasopressin. These results imply that adenosine receptors are more numerous or more effective on the apical membrane than on the basolateral membrane. Inhibition of Na+ transport was probably not mediated by an adenosine receptor as evidenced by (i) a lack of effect of adenosine and other adenosine analogues on Na+ transport; (ii) a lack of effect of nonmetabolizable cyclic nucleotides on Na+ transport; and (iii) a clear discrepancy in the temporal course of 2-CA effects on a second messenger system (cAMP) and 2-CA inhibition of Na+ transport. Dipyridimole, an inhibitor of adenosine transport, also reduced Na+ transport. Taken together, the data suggest that 2-CA inhibits Na+ transport by interfering with adenosine transport or metabolism.     

Interaction between the bound Mg.ATP and the Walker A serine residue in NBD2 of multidrug resistance-associated protein MRP1 plays a crucial role for the ATP-dependent leukotriene C4 transport

Structural analysis of human MRP1-NBD1 revealed that the Walker A S685 forms a hydrogen bond with the Walker B D792 and interacts with the Mg (2+) cofactor and the beta-phosphate of the bound Mg.ATP. We have found that substitution of the S685 with an amino acid that potentially prevents the formation of the hydrogen bond resulted in misfolding of the protein and significantly affect the ATP-dependent leukotriene C4 (LTC4) transport. In this report we tested whether the corresponding substitution in NBD2 would also result in misfolding of the protein. In contrast to the NBD1 mutations, none of the mutations in NBD2, including S1334A, S1334C, S1334D, S1334H, S1334N, and S1334T, caused misfolding of the protein. However, elimination of the hydroxyl group at S1334 in mutations including S1334A, S1334C, S1334D, S1334H, and S1334N drastically reduced the ATP binding and the ATP-enhanced ADP trapping at the mutated NBD2. Due to this low efficient ATP binding at the mutated NBD2, the inhibitory effect of ATP on the LTC4 binding is significantly decreased. Furthermore, ATP bound to the mutated NBD2 cannot be efficiently hydrolyzed, leading to almost completely abolishing the ATP-dependent LTC4 transport. In contrast, S1334T mutation, which retained the hydroxyl group at this position, exerts higher LTC4 transport activity than the wild-type MRP1, indicating that the hydroxyl group at this position plays a crucial role for ATP binding/hydrolysis and ATP-dependent solute transport.     

RF3:GTP promotes rapid dissociation of the class 1 termination factor

Translation termination is promoted by class 1 and class 2 release factors in all domains of life. While the role of the bacterial class 1 factors, RF1 and RF2, in translation termination is well understood, the precise contribution of the bacterial class 2 release factor, RF3, to this process remains less clear. Here, we use a combination of binding assays and pre-steady state kinetics to provide a kinetic and thermodynamic framework for understanding the role of the translational GTPase RF3 in bacterial translation termination. First, we find that GDP and GTP have similar affinities for RF3 and that, on average, the t_1/2 for nucleotide dissociation from the protein is 1–2 min. We further show that RF3:GDPNP, but not RF3:GDP, tightly associates with the ribosome pre- and post-termination complexes. Finally, we use stopped-flow fluorescence to demonstrate that RF3:GTP enhances RF1 dissociation rates by over 500-fold, providing the first direct observation of this step. Importantly, catalytically inactive variants of RF1 are not rapidly dissociated from the ribosome by RF3:GTP, arguing that a rotated state of the ribosome must be sampled for this step to efficiently occur. Together, these data define a more precise role for RF3 in translation termination and provide insights into the function of this family of translational GTPases.     

Nucleotide triphosphatase activity of the N-terminal nucleotide-binding domains of the multidrug resistance proteins P-glycoprotein and MRP1

The multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 are drug-efflux pumps. In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRP1, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRP1 have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K(m) of MRP1 NBD1 for ATP is lower than that for GTP, and (3) phosphorylation of MLD by PKA or PKC produces a marginal increase of V(max) for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRP1, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity.     

Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a tri-glutathione conjugate is required

Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C(4)) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (As(III)) only in the presence of GSH but does not transport arsenate (As(V)) (with or without GSH). The non-reducing GSH analogs L-gamma-glutamyl-L-alpha-aminobutyryl glycine and S-methyl GSH did not support As(III) transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of As(III) was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)(3)). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport As(III) even in the presence of GSH but did transport synthetic As(GS)(3). The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent As(III) transport. The apparent K(m) of As(GS)(3) for MRP1 was 0.32 microM, suggesting a remarkably high relative affinity. As(GS)(3) transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (K(i) 2.8 microM). As(GS)(3) transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)(3) and leukotriene C(4). Finally, human MRP2 also transported As(GS)(3). In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process.