Towards the molecular mechanism of prokaryotic and eukaryotic multidrug transporters

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 12, 2001: pp. 239–245 doi:10.1006/scdb.2000.0249, available online at http://www.idealibrary.com on

Towards the molecular mechanism of prokaryotic and eukaryotic multidrug transporters Hendrik W. van Veen

utilize the release of phosphate bond-energy by ATP hydrolysis to catalyse the extrusion of drugs. 5,6 Secondary multidrug transporters are proton or sodium motive force-driven antiporters that mediate the extrusion of drugs from the cell in a coupled exchange with protons or sodium ions, respectively. In general, these transport proteins belong to the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, the resistance nodulation cell division (RND) family, or the multidrug and toxic compounds extrusion (MATE) family. 7–9 Secondary multidrug transporters are often responsible for drug resistance in bacteria, whereas ABC multidrug transporters play an important role in drug resistance in eukaryotic cells, including human cells. Three aspects of multidrug transporters deserve our attention. First, in spite of the significant variations that exist between the primary protein structures of multidrug transporters in prokaryotic and eukaryotic cells, many of these systems show specificity for similar dyes, toxic ions and clinically relevant antibiotics and anticancer drugs. For example, the lactic acid bacterium Lactococcus lactis possesses the ABC multidrug transporter LmrA 10 and the MFS multidrug transporter LmrP 11 which have overlapping specificities for amphiphilic drugs (e.g. ethidium bromide, Hoechst 33342, anthracyclines, tetracyclines, macrolides) and modulators (e.g. calcium channel blockers, 1,4-dihydropyridines, and antimalarials). 12 Interestingly, many of these drugs and modulators are also recognized by the human multidrug resistance P-glycoprotein, 13 overexpression of which is one of the main causes of multidrug resistance in human cancer cells. 14 The molecular basis of these similarities is not well known at the present. Second, there is an urgent need to understand the structure–function relationships in multidrug transporters that underlie the transport mechanism of these proteins. Knowledge about the architecture of drug- and modulator-binding sites and the link between energy-generating and drug-

Due to their ability to extrude structurally dissimilar cytotoxic drugs out of the cell, multidrug transporters are able to reduce the cytoplasmic drug concentration, and, hence, are able to confer drug resistance on human cancer cells and pathogenic microorganisms. This review will focus on the molecular properties of two bacterial multidrug transporters, the ATPbinding cassette transporter LmrA and the proton motive force-dependent major facilitator superfamily transporter LmrP, which each represent a major class of multidrug transport proteins encountered in pro- and eukaryotic cells. In spite of the structural differences between LmrA and LmrP, the molecular bases of their drug transport activity may turn out to be more similar than might currently appear. Key words: LmrA / LmrP / membrane protein / multidrug transporter / transport mechanism c 2001 Academic Press

Introduction The extrusion of cytotoxic drugs from multidrug resistant cells by overexpressed multidrug transporters is an important cause of failure of the drug-based treatment of patients with cancers 1 or infections by pathogenic microorganisms. 2 Even in non-overexpressing cells, drug efflux by multidrug transporters may interfere with the effective dosing of potentially efficacious drugs. Two major classes of multidrug transport proteins are found in proand eukaryotic organisms. 3,4 ATP-binding cassette transporters are primary transport proteins that

From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK. E-mail: [email protected] c

2001 Academic Press 1084–9521 / 01 / 030239+ 07 / $35.00 / 0

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translocating functions of multidrug transporters may allow us to rationally design new drugs that can poison or circumvent the activity of these transport proteins. Third, if we are to inhibit multidrug transporters in human cells, we should know more about their physiological functions and the factors that affect their expression. This paper will certainly not provide the final answer to these intriguing questions, but will highlight key recent advances in our understanding of the molecular mechanisms of LmrA and LmrP that may lift a corner of the veil for some of these questions. Basic research discoveries on the fundamental mechanisms responsible for a disease state often lead to the most direct pharmaceutical approaches to manage the disease. 15 Therefore, I do hope that the topics emphasized in this article will offer perspectives for future research.

protein from lactococcal membranes using dodecyl maltoside, purified by Ni-affinity chromatography, and reconstituted in dodecyl maltoside-destabilized preformed liposomes prepared from Escherichia coli or L. lactis lipids and egg phosphatidyl choline. 20 Interestingly, LmrA and LmrP both mediate drug and antibiotic resistance by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, 18,21,22 as has been suggested for P-glycoprotein. 23 Extrusion from the membrane could, in principle, explain the broad drug specificity of these multidrug transporters. The ability of drugs to intercalate into the phospholipid bilayer would pre-select drugs to be transported from other cellular compounds which are not to be transported. The interaction between the pre-selected drug and the transporter is another important determinant of specificity. 24 LmrA mediates drug transport at the expense of ATP hydrolysis. Similar to P-glycoprotein, the in vitro basal rate of ATP hydrolysis of LmrA is stimulated three to four-fold by transport substrates, and the two proteins have a similar apparent affinity for MgATP. 16,25 For P-glycoprotein, both ABC domains have been shown to bind and hydrolyse ATP, and several observations provide strong support to an alternating catalytic sites model in which the ABC domains of Pglycoprotein act alternately to hydrolyse ATP. 26 However, the mechanism by which P-glycoprotein couples the hydrolysis of ATP to the movement of drugs across the membrane is not known at the molecular level. Indeed, the number and nature of the drug-binding site(s) in P-glycoprotein are ill-defined.

General properties of LmrA and LmrP L. lactis contains a number of interesting multidrug transporters. One of these systems, LmrA, is a 590-amino acid ABC half-transporter containing an amino-terminal membrane domain, consisting of six transmembrane segments, followed by a hydrophilic nucleotide-binding domain. LmrA was the first ABC multidrug transporter identified in bacteria. 10 The protein is homologous to each of the two halves of the human multidrug resistance P-glycoprotein. Surprisingly, LmrA could functionally substitute for P-glycoprotein in human lung fibroblast cells. 16 The LmrA transporter is a homodimeric protein with a similar domain organization as monomeric P-glycoprotein, as suggested by the negative dominance of a transport inactive LmrA mutant over the transport-active wildtype LmrA protein in a co-reconstituted liposomal system, and by the observation that the covalent fusion of two wildtype LmrA monomers, but not of a mutant and wildtype monomer, yields a functional transporter. 17 In addition to LmrA, L. lactis possesses a secondary multidrug transporter LmrP. This protein is composed of 408 amino acid residues, contains 12 putative membrane-spanning segments, and is a member of the MFS. 11 LmrP-mediated drug transport is driven by the transmembrane potential and transmembrane chemical proton gradient, and, hence, is based on an electrogenic nH+ /drugs antiport reaction (n ≥ 2). 18 Like LmrA, 19 LmrP has been solubilized as a histidine-tagged

Two-cylinder engine mechanism To mediate the ATP-dependent extrusion of drugs, LmrA has to couple conformational changes in the nucleotide-binding domains, induced by ATP binding and/or hydrolysis, to conformational changes in drug-binding domains that allow the binding and release of drug molecules. Conformational changes in LmrA have been demonstrated by infrared spectroscopy and trytophan fluorescence quenching analysis. 27 Based on vinblastine equilibrium binding experiments, photoaffinity labelling experiments and drug transport assays, the results of which will not be recited in the present discussion, it has been proposed that homodimeric LmrA mediates drug transport by a two-cylinder engine mechanism. 17 240

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In this transport model (Figure 1), the hydrolysis of ATP by the nucleotide-binding domain in one half of the transporter is coupled to drug efflux through the movement of an inside-facing, highaffinity drug-binding site (which binds the drug molecule at the inner leaflet of the membrane) to the outside surface of the membrane with a concomitant change to low affinity (resulting in the release of the drug molecule into the extracellular medium). In the ADP/vanadate trapped transition state conformation of LmrA, the high-affinity drugbinding site is occluded. In addition, ATP binding and/or hydrolysis by the nucleotide-binding domain in one half of the transporter must facilitate the return of an unliganded, outside-facing low-affinity site at the membrane domain in the other halfmolecule to an inside-facing high-affinity site. Two site transport mechanisms proposed for LmrA may also apply to other ABC transporters. First, a number of observations suggest that P-glycoprotein does possess at least two separate drug-binding sites, which each may be composed of multiple drug interaction sites to account for the wide range of compounds that are transported (for review see Reference 24). Second, a two-site transport mechanism may be relevant for mammalian MRP proteins. These drug pumps are able to efflux glutathione, glucoronide and sulfate conjugates, as well as non-conjugated drugs in cotransport with reduced glutathione. 28 The stimulation of the transport of non-conjugated drugs by glutathione and vice versa 29–31 may indicate the presence of at least two positively cooperative transport-competent substratebinding sites in the protein, one for glutathione and the other for unconjugated drugs. 31 Third, the two-cylinder engine mechanism, as proposed for LmrA may also be relevant for prokaryotic-binding protein-dependent ABC transporters involved in the uptake of solutes (e.g. amino acids and sugars). In general, these transporters contain four core domains in the translocator, analogous to the domains of P-glycoprotein, and an additional extracellular solute-binding protein. 32 Surprisingly, the osmoregulated ABC transporter OpuA in L. lactis possesses two extracellular solute-binding proteins of which one is covalently linked to the N-terminal membrane domain and the other to the C-terminal domain. 33 The stoichiometry of two binding proteins per translocator complex raises questions about the observations for the bacterial periplasmic histidine, ribose and maltose permeases, each of which makes use of a soluble periplasmic solute-binding

Figure 1. Alternating two-site (two-cylinder engine) transport model. Rectangles represent the transmembrane domains of LmrA. Circles, squares and hexagons represent different conformations of the nucleotide-binding domains. The ATP-bound (circle) state is associated with a high-affinity drug-binding site on the inside of the transporter. The ADP-bound (square) state is associated with a low-affinity drug-binding site on the outside of the transporter. The ADP-Pi (hexagonal) state is associated with an occluded drug binding site, and represents the ADP/vanadate-trapped form of the ABC domain. In and Out refer to the inside and outside of the phospholipid bilayer, respectively. In the presence of ATP, the transport cycle turns counter clockwise (as indicated by Arrows 1 and 2). Arrow 1, ATP binding/hydrolysis at the second ABC domain in the LmrA dimer is coupled to: (i) drug efflux at the second membrane domain through the movement of a liganded inside-facing high affinity site to the outside of the membrane with the concomitant change to low-affinity, via a catalytic transition intermediate in which the transport site at the second membrane domain is inaccessible; (ii) the reorientation of an empty outside-facing low-affinity site at the first membrane domain to an inside-facing high-affinity site; and (iii) ATP binding at the first ABC domain. Arrow 2, the first and second LmrA molecule in the dimer have reversed their relationship, and the next ATP hydrolysis step will occur at the first ABC domain. Thus, in a complete drug transport cycle, the LmrA dimer exposes four drugbinding sites in two pairs of two sites via an alternating twosite mechanism. 17

protein, that only a single binding protein interacts with the dimeric membrane complex and that each of the two lobes of a single binding protein interacts with a separate membrane domain in these transporters. 34–36 However, if OpuA would operate 241

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by an alternating two-site mechanism, each of the two binding proteins would interact with the translocator complex in one half of the catalytic cycle, giving two interactions per cycle, and this would be consistent with the available data for the periplasmic permeases.

characterizes most secondary transporters, and that the minimal unit of biochemical function has a hydrophobic core consisting of at least eight transmembrane segments. In this context, the two drug-interaction sites in LmrP may be located within a single drug-binding region or may represent separate binding sites in the transporter. Neither of these views is unprecedented. For example, it has been suggested that bacterial MFS transporters involved in Pi -linked antiport (e.g. the E. coli UhpT system) contain a single bifunctional substrate-binding site that either accepts two monovalent Pi anions or a single divalent glucose-6-phosphate anion. 43 On the other hand, studies by Carruthers and coworkers demonstrate that the human erythrocyte MFS transporter GluT1 presents distinct sugar influx and release sites simultaneously. 44 In addition, some biochemical evidence suggests the presence of two kinetically distinguishable lactose-binding sites in the MFS transporter LacY in E. coli with high and low affinity for the substrate. 45 It is also noteworthy that secondary transport proteins, such as the 12 transmembrane segment-containing human GluT1 transporter and lactose transporter LacS in Streptococcus thermophilus, and the four transmembrane segment-containing multidrug transporter EmrE in E. coli appear to self-associate into a homodimeric complex. 44,46–48 Two monomers in these dimeric complexes may each provide the transporter with a separate binding site of which one is involved in the influx and the other in the release of substrate. Possibly, these two sites are interconverted during the operation of the transport cycle. Obviously, the link between the oligomeric state of secondary transporters and the number of substrate binding sites exposed will be an intriguing new area of research.

Two drug-binding sites in secondary multidrug transporters? There is increasing evidence for the presence of more than one drug-interaction site in secondary multidrug transporters. For LmrP, transport studies in membrane vesicles of L. lactis suggest that some drugs inhibit the LmrP-mediated transport of Hoechst 33342 through competition with Hoechst 33342 for binding to the same drug-binding site on LmrP, whereas other drugs inhibit LmrP-mediated transport of Hoechst 33342 by non-competitive inhibition, through binding to a drug-interaction site different from the Hoechst 33342 binding site. 37 Similar observations have been reported for the staphylococcal MFS transporter QacA. Competition studies demonstrated that the QacA-mediated transport of ethidium bromide is competitively inhibited by monovalent ions and non-competitively inhibited by divalent cations. Thus, monovalent and divalent cations appear to bind to separate sites on the transporter. 38 Recently, the analyses of a threedimensional structure analysis of the Bacillus subtilis transcriptional regulator BmrR 39 and of site-directed mutants of the E. coli MFS transporter MdfA 40 and SMR transporter EmrE 41 have revealed that negatively charged amino acid residues can play a key role in cation selectivity of multidrug transporters (see reviews by others in this issue for more details). Similarly, the three negatively charged residues in putative transmembrane segments of LmrP (D142 in TM5, E327 in TM10, and E388 in TM12) may play a key role in the binding of cationic drugs by LmrP. Recent experiments suggest that the presence of an acidic residue at position 142 and 327, but not 388, is critical for the ability of LmrP to transport cationic drugs (H.W.v.V., unpublished). Interestingly, LmrA and P-glycoprotein do not contain negatively charged residues in transmembrane segments. Hence, the cation specificity of these transporters must be based on other principles, such as the interaction between cations and the π-electron systems of aromatic residues located in the hydrophobic environment of the phospholipid bilayer. 42 It is feasible that a single organizational principle

LmrA mediates bidirectional drug transport It is generally assumed that ABC transporters are unidirectional pumps, which are dedicated to mediating substrate transport in a single direction (uptake or efflux). In contrast, secondary transporters are reversible systems that mediate transport in a direction that depends on the transmembrane orientation of electrochemical ion gradients and the ion/substrate coupling mechanism of the transporter. Surprisingly, new evidence suggests that LmrA is able to catalyse both the outward and the inward movement of drugs. In one 242

Towards the molecular mechanism of prokaryotic and eukaryotic multidrug transporters

Figure 2. Uptake of a fluorescent lipid analogue in L. lactis NZ9000 expressing wildtype LmrA (a) or the transport-inactive K388M mutant form of LmrA (b) in the absence (solid circles) or presence (open circles) of ortho-vanadate. Transport of 1-myristoyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine (C6 -NBD-PE) was essentially measured as described. 17,19 Since ortho-vanadate inhibits glycolysis in L. lactis, metabolic energy for transport was generated through the ortho-vanadate insensitive arginine deiminase pathway, as described previously. 51 At the onset of the experiment (t = 0 s), L. lactis cells were diluted to an OD660 of 0.025 in 2 ml of prewarmed 50 (K)HEPES pH 7.4 containing 0.1 mM MgSO4 , 20 mM L-arginine and 0.5 mM sodium ortho-vanadate, and subsequently preincubated at 30 ◦ C to allow the accumulation of vanadate in the cytoplasm. Control cells were preincubated in the absence of vanadate. Transport of C6 -NBD-PE from the quenched environment of donor liposomes to the non-quenched environment of the cytoplasmic membrane of L. lactis was initiated at t = 238 s by the addition of donor liposomes containing 3 mol % C6 -NBD-PE, 6 mol % NBD fluorescence quencher L-α-phosphatidylethanolamine-N -(lissamine rhodamine B sulfonyl) (egg) (N-RhPE) and 91 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to a concentration of 35 g lipid ml−1 . NBD fluorescence was monitored at an excitation wavelength of 475 nm (15 nm slit width) and an emission wavelength of 530 nm (4 nm slit width). L. lactis expressed the wild-type and mutant form of LmrA at equal levels (data not shown).

form of LmrA. The data obtained suggest that LmrA mediates net uptake of C6 -NBD-PE in L. lactis cells during the first 10 s after the addition of C6 -NBD-PE. The two-cylinder engine model, mentioned above, proposes the presence of two general drug-binding sites in homodimeric LmrA: a high-affinity drugbinding site exposed at the inner membrane surface, and a low-affinity drug-binding site exposed at the outer membrane surface. In this two-site model, the bidirectional transport of C6 -NBD-PE by LmrA can be explained by the involvement of the low-affinity drugbinding site in the reversed transport of C6 -NBD-PE from the outer leaflet to the inner leaflet of the phospholipid bilayer (equivalent to uptake) when the local drug concentration is high in the outer leaflet and low in the inner leaflet, and the involvement of the high-affinity drug-binding site in LmrA-mediated outward movement of C6 -NBD-PE from the inner leaflet to the outer leaflet (equivalent to efflux) when the local drug concentration is high in the inner leaflet. Hence, the direction of drug transport by LmrA will depend on the relative affinities of the two binding

type of experiment, presented in Figure 2, the uptake of the fluorescent substrate 1-myristoyl-2-[6-[(7-nitro2,1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3phosphoethanolamine (C6 -NBD-PE) in L. lactis was monitored in cells expressing either transport-active wildtype LmrA [Figure 2(a)] or transport-inactive mutant LmrA containing the K388M mutation in the Walker A region of the protein [Figure 2(b)]. After 40 s of uptake, the accumulation level of C6 -NBD-PE in cells expressing wildtype LmrA was significantly enhanced if LmrA-mediated transport was inhibited by ortho-vanadate. In contrast, ortho-vanadate did not influence the accumulation level of C6 -NBD-PE in cells expressing the transport-inactive mutant form of LmrA. These data demonstrate the LmrA-mediated efflux of C6 -NBD-PE from L. lactis cells. Surprisingly, the initial uptake of C6 -NBD-PE (during the first 10 s) in cells expressing wildtype LmrA was significantly reduced if LmrA-mediated transport was inhibited by ortho-vanadate. The inhibitory effect of ortho-vanadate on the initial uptake of C6 -NBD-PE was not observed in cells expressing the transport-inactive mutant 243

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sites for the drug transported, and the local drug concentrations in the inner and outer leaflet of the membrane. Currently, it is not known whether the LmrAmediated uptake of C6 -NBD-PE reflects heterologous exchange between C6 -NBD-PE and an endogenous substrate in L. lactis cells (e.g. lipid), or whether uptake of C6 -NBD-PE involves the return of unliganded binding sites to the outside surface of the membrane. The surprising suggestion that LmrA is able to mediate bidirectional transport has also been reported for other ABC transporters, such as the binding protein-dependent general amino acid permease in Rhizobium leguminosarum 49 and the histidine permease in Salmonella typhimurium 50 and may relate to observations that, for certain drugs, the expression of ABC multidrug transporters is associated with an increased drug sensitivity rather than an increased drug resistance. Further analysis of the molecular basis of this phenomenon may enable the development of cytotoxic drugs that are preferentially accumulated in multidrug resistant cells in which (multi)drug transporters are overexpressed.

4. van Veen HW, Konings WN (1997) Multidrug transporters from bacteria to man: similarities in structure and function. Semin Cancer Biol 8:183–191 5. Higgins CF (1992) ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8:67–113 6. van Veen HW, Konings WN (1998) The ABC family of multidrug transporters in microorganisms. Biochim Biophys Acta 1365:31–36 7. Paulsen IT, Brown MH, Skurray RA (1996) Proton dependent multidrug efflux systems. Microbiol Rev 60:575–608 8. Putman M, van Veen HW, Konings WN (2000) Molecular properties of bacterial multidrug transporters. Microbiol Mol Biol Rev 64:672–693 9. Lewis K (1994) Multidrug resistance pumps in bacteria: variations on a theme. Trends Biochem Sci 19:119–123 10. van Veen HW, Venema K, Bolhuis B, Oussenko I, Kok J, Poolman B, Driessen AJM, Konings WN (1996) Multidrug resistance mediated by a bacterial homolog of the human drug transporter MDR1. Proc Natl Acad Sci USA 93:10668–10672 11. Bolhuis B, Poelarends G, van Veen HW, Poolman B, Driessen AJM, Konings WN (1995) The lactococcal lmrP gene encodes a proton motive force-dependent drug transporter. J Biol Chem 270:26092–26098 12. van Veen HW, Putman M, Margolles M, Sakomoto K, Konings WN (2000) Molecular pharmacological characterization of two multidrug transporters in Lactococcus lactis. Pharmacol Ther 85:245–249 13. Orlowski S, Garrigos M (1999) Multiple recognition of various amphiphilic molecules by the multidrug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs. Anticancer Res 19:3109–3124 14. Nooter K, Sonneveld P (1994) Clinical relevance of Pglycoprotein expression in haematological malignancies. Leuk Res 18:233–243 15. Gibbs JB (2000) Mechanism-based target identification and drug discovery in cancer research. Science 287:1969–1973 16. van Veen HW, Callaghan R, Soceneantu L, Sardini A, Konings WN, Higgins CF (1998) A bacterial antibioticresistance gene that complements the human multidrugresistance P-glycoprotein gene. Nature 391:291–295 17. van Veen HW, Margolles A, Müller M, Higgins CF, Konings WN (2000) The homodimeric ATP-binding transporter LmrA mediates multidrug transport by an alternating two-site (twocylinder engine) mechanism. EMBO J 19:2503–2514 18. Bolhuis H, van Veen HW, Brands JR, Putman M, Poolman B, Driessen AJM, Konings WN (1996) Energetics and mechanism of drug transport by the lactococcal multidrug transporter LmrP. J Biol Chem 271:24123–24128 19. Margolles A, Putman M, van Veen HW, Konings WN (1999) The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids. Biochemistry 38:16298–16306 20. Putman M, van Veen HW, Poolman B, Konings WN (1999) Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP. Biochemistry 38:1002–1008 21. Putman M, van Veen HW, Degener JE, Konings WN (2000) Antibiotic resistance: era of the multidrug pump. Mol Microbiol 36:772–774 22. Bolhuis H, van Veen HW, Molenaar D, Poolman B, Driessen AJM, Konings WN (1996) Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane. EMBO J

Future prospects It is still far from clear how multidrug transporters are able transport such a diversity of compounds with different structures, and there is still much to be unravelled about the endogenous substrates and the role of these proteins in physiological and pathological processes. However, the current evidence suggests that ABC multidrug transporters and secondary multidrug transporters have much more in common than was originally anticipated. If so, the presence of a common denominator makes it possible that contributions to the general field of transport can continue to arise from diverse sources. The structure–function relationships that dictate substrate recognition and transport in pro- and eukaryotic multidrug transporters will represent an intriguing area of future research.

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