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1.
Biochim Biophys Acta ; 1768(11): 2698-713, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17662239

RESUMO

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Colesterol/fisiologia , Lipídeos de Membrana/fisiologia , Proteínas de Neoplasias/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Estradiol/análogos & derivados , Estradiol/farmacocinética , Humanos , Metotrexato/farmacocinética , Spodoptera , beta-Ciclodextrinas/farmacologia
2.
FEBS Lett ; 580(26): 6139-44, 2006 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-17055487

RESUMO

Mutations in the ATP-binding cassette (ABC) proteins ABCG5 or ABCG8 cause sitosterolemia, a condition with increased accumulation of plant sterols. Upon high level expression of the ABCG5 and ABCG8 proteins in baculovirus-Sf9 cell expression system we found a distinct, vanadate sensitive ATPase activity in isolated membrane preparations only when the two proteins were co-expressed. This ATPase activity was significantly stimulated by the addition of certain androgen hormones and analogs, and was effectively inhibited by progesterone. Our results provide a new aspect of biochemical and functional characterization of the ABCG5/ABCG8 proteins and their possible involvement in steroid hormone transport or regulation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/metabolismo , Androstanos/farmacologia , Lipoproteínas/fisiologia , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Androgênios/farmacologia , Animais , Linhagem Celular , Hormônios/metabolismo , Humanos , Insetos , Lipoproteínas/genética , Proteínas de Membrana , Progesterona/farmacologia , Transdução Genética , Vanadatos
3.
Biochemistry ; 45(24): 7605-16, 2006 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-16768456

RESUMO

Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP).


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/metabolismo , Tirosina/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/análise , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Sequência Conservada , Dimerização , Células HeLa , Humanos , Hidrólise , Insetos/citologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência do Ácido Nucleico , Tripsina/farmacologia , Vaccinia virus/metabolismo
4.
Biochemistry ; 41(47): 13989-4000, 2002 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-12437356

RESUMO

The human MDR1 (ABCB1) gene product, P-glycoprotein (Pgp), functions as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. In this study, we assessed the role of conserved glutamate residues in the Walker B domain of the two ATP sites (E556 and E1201, respectively) during the catalytic cycle of human Pgp. The mutant Pgps (E556Q, E556A, E1201Q, E1201A, E556/1201Q, and E556/1201A) were characterized using a vaccinia virus based expression system. Although steady-state ATP hydrolysis and drug transport activities were abrogated in both E556Q and E1201Q mutant Pgps, [alpha-(32)P]-8-azidoADP was trapped in the presence of vanadate (Vi), and the release of trapped [alpha-(32)P]-8-azidoADP occurred to a similar extent as in wild-type Pgp. This indicates that these mutations do not affect either the first hydrolysis event or the ADP release step. Similar results were also obtained when Glu residues were replaced with Ala (E556A and E1201A). Following the first hydrolysis event and release of [alpha-(32)P]-8-azidoADP, both E556Q and E1201Q mutant Pgps failed to undergo another cycle of Vi-induced [alpha-(32)P]-8-azidoADP trapping. Interestingly, the double mutants E556/1201Q and E556/1201A trapped [alpha-(32)P]-8-azidoADP even in the absence of Vi, and the occluded nucleotide was not released after incubation at 37 degrees C for an extended period. In addition, the properties of transition state conformation of the double mutants generated in the absence of Vi were found to be similar to that of the wild-type protein trapped in the presence of Vi (Pgp x [alpha-(32)P]-8-azidoADP xVi). Thus, in contrast to the single mutants, the double mutants appear to be defective in the ADP release step. In aggregate, these data suggest that E556 and E1201 residues in the Walker B domains may not be critical as catalytic carboxylates for the cleavage of the bond between the gamma-P and the beta-P of ATP during hydrolysis but are essential for the second ATP hydrolysis step and completion of the catalytic cycle.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Ácido Glutâmico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Catálise , Sequência Conservada , Primers do DNA , Humanos , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vanadatos/farmacologia
5.
Biochemistry ; 41(31): 10123-32, 2002 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-12146977

RESUMO

To enable cell surface localization of the human multidrug resistance protein (MRP1, ABCC1) and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of the three membrane-spanning domains (MSDs) of the transporter. We constructed and expressed various partially and fully glycosylation-deficient, FLAG-tagged MRP1 proteins in a Vaccinia virus-based transient expression system, and the cell surface expression level of MRP1 on intact cells was followed by flow cytometry, using the FLAG tag specific monoclonal antibody M2. We also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in stably transfected HEK293 cells, and followed the cell surface expression and the transport function of MRP1 both by monitoring the efflux of fluorescent substrate and by their ability to confer resistance to HEK293 transfectants to anticancer agents such as daunorubicin and etoposide. When we inserted the FLAG epitope in extracellular loops of the MSD1 or MSD3, the tag was accessible upon removal of N-glycosylation sites (N --> Q at positions 17, 23, and 1006, respectively), whereas the FLAG epitope placed in the MSD2 was not accessible even after removal of all three N-glycosylation sites, indicating that MSD2 region is deeply buried in the plasma membrane. However, all FLAG tagged MRP1 mutants were expressed at the cell surface to the same extent as the wild-type protein and also exhibited normal transport function. Our results demonstrate that the accessibility of the external FLAG epitope is strongly dependent on the position of the tag and the glycosylation state of the different FLAG-tagged MRP1s, and the conformation of extracellular loops in MSD1 and MDS3 does not appear to contribute to the functional status of MRP1.


Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Glicosilação , Células HeLa , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Mutagênese Sítio-Dirigida , Plasmídeos , Transfecção
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