ATP-binding Cassette Half-transporter Research Articles

Association of ABCG5 and ABCG8 Transporters with Sitosterolemia.

Sitosterolemia is a rare genetic lipid disorder, mainly characterized by the accumulation of dietary xenosterols in plasma and tissues. It is caused by inactivating mutations in either ABCG5 or ABCG8 subunits, a subfamily-G ATP-binding cassette (ABCG) transporters. ABCG5/G8 encodes a pair of ABC half transporters that form a heterodimer (G5G8). This heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the hepatobiliary and transintestinal secretion of cholesterol and dietary plant sterols to the surface of hepatocytes and enterocytes, promoting the secretion of cholesterol and xenosterols into the bile and the intestinal lumen. In this way, ABCG5/G8 function in the reverse cholesterol transport pathway and mediate the efflux of cholesterol and xenosterols to high-density lipoprotein and bile salt micelles, respectively. Here, we review the biological characteristics and function of ABCG5/G8, and how the mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis, among other features.

ABCD subfamily of peroxisomal ABC transporters in GtoPdb v.2023.1

Peroxisomes are indispensable organelles in higher eukaryotes. They are essential for the oxidation of a wide variety of metabolites, which include: saturated, monounsaturated and polyunsaturated fatty acids, branched-chain fatty acids, bile acids and dicarboxylic acids [5]. However, the peroxisomal membrane forms an impermeable barrier to these metabolites. The mammalian peroxisomal membrane harbours three ATP-binding cassette (ABC) half-transporters, named ABCD1, -2 and -3. The ABCD transporters predominantly act as homodimers to transport different acyl-CoAs.

Identification of Functional Transcriptional Binding Sites within Chicken Abcg2 Gene Promoter and Screening Its Regulators.

Breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) half transporter encoded by the Abcg2 gene, is reported to influence the pharmacokinetics of substrate drugs during clinical therapy. The aim of this study was to clarify the mechanisms that regulate the transcription of the chicken Abcg2 gene through cloning and characterization of its promoter region. Results showed that the Abcg2 gene is transcribed by a TATA-less promoter with several putative Sp1 sites upstream from two putative CpG islands. A luciferase reporter assay conducted both in chicken leghorn male hepatoma (LMH) cells and chicken primary hepatocytes mapped a basal promoter to nucleotides −110 to +30, which is responsible for the constitutive expression of Abcg2. The 5′-region upstream of the basal promoter was characterized by both positive and negative regulatory domains. Further, using the cell-based reporter gene assay combined with RT-PCR and drug accumulation analysis, we found that four xenobiotics, daidzein, clotrimazole, ivermectin, and lipopolysaccharide (LPS), influence the expression and function of BCRP through significant regulation of the Abcg2 gene promoter. Interaction sites with the Abcg2 gene promoter of these four selected regulators were clarified by progressive deletions and mutation assays. This study shed some light on the regulatory mechanisms involved in chicken Abcg2 gene expression and the results may have far-reaching significance regarding the usage and development of veterinary drugs.

ABCD subfamily of peroxisomal ABC transporters (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Peroxisomes are indispensable organelles in higher eukaryotes. They are essential for the oxidation of a wide variety of metabolites, which include: saturated, monounsaturated and polyunsaturated fatty acids, branched-chain fatty acids, bile acids and dicarboxylic acids [3]. However, the peroxisomal membrane forms an impermeable barrier to these metabolites. The mammalian peroxisomal membrane harbours three ATP-binding cassette (ABC) half-transporters, which act as homo- and/or heterodimers to transport these metabolites across the peroxisomal membrane.

Peroxisomal ATP-binding cassette transporters form mainly tetramers

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

Modulation of Expression and Activity of ABC Transporters by the Phytoestrogen Genistein. Impact on Drug Disposition.

ATP binding cassette (ABC) transporters are involved in drug absorption, distribution and elimination. They also mediate multidrug resistance in cancer cells. Isoflavones, such as genistein (GNT), belong to a class of naturally-occurring compounds found at high concentrations in commonly consumed soya based-foods and dietary supplements. GNT and its metabolites interact with ABC transporters as substrates, inhibitors and/or modulators of their expression. This review compiles information about regulation of ABC transporters by GNT with special emphasis on the three major groups of ABC transporters involved in excretion of endo- and xenobiotics as follows: Pglycoprotein (MDR1, ABCB1), a group of multidrug resistance associated proteins (MRPs, ABCC subfamily) and ABCG2 (BCRP), an ABC half-transporter. The impact of these regulations on potential GNT-drug interactions is further considered.

LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis.

BackgroundMitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host.MethodsLmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software.ResultsLmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis.ConclusionsAltogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1284-5) contains supplementary material, which is available to authorized users.

The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter.

Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.

A New ABC Half-Transporter in Leishmania major Is Involved in Resistance to Antimony

The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania

Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite.

Post-meiotic deficient anther1 (PDA1) encodes an ABC transporter required for the development of anther cuticle and pollen exine in rice

The tapetum of the anther locule encloses the male reproductive cells and plays a supportive role for normal pollen development. However, the underlying mechanism remains less understood. Previously, we identified a complete recessive male sterile mutant, post-meiotic deficient anther1 (pda1), with abnormal postmeiotic tapetal development. In this study we comprehensively characterized pda1. Chemical analysis uncovered that pda1 anther had significant lower levels of cutin monomers and cuticular waxes. PDA1 gene encodes an ATP-binding cassette (ABC) half-transporter, namely OsABCG15, which is conserved from algae to higher plants. In situ RNA hybridization assay showed that PDA1 is strongly expressed in tapetal cells, and weakly in microspores during the anther development. Additionally, the expression of two pollen exine biosynthetic genes CYP704B2 and CYP703A3 was dramatically reduced in pda1 mutant anthers. Altogether, these observations suggest that the tapetum-expressed ABC transporter PDA1 plays a crucial role in secreting lipidic precursors from the tapetum to developing microspores and the anther epidermis.

Androgen receptor decreases the cytotoxic effects of chemotherapeutic drugs in upper urinary tract urothelial carcinoma cells

Upper urinary tract urothelial carcinomas (UUTUCs) represent relatively uncommon yet devastating tumors that affect more males than females. However, the correlation between gender difference and disease progression remains unclear. Androgen and the androgen receptor (AR) were previously hypothesized to account for the gender difference in the incidence of urothelial carcinomas; however, the role of AR in the development and progression of UUTUCs is not well understood. In addition, although UUTUCs are responsive to chemotherapy, various responses are presented among patients. Therefore, the aim of the present study was to determine the role of AR in the response of UUTUC cells to chemotherapeutic drugs. In this study, AR overexpression in UUTUC cells (BFTC 909) was identified to reduce the cytotoxic effect of chemotherapeutic drugs, including doxorubicin, cisplatin and mitomycin C and protected cells from drug-induced death. The expression of ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, was increased in AR-overexpressing BFTC cells. In addition, use of the AR degradation enhancer, ASC-J9®, repressed the AR effect on increasing cell viability under drug treatment. In summary, results of the present study indicate that the status of AR expression levels in UUTUCs may be a significant factor in affecting the efficacy of chemotherapy and classic chemotherapeutic drugs and AR targeted therapy may provide a novel potential therapeutic approach to improve treatment of UUTUCs.

Identification of a New Fatty Acid Synthesis-Transport Machinery at the Peroxisomal Membrane

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.

Abstract 2610: Histone deacetylase inhibitors mediate pharmacological rescue of the ABCG2 Q141K variant: Potential for therapeutics in cancer and gout

Abstract ABCG2 is an ATP-binding cassette half-transporter that has garnered interest in pharmacogenomics as a modulator of oral drug absorption, drug excretion, and drug distribution through expression at the blood-brain barrier and the maternal-fetal barrier. A single nucleotide polymorphism (SNP) C421A in ABCG2, resulting in a glutamic acid to lysine mutation at amino acid 141 (Q141K), confers impaired protein expression and function. Pharmacogenomic studies have linked the SNP to increased exposure to substrate drugs including irinotecan, topotecan, sunitinib and gefitinib. Moreover, it has been shown that 10 % of gout cases can be attributed to the Q141K variant. We have studied the biology underlying altered expression and function of the ABCG2 variant. Q141K protein was resistant to degradation by Endo H, suggesting that it was fully processed and folded. Q141K ABCG2 expression was increased by the lysosome inhibitor bafilomycin, indicating that some of the protein reached the cell surface, although at reduced levels. But the largest fraction of the Q141K variant was retained in an intracellular compartment where a part was eliminated through the proteasomal pathway while the undegraded ABCG2 accumulated in the aggresomes, as shown by colocalization of the transporter with the centrosome marker, γ-tubulin. The ability of several agents to impact ABCG2 trafficking to the cell surface was evaluated. Mitoxantrone, an ABCG2 substrate previously noted to act as a pharmacological chaperone, induced a 2-fold increase in Q141K ABCG2 expression at the cell surface but only slightly enhanced ABCG2-related efflux. Romidepsin and other histone deacetylase inhibitors (HDIs), known to increase levels of ABCG2 mRNA, were also assayed. A significant increase in total protein, surface expression, and function was seen in Q141K ABCG2 variant when exposed to various HDIs. Immunofluorescence analysis by confocal microscopy showed a dramatic shift to the plasma membrane following exposure to the HDIs, whereas the localization of α-tubulin, HDAC6 and vimentin, involved in aggresome formation and structure, were unchanged. These results support the notion that Q141K ABCG2 variant is trapped in the aggresome and that HDIs aid localization to the surface. Some supplementary experiments showed that the Q141K ABCG2 states of phosphorylation, acetylation and dimerization were unmodified after HDIs treatment, and that the chaperones BiP, Hsp70 or Hsp90 were not involved in rescue. Investigations into the HDI-mediated mechanisms are underway. In addition to non-oncologic indications, the restoration of ABCG2 function has potential applications in oncology, in improving normal tissue protection and drug elimination, as well as in cancer prevention since several carcinogens are substrates for ABCG2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2610. doi:10.1158/1538-7445.AM2011-2610

The Challenge of Exploiting ABCG2 in the Clinic

ABCG2, or breast cancer resistance protein (BCRP), is an ATP-binding cassette half transporter that has been shown to transport a wide range of substrates including chemotherapeutics, antivirals, antibiotics and flavonoids. Given its wide range of substrates, much work has been dedicated to developing ABCG2 as a clinical target. But where can we intervene clinically and how can we avoid the mistakes made in past clinical trials targeting P-glycoprotein? This review will summarize the normal tissue distribution, cancer tissue expression, substrates and inhibitors of ABCG2, and highlight the challenges presented in exploiting ABCG2 in the clinic. We discuss the possibility of inhibiting ABCG2, so as to increase oral bioavailability or increase drug penetration into sanctuary sites, especially the central nervous system; and at the other end of the spectrum, the possibility of improving ABCG2 function, in the case of gout caused by a single nucleotide polymphism. Together, these aspects of ABCG2/BCRP make the protein a target of continuing interest for oncologists, biologists, and pharmacologists.

Asymmetric ATP Hydrolysis Cycle of the Heterodimeric Multidrug ABC Transport Complex TmrAB from Thermus thermophilus

ATP-binding cassette (ABC) systems translocate a wide range of solutes across cellular membranes. The thermophilic gram-negative eubacterium Thermus thermophilus, a model organism for structural genomics and systems biology, discloses ∼46 ABC proteins, which are largely uncharacterized. Here, we functionally analyzed the first two and only ABC half-transporters of the hyperthermophilic bacterium, TmrA and TmrB. The ABC system mediates uptake of the drug Hoechst 33342 in inside-out oriented vesicles that is inhibited by verapamil. TmrA and TmrB form a stable heterodimeric complex hydrolyzing ATP with a K(m) of 0.9 mm and k(cat) of 9 s(-1) at 68 °C. Two nucleotides can be trapped in the heterodimeric ABC complex either by vanadate or by mutation inhibiting ATP hydrolysis. Nucleotide trapping requires permissive temperatures, at which a conformational ATP switch is possible. We further demonstrate that the canonic glutamate 523 of TmrA is essential for rapid conversion of the ATP/ATP-bound complex into its ADP/ATP state, whereas the corresponding aspartate in TmrB (Asp-500) has only a regulatory role. Notably, exchange of this single noncanonic residue into a catalytic glutamate cannot rescue the function of the E523Q/D500E complex, implicating a built-in asymmetry of the complex. However, slow ATP hydrolysis in the newly generated canonic site (D500E) strictly depends on the formation of a posthydrolysis state in the consensus site, indicating an allosteric coupling of both active sites.

Diet-induced Lipid Accumulation in Liver Enhances ATP-binding Cassette Transporter g5/g8 Expression in Bile Canaliculi

The ATP-binding cassette half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterols into bile. Studies have demonstrated the diet-induced expression of these transporters in liver, but precisely where this occurs remains to be elucidated. This study investigated the changes in the expression of these transporters in bile canaliculi in cholesterol-loaded livers. Mice were fed either a standard (SD) diet or a high-fat and high-sucrose (HF/HS) diet for 3 weeks. Bile canaliculi proteins and cryosections were prepared from the liver, and the protein levels and distribution of Abcg5/Abcg8 were determined. The high-calorie diet induced a marked accumulation of lipids in mouse liver. Protein levels of Abcg5 and Abcg8 in bile canaliculi were significantly increased by the HF/HS diet compared to the SD diet. No significant differences in Abca1, Abcb4 (Mdr2), Abcb11 (Bsep), or Abcc2 (Mrp2) levels were observed. Immunohistochemical analyses confirmed that these increases occurred in bile canaliculi. These results suggest that diet-induced lipid loading of the liver causes a significant increase in the expression of Abcg5 and Abcg8 in bile canaliculi.

The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes

ABCG2 is a human membrane ATP-binding cassette half-transporter that hydrolyzes ATP to efflux a large number of chemotherapeutic agents. Several oligomeric states of ABCG2 from homodimers to dodecamers have been reported depending on the overexpression systems and/or the protocols used for purification. Here, we compared the oligomeric state of His 6-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His 6-ABCG2 was purified through a new approach involving its specific recognition onto a functionalized lipid layer containing a Ni-NTA lipid. This approach allowed the purification of His-ABCG2 in presence of all solubilized membrane components that might be involved in the stabilisation of native oligomers and without requiring any additional washing or concentration passages. ABCG2 purified onto the NiNTA lipid surfaces were directly analyzed by electron microscopy and by biochemical assays. Altogether, our data are consistent with a tetrameric organization of ABCG2 when expressed in either heterologous Sf9 insect cells or in human homologous cells.

Abstract 3541: Pharmacological chaperones increase membrane expression of ABCG2 harboring the Q141K single nucleotide polymorphism

Abstract ABCG2 is an ATP-binding cassette half-transporter that functions as a xenobiotic transporter. Since ABCG2's discovery in multidrug-resistant cancer cells, the transporter has garnered interest in pharmacogenomics as a component of the blood-brain barrier and the maternal-fetal barrier as well as a modulator of oral drug absorption. A single nucleotide polymorphism (SNP) C421A in ABCG2, resulting in a glutamic acid to leucine mutation at amino acid 141 (Q141K), has been extensively studied and has been found to affect protein expression and ATPase activity, but may also affect protein folding. As misfolded ABCG2 has been shown to be degraded through the endoplasmic reticulum associated protein degradation (ERAD) system (T. Furukawa et al., Pharm Res., 26(2): 469-479, 2008), we postulated that pharmacological chaperones such as mitoxantrone, a known substrate of ABCG2, might increase surface expression of Q141K ABCG2. In this study, we used HEK293 stably transfected with Q141K ABCG2 to examine whether incubating cells expressing the mutated transporter with mitoxantrone or other ABCG2 substrates would serve to “rescue” Q141K ABCG2 and improve trafficking to the cell surface, as we have demonstrated in other mutations that impair trafficking (O. Polgar et al., Biochemistry, 43(29): 9448-9456, 2004). Expression of ABCG2 in cells transfected with wild-type or Q141K ABCG2 was measured by immunoblot analysis and flow cytometry. After treatment with 5 μM of mitoxantrone, cell surface expression of ABCG2 in cells transfected with vector containing Q141K ABCG2 increased, while cells transfected with wild-type ABCG2 showed no change. Increased ABCG2 expression was also observed in membrane preparations from mitoxantrone-treated cells transfected with Q141K ABCG2 compared to wild-type ABCG2 transfected cells. Interestingly, immunoblot analysis showed that when cells transfected with Q141K ABCG2 were incubated with 50 μM of the dietary compounds tert-butyl hydroquinone (TBHQ) and dibenzoylmethane (DBM), increased expression of ABCG2 was observed in membrane preparations, while no change was observed for cells expressing wild-type ABCG2. These results suggest that some dietary compounds may promote proper folding and membrane localization of mutant forms of ABCG2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3541.