Energy-dependent Transporter Research Articles

DNA Copy Number Aberrations and Expression of ABC Transporter Genes in Breast Tumour: Correlation with the Effect of Neoadjuvant Chemotherapy and Prognosis of the Disease

One of the important reasons for the ineffectiveness of chemotherapy in breast cancer (BC) is considered to be the formation of a multidrug resistance phenotype in tumour cells, which is caused by the expression of energy-dependent ABC transporters. The aim of this work was to assess chromosomal aberrations and the level of transcripts of all 49 known ABC transporter genes in breast tumours. Materials and Methods. The study included 129 patients with breast cancer. A microarray study of all tumour samples was carried out on microchips. Results. This study established that the presence of a deletion in genes ABCB1, ABCB4, ABCB8, ABCC7, ABCC11, ABCC12, ABCF2, and ABCG4 is associated with an objective response to treatment (p ≤ 0.05). A decrease in the expression of genes was associated with a good response to chemotherapy, whereas an increase in expression caused the progression and stabilization of the tumour. Analysis of metastatic-free survival rates showed that the presence of ABCB1/4 and ABCC1/6 deletions was associated with 100% survival (log-rank test p = 0.01 and p = 0.03). Conclusions. The study showed that the aberrant state of ABC transporter genes, as well as a decrease in the expression of these genes, is a predictor of the effectiveness of therapeutic treatment and a potential prognostic marker of metastatic survival.

Recent Findings on Thymoquinone and Its Applications as a Nanocarrier for the Treatment of Cancer and Rheumatoid Arthritis.

Cancer causes a considerable amount of mortality in the world, while arthritis is an immunological dysregulation with multifactorial pathogenesis including genetic and environmental defects. Both conditions have inflammation as a part of their pathogenesis. Resistance to anticancer and disease-modifying antirheumatic drugs (DMARDs) happens frequently through the generation of energy-dependent transporters, which lead to the expulsion of cellular drug contents. Thymoquinone (TQ) is a bioactive molecule with anticancer as well as anti-inflammatory activities via the downregulation of several chemokines and cytokines. Nevertheless, the pharmacological importance and therapeutic feasibility of thymoquinone are underutilized due to intrinsic pharmacokinetics, including short half-life, inadequate biological stability, poor aqueous solubility, and low bioavailability. Owing to these pharmacokinetic limitations of TQ, nanoformulations have gained remarkable attention in recent years. Therefore, this compilation intends to critically analyze recent advancements in rheumatoid arthritis and cancer delivery of TQ. This literature search revealed that nanocarriers exhibit potential results in achieving targetability, maximizing drug internalization, as well as enhancing the anti-inflammatory and anticancer efficacy of TQ. Additionally, TQ-NPs (thymoquinone nanoparticles) as a therapeutic payload modulated autophagy as well as enhanced the potential of other drugs when given in combination. Moreover, nanoformulations improved pharmacokinetics, drug deposition, using EPR (enhanced permeability and retention) and receptor-mediated delivery, and enhanced anti-inflammatory and anticancer properties. TQ’s potential to reduce metal toxicity, its clinical trials and patents have also been discussed.

Changes in Gene Expression Profiling and Phenotype in Aged Multidrug Resistance Protein 4-Deficient Mouse Retinas.

Multidrug resistance protein 4 (MRP4) is an energy-dependent membrane transporter responsible for cellular efflux of a broad range of xenobiotics and physiological substrates. In this trial, we aimed to investigate the coeffects of aging and MRP4 deficiency using gene expression microarray and morphological and electrophysiological analyses of mouse retinas. Mrp4-knockout (null) mice and wild-type (WT) mice were reared in the same conditions to 8–12 weeks (young) or 45–55 weeks (aged). Microarray analysis identified 186 differently expressed genes from the retinas of aged Mrp4-null mice as compared to aged WT mice, and subsequent gene ontology and KEGG pathway analyses showed that differently expressed genes were related to lens, eye development, vision and transcellular barrier functions that are involved in metabolic pathways or viral infection pathways. No significant change in thickness was observed for each retinal layer among young/aged WT mice and young/aged Mrp4-null mice. Moreover, immunohistochemical analyses of retinal cell type did not exhibit an overt change in the cellular morphology or distribution among the four age/genotype groups, and the electroretinogram responses showed no significant differences in the amplitude or the latency between aged WT mice and aged Mrp4-null mice. Aging would be an insufficient stress to cause some damage to the retina in the presence of MRP4 deficiency.

Short chain fatty acids and monocarboxylate transporters in irritable bowel syndrome.

Gut microbiota ferments indigestible food that rests in the colon to produce short-chain fatty acids (SCFAs) acetate, propionate, and butyrate. Colonic SCFA stimulate the synthesis of serotonin which is central in irritable bowel syndrome (IBS) pathophysiology. Reduced SCFA have been linked to specific IBS symptoms like colonic hyperalgesia and hypersensitivity. SCFA enter the colonocyte mainly via 2 energy-dependent monocarboxylate transporters, MCT1 (SLC16A1) and SMCT1 (SLC5A8). We investigated specific gut microbiota, SCFA concentrations, and monocarboxylate transporter mRNA expression in patients with IBS. A total of 30 IBS patients-15 constipation-predominant (C-IBS) and 15 diarrhoea-predominant (D-IBS)-and 15 healthy controls were recruited. Bacteroidetes and Bifidobacterium species were analyzed using quantitative polymerase chain reaction (qPCR) on stool samples. SCFA concentrations were determined by gas chromatography/mass spectroscopy of stool samples. Monocarboxylate transporter mRNA was quantified by qPCR on colon biopsy specimens. Bacteroides was significantly increased in the D-IBS group compared with the C-IBS group and healthy controls. Bifidobacterium was significantly reduced in both IBS groups. SCFA ratios were altered in both IBS groups with a reduction of all 3 measured SCFA in C-IBS and acetic acid in D-IBS. MCT1 and SMCT1 were significantly reduced in C-IBS and D-IBS. In agreement with findings of previous studies, the microbiota assessed were significantly altered inferring dysbiosis in IBS. SCFA and their ratios were significantly altered in both IBS groups. SCFA transporters, MCT1 and SMCT1 were significantly reduced in both IBS groups, suggesting reduced colonocyte SCFA transfer. SCFA availability and transfer into the colonocytes may be important in IBS pathogenesis and should be prospectively studied.

The Boron & De Weer Model of Intracellular pH Regulation

The classic Boron & De Weer (1976) paper provided the first evidence of active regulation of pH} in cells by an energy-dependent acid-base transporter. These authors also developed a quantitative model --- comprising passive fluxes of acid-base equivalents across the cell membrane, intracellular reactions, and an active transport mechanism in the cell membrane (modelled as a proton pump) --- to help interpret their measurements of intracellular pH under perturbations of both extracellular CO2/HCO3- and extracellular NH3/NH4+. This Physiome paper seeks to make that model, and the experimental conditions under which it was developed, available in a reproducible and well-documented form, along with a software implementation that makes the model easy to use and understand. We have also taken the opportunity to update some of the units used in the original paper, and to provide a few parameter values that were missing in the original paper. Finally, we provide an historical background to the Boron & De Weer (1976) proposal for active pH regulation and a commentary on subsequent work that has enriched our understanding of this most basic aspect of cellular physiology.

The Boron & De Weer Model of Intracellular pH Regulation

The classic Boron & De Weer (1976) paper provided the first evidence of active regulation of pH} in cells by an energy-dependent acid-base transporter. These authors also developed a quantitative model --- comprising passive fluxes of acid-base equivalents across the cell membrane, intracellular reactions, and an active transport mechanism in the cell membrane (modelled as a proton pump) --- to help interpret their measurements of intracellular pH under perturbations of both extracellular CO2/HCO3- and extracellular NH3/NH4+. This Physiome paper seeks to make that model, and the experimental conditions under which it was developed, available in a reproducible and well-documented form, along with a software implementation that makes the model easy to use and understand. We have also taken the opportunity to update some of the units used in the original paper, and to provide a few parameter values that were missing in the original paper. Finally, we provide an historical background to the Boron & De Weer (1976) proposal for active pH regulation and a commentary on subsequent work that has enriched our understanding of this most basic aspect of cellular physiology.

Mechanism of the Intestinal Absorption of Six Flavonoids from Zizyphi Spinosi Semen Across Caco-2 Cell Monolayer Model.

Flavonoid compounds are one kind of active ingredients isolated from a traditional Chinese herb Zizyphi spinosae semen (ZSS). Studies have shown that ZSS flavonoids have significant antioxidant effects. In this study, the Caco-2 cell monolayer model was constructed to investigate the intestinal absorption characteristics and mechanism of Isovitexin (IV), Swertisin (ST), Isovitexin-2''-O-β-D-glucopyranoside (IVG), Spinosin (S), 6'''-p-coumaroylspinosin (6-CS) and 6'''-feruloylspinosin (6-FS). The results of the bidirectional transport assay showed that the six flavonoids have good intestinal absorption in a near-neutral and 37°C environment, and the absorbability in descending order was 6-FS>6- CS>IVG>S>IV>ST. The results of carrier inhibition experiments and transport kinetics indicated that the absorption mechanism of six flavonoids was energy-dependent monocarboxylate transporter (MCT)-mediated active transport. In particular, the para-cellular pathway also participated in the transport of IV, ST, IVG and S. Furthermore, the efflux process of six flavonoids was mediated by P-glycoprotein (P-gp) and multidrug resistance protein (MRP), which may result in a decrease of bioavailability. Our findings provide significant information for revealing the relationship between the intestinal absorption mechanism of flavonoids and its structure as well as laying a basis for the research of flavonoid preparations.

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

SummarySiderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Donepezil, a cholinesterase inhibitor used in Alzheimer's disease therapy, is actively exported out of the brain by abcb1ab p-glycoproteins in mice

Polymorphisms in the drug transporter gene ABCB1 predict the treatment response of selected antidepressants and limit anticonvulsive medication's effectiveness. The ABCB1 locus encodes the energy-dependent transporter P-glycoprotein (P-gp) of the blood brain barrier (BBB), which serves as an efflux pump of its substrates in the expressing tissues of vertebrates. One experimental setup to determine a posteriori the P-gp substrate status is the use of the double abcb1ab knock-out (KO) mice model. Since so far, P-gp substrate status of donepezil, a cholinesterase inhibitor wildly used in Alzheimer's disease therapy was inconclusive, we performed subcutanous (s.c.), continuous injections over 11 days in double abcb1ab KO and P-gp competent wildtype (WT) mice. Both in brain and in testis concentrations of donepezil were significantly higher in P-gp deficient mice compared to WT controls (2.39 and 2.24 times respectively). In conclusion, in mice donepezil's brain bioavailability depends on P-gp.

Acyl-CoA synthetase-4 is implicated in drug resistance in breast cancer cell lines involving the regulation of energy-dependent transporter expression

Acyl-CoA synthetase-4 (ACSL4) is an enzyme implicated in estrogen receptor α (ERα) negative regulation and hormone therapy resistance in breast cancer. In addition, ACSL4 has been associated to certain types of hormone resistance in prostate cancer.Chemotherapeutic treatment of disseminated breast cancer is usually faced with therapy resistance associated to ATP-binding cassette (ABC) transporter expression, which detect and eject anti-cancer drugs from cells. In this context, the aim of the present work was to study the role of ACSL4 in anti-cancer drug resistance and the involvement of ABC transporters in the underlying mechanisms.To this end, we used MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, which overexpress ACSL4, and control line MCF-7 Tet-Off empty vector, MDA-MB-231 shRNA ACSL4 and MDA-MB-231 wild type cells. Assays were conducted on cell viability (MTT), cell proliferation (BrdU), drug efflux (flow cytometry), ACSL4-responsive drug resistance ABC transporter genes (RNA-Seq), transporter mRNA expression, protein levels and signaling pathway participation (real-time PCR and Western blot).Higher survival rates upon chemotherapeutic treatment were obtained in MCF-7 Tet-Off/ACSL4 and MDA-MB-231 mock cells, an effect counteracted by doxycycline- or shRNA-induced ACSL4 inhibition, respectively. A synergic effect of ACSL4 inhibitor triacsin C and chemotherapeutic drugs was observed on the inhibition of MDA-MB-231 wild type cell proliferation. MCF-7 Tet-Off/ACSL4 cells showed greater doxorubicin, Hoechst 33342 and calcein AM efflux. In contrast, MDA-MB-231 shRNA ACSL4 cells evidenced inhibition of chemotherapeutic drug efflux. ABCG2, ABCC4, and ABCC8 were identified as ACSL4-responsive drug resistance genes whose expression was increased in MCF-7 Tet-Off/ACSL4 cells but inhibited in MDA-MB-231 shRNA ACSL4 cells. Further cell survival assays in the presence of Ko 143 and Ceefourin 1, inhibitors of ABCG2 and ABCC4, respectively, upon chemotherapeutic treatment showed greater participation of ABCG2 in anti-cancer drug resistance in cells overexpressing ACSL4. In addition, ACSL4 inhibition and chemotherapeutic treatment combined with rapamycin-induced mTOR inhibition synergically inhibited proliferation and reduced ABCG2 expression in cells overexpressing ACSL4.In sum, ACSL4 may be regarded as a novel therapeutic target regulating the expression of transporters involved in anticancer drug resistance through the mTOR pathway to restore drug sensitivity in tumors with poor prognosis for disease-free and overall survival.

A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis.

ABSTRACTCa2+ plays an important role in the physiology of bacteria. Intracellular Ca2+ concentrations are tightly maintained in the nanomolar range. Molecular mechanisms of Ca2+ uptake in bacteria remain elusive. Here we show that CtpE is responsible for Ca2+ uptake in Mycobacterium smegmatis. It represents a previously uncharacterized P-type ATPase family in bacteria. Disruption of ctpE in M. smegmatis resulted in a mutant with impaired growth under Ca2+-deficient conditions. The growth defect of the mutant could be rescued by Ca2+ or by ectopic expression of ctpE from M. smegmatis or the orthologous gene (Rv0908) from Mycobacterium tuberculosis H37Rv. Radioactive transport assays revealed that CtpE is a Ca2+-specific transporter. Ca2+ deficiency increased expression of ctpE, resulting in increased 45Ca2+ accumulation in cells. ctpE is a gene that is part of an operon, which is negatively regulated by Ca2+. The ctpE mutant also showed hypersensitivity to polymyxin B, increased biofilm formation, and higher cell aggregation, indicating cell envelope defects. Our work establishes, for the first time, the presence of Ca2+ uptake pumps of the energy-dependent P-type ATPase superfamily in bacteria and also implicates that intracellular Ca2+ is essential for growth and cell envelope integrity in M. smegmatis.

SN38-PEG-PLGA-verapamil nanoparticles inhibit proliferation and downregulate drug transporter ABCG2 gene expression in colorectal cancer cells

Nowadays, nanoparticle-based drug delivery systems are recognized to reduce the therapeutic side effects. One of the common problems in cancer treatment is cancer drug resistance, resulting from the over-expression of one energy-dependent transporter that enhances drug efflux. Irinotecan is used for metastatic colorectal cancer. The involvement of ABCG2 transporter in irinotecan resistance has been established. The current study was designed to characterize SN38-loaded pegylated (polyethylene glycol) PLGA [poly(lactic-co-glycolic acid)]-verapamil nanoparticles (NPs), and to distinguish the cytotoxic effect of SN38-PEG-PLGA-Ver NPs and the ability of SN38-PEG-PLGA-Ver NPs to inhibit drug resistance through the inhibition of ABCG2 expression. The surface morphology of nanoparticles was determined by scanning electron microscopy. The drug cytotoxicity of SN38-PEG-PLGA-verapamil nanoparticles was measured by MTT assay with desired concentrations and SN38-PEG-PLGA-Ver at different incubation times. Real-time PCR was used to determine the mRNA level of ABCG2, BAX, and BCL2. The cellular uptake assay was performed to show the cellular uptake of nanoparticles. The size of NPs used in this study was about 179 nm with surface charge of −17.1 mV. MTT assay results showed that 1 μmol/L of free drug and 3 μmol/L of NPs could reduce HT29 cells by half (IC50) after 48 and 96 h, respectively. An increase in expression of BAX and a decrease in expression of ABCG2 were observed according to the real-time PCR. No significant change was detected in expression of BCL2. In conclusion, sufficient uptake of SN38-PEG-PLGA-Ver NPs and a significant decrease in expression of ABCG2 and an increase in expression of BAX and BAX/BCL2 ratio was observed after treatment with nanoparticles compared with free SN38. These results reveal that SN38-PEG-PLGA-Ver NPs can be an effective therapeutic method in colon cancer treatments and also may prevent anticancer drug resistance.

Abstract 5217: Mechanism of polyspecificity of P-glycoprotein: Substitution of fifteen residues in the drug-binding pocket with tyrosine reveals a negative correlation between substrate size and transport

Abstract P-glycoprotein (P-gp) belongs to the superfamily of ATP-binding cassette (ABC) transporters. This energy-dependent cell surface transporter can pump various chemically dissimilar amphipathic or hydrophobic compounds out of cells. In cancer cells, the expression of P-gp is often linked to the development of multidrug resistance (MDR). Several studies have demonstrated that P-gp can recognize and transport various chemotherapeutic drugs and limit their bioavailability and effectiveness. The drug-substrate binding sites, translocation mechanism, and the signal transduction between drug-binding sites and ATP-hydrolysis at nucleotide-binding domains of this clinically important transporter are not yet well defined. Utilizing molecular modeling and mutagenesis, we previously identified key amino acid residues that interact with the most potent inhibitors of P-gp (zosuquidar, tariquidar, and elacridar). We found that replacing key interacting amino acids (Y307, Y953, and Q725) with alanine in the drug-binding pocket switches inhibition by these modulators of ATPase activity to stimulation. The data demonstrated that hydrogen bond interactions are critical for inhibition of ATP hydrolysis of P-gp by modulators. As tyrosine and glutamine residues can function as both hydrogen bond donors and acceptors, we hypothesized that increasing the potential for hydrogen bond interactions by introducing more tyrosine residues into the drug-binding pocket would increase the affinity of substrates for P-gp. To investigate this, we first identified residues in the transmembrane domains that interact with a number of substrates by employing a molecular modeling technique. Then we substituted fifteen conserved residues (11 Phe, 2 Leu, 2 Ilu and 1 Met) with tyrosine, creating a mutant we named 15Y. Compared to WT, for the majority of tested substrates, we observed normal transport of thirteen fluorescent substrates by the 15Y P-gp mutant demonstrating that fifteen mutations in the transmembrane domains is well tolerated in P-gp. Additionally, these changes have no effect on the cell surface expression or total level of the mutant protein and normal conformational changes occur during transport, further confirming flexible nature of transmembrane domains of P-gp. Interestingly, transport of three large-size substrates was significantly decreased. Physiochemical characterization of the substrates revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant, possibly due to increased hydrogen bond interactions. These data also demonstrate that hydrogen bond interactions are substrate- or modulator-specific. Citation Format: Shahrooz Vahedi, Eduardo Chufan, Suresh V. Ambudkar. Mechanism of polyspecificity of P-glycoprotein: Substitution of fifteen residues in the drug-binding pocket with tyrosine reveals a negative correlation between substrate size and transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2017-5217

Amino acid transport system - A substrate predicts the therapeutic effects of particle radiotherapy.

L-[methyl-11C]Methionine (11C-Met) is useful for estimating the therapeutic efficacy of particle radiotherapy at early stages of the treatment. Given the short half-life of 11C, the development of longer-lived 18F- and 123I-labeled probes that afford diagnostic information similar to 11C-Met, are being sought. Tumor uptake of 11C-Met is involved in many cellular functions such as amino acid transport System-L, protein synthesis, and transmethylation. Among these processes, since the energy-dependent intracellular functions involved with 11C-Met are more reflective of the radiotherapeutic effects, we evaluated the activity of the amino acid transport System-A as an another energy-dependent cellular function in order to estimate radiotherapeutic effects. In this study, using a carbon-ion beam as the radiation source, the activity of System-A was evaluated by a specific System-A substrate, alpha-[1-14C]-methyl-aminoisobutyric acid (14C-MeAIB). Cellular growth and the accumulation of 14C-MeAIB or 14C-Met were evaluated over time in vitro in cultured human salivary gland (HSG) tumor cells (3-Gy) or in vivo in murine xenografts of HSG tumors (6- or 25-Gy) before and after irradiation with the carbon-ion beam. Post 3-Gy irradiation, in vitro accumulation of 14C-Met and 14C-MeAIB decreased over a 5-day period. In xenografts of HSG tumors in mice, tumor re-growth was observed in vivo on day-10 after a 6-Gy irradiation dose, but no re-growth was detected after the 25-Gy irradiation dose. Consistent with the growth results, the in vivo tumor accumulation of 14C-MeAIB did not decrease after the 6-Gy irradiation dose, whereas a significant decrease was observed after the 25-Gy irradiation dose. These results indicate that the activity of energy dependent System-A transporter may reflect the therapeutic efficacy of carbon-ion radiotherapy and suggests that longer half-life radionuclide-labeled probes for System-A may also provide widely available probes to evaluate the effects of particle radiotherapy on tumors at early stage of the treatment.

Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence.

ABSTRACTCandida glabrata causes persistent infections in patients treated with fluconazole and often acquires resistance following exposure to the drug. Here we found that clinical strains of C. glabrata exhibit cell-to-cell variation in drug response (heteroresistance). We used population analysis profiling (PAP) to assess fluconazole heteroresistance (FLCHR) and to ask if it is a binary trait or a continuous phenotype. Thirty (57.6%) of 52 fluconazole-sensitive clinical C. glabrata isolates met accepted dichotomous criteria for FLCHR. However, quantitative grading of FLCHR by using the area under the PAP curve (AUC) revealed a continuous distribution across a wide range of values, suggesting that all isolates exhibit some degree of heteroresistance. The AUC correlated with rhodamine 6G efflux and was associated with upregulation of the CDR1 and PDH1 genes, encoding ATP-binding cassette (ABC) transmembrane transporters, implying that HetR populations exhibit higher levels of drug efflux. Highly FLCHR C. glabrata was recovered more frequently than nonheteroresistant C. glabrata from hematogenously infected immunocompetent mice following treatment with high-dose fluconazole (45.8% versus 15%, P = 0.029). Phylogenetic analysis revealed some phenotypic clustering but also variations in FLCHR within clonal groups, suggesting both genetic and epigenetic determinants of heteroresistance. Collectively, these results establish heteroresistance to fluconazole as a graded phenotype associated with ABC transporter upregulation and fluconazole efflux. Heteroresistance may explain the propensity of C. glabrata for persistent infection and the emergence of breakthrough resistance to fluconazole.

Structure of the AcrABZ-TolC Multidrug Efflux Pump in a Drug-Bound State

The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative species, the transporters are components of nanomachines that span the cell envelope, and a well characterized representative of such assemblies is the AcrABZ-TolC complex, a acridine resistance complex pump of Escherichia coli. The AcrABZ-TolC assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, the periplasmic AcrA which bridges these two integral membrane proteins, and a 49 amino acid residue protein, AcrZ. The entire AcrABZ-TolC complex has a molecular mass of ∼850 kDa. In E. coli, the AcrABZ-TolC pump transports vectorially chemically similar compounds thereby conferring bacterial resistance to a broad spectrum of antibiotics.In our previous study, we have determined a substrate-free structure of the entire AcrABZ-TolC pump at 16 A resolution using single-particle cryo-EM approach (Du, Wang, et al Nature 509, 512-5, 2014). We were able to build a pseudo-atomic model of the pump utilizing available X-ray crystal structures of its components. This model defines the quaternary arrangement of the pump components and identifies key domain interaction interfaces critical for the channel assembly and opening.Recently, using single-particle cryo-EM we have resolved the structure of AcrABZ-TolC pump bound to puromycin to subnanometer resolution. The cryo-EM density map resolves the secondary structural elements of the components and clearly identifies molecular boundaries between individual components of the pump. The new structure provides insights into conformational changes that might be involved in substrate binding and transport. Further studies are being pursued to decipher the molecular mechanisms underlying assembly and function of the AcrABZ-TolC pump.

A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1–2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

Abstract 552: Genetic polymorphisms of the drug transporter ABCB5 associated with advanced liver cancer

Abstract Introduction: ATP-Dependent Binding Cassette B5 (ABCB5) is a transmembrane protein which acts as energy-dependent drug efflux transporter to mediate multidrug resistance of human cancer. Genetic polymorphisms in different gene regions of ABC transporters had been reported to associate with different clinical significances. Our lab has reported that ABCB5 mRNA expression levels were associated with recurrence-free survival of hepatocellular carcinoma patients (Cheung et al Gastroenterology 2011). Aims: To examine the gene sequence of ABCB5 in human liver cancer and investigate the correlation of genetic polymorphisms with expressions and clinico-pathological features. Methods: Polymerase chain reaction (PCR) amplification and Sanger sequencing approach was used. PCR was performed for a total of 28 exons and exon-intron boundaries of ABCB5 gene using genomic DNA from liver cancer tissues. Genomic blood DNA from the same patient cohort was used to delineate the genetic polymorphism in the tumor as mutation or germline sequence variation. Results: Twenty liver cancer tissues and blood DNA samples were examined. A total of 40 genetic polymorphisms were observed in human ABCB5 gene, where 4 were novel genetic polymorphisms (G IVS6+17 T in intron 6; T IVS14+50 C in intron 14; T2166 G in exon 18; A4609G in 3’untranslated region of Exon28) and 36 had been reported in NCBI dbSNP database and SNP consortium. The genetic polymorphisms observed in the tumors were identical to their paralleled blood specimens which suggested that ABCB5 polymorphisms were germline sequence variations. An association of the genetic polymorphism (C784A) in Exon8 of ABCB5 gene with expression level of ABCB5 was observed (P=0.025). Variant genotypes (heterozygous C/A and homozygous A/A) were associated with significantly lower mRNA level of ABCB5 compared to wild type genotype (homozygous C/C). Importantly, polymorphism C784A was associated with tumor stage (P=0.002). Variant genotypes (C/A and A/A) were associated with early stage whereas wild type genotype (C/C) was associated with late stage liver cancer. Summary: ABCB5 polymorphisms were germline sequence variations in liver cancer patients, as identical genetic sequences were demonstrated in the tumors and their paralleled blood samples. Genetic polymorphism C784A was significantly associated with the transcript levels and tumor stage. Further investigation would increase HCC sample size, inclusion of healthy population, and elucidate the functional implication of the polymorphisms. Citation Format: Idy C.Y. Leung, Wallace C.W. Yip, Phyllis. F.Y. Cheung, Tan To Cheung, Ronnie T.P. Poon, Sheung Tat Fan, Siu Tim Cheung. Genetic polymorphisms of the drug transporter ABCB5 associated with advanced liver cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 552. doi:10.1158/1538-7445.AM2014-552

Structure of the AcrAB-TolC multidrug efflux pump.

The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent ‘pumps’ that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component1-7. A model system for such a pump is the acridine resistance complex of Escherichia coli1. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to vectorially transport a diverse array of compounds with little chemical similarity, and accordingly confers resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including pathogens of animals and plants. Crystal structures are available for the individual pump components2-7 and these have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. How the subunits are organised in the pump, their stoichiometry and the details of their interactions are not known and are under debate. In this manuscript, we present the pseudoatomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner8. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.

Reduced Antimony Accumulation in ARM58 -Overexpressing Leishmania infantum

Antimony-based drugs are still the mainstay of chemotherapy against Leishmania infections in many countries where the parasites are endemic. The efficacy of antimonials has been compromised by increasing numbers of resistant infections, the basis of which is not fully understood and likely involves multiple factors. By using a functional cloning strategy, we recently identified a novel antimony resistance marker, ARM58, from the parasite Leishmania braziliensis that protects the parasites against antimony-based antileishmanial compounds. Here we show that the Leishmania infantum homologue also confers resistance against antimony but not against other antileishmanial drugs and that its function depends critically on one of four conserved domains of unknown function. This critical domain requires at least two hydrophobic amino acids and is predicted to form a transmembrane structure. Overexpression of ARM58 in antimony-exposed parasites reduces the intracellular Sb accumulation by over 70%, indicating a role for ARM58 in Sb extrusion pathways, but without involvement of energy-dependent transporter proteins.