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Domperidone and metoclopramide are widely prescribed antiemetic drugs with distinct neurological side effects. The impact of P-glycoprotein (P-gp)-mediated efflux at the blood–brain barrier (BBB) on brain exposure and BBB permeation was compared in vitro and in vivo using positron emission tomography (PET) imaging in rats with the radiolabeled analogs [11C]domperidone and [11C]metoclopramide. In P-gp-overexpressing cells, the IC50 of tariquidar, a potent P-gp inhibitor, was drastically different using [11C]domperidone (221 nM [198–248 nM]) or [11C]metoclopramide (4 nM [2–8 nM]) as the substrate. Complete P-gp inhibition led to a 1.8-fold higher increase in the cellular uptake of [11C]domperidone compared with [11C]metoclopramide (p < 0.0001). Brain PET imaging revealed that the baseline brain exposure (AUCbrain) of [11C]metoclopramide was 2.4-fold higher compared with [11C]domperidone (p < 0.001), consistent with a 1.8-fold higher BBB penetration (AUCbrain/AUCplasma). The maximal increase in the brain exposure (2.9-fold, p < 0.0001) and BBB penetration (2.9-fold, p < 0.0001) of [11C]metoclopramide was achieved using 8 mg/kg of tariquidar. In comparison, neither 8 nor 15 mg/kg of tariquidar increased the brain exposure of [11C]domperidone (p > 0.05). Domperidone is an avid P-gp substrate that was in vitro compared with metoclopramide. Domperidone benefits from a lower brain exposure and a limited risk for P-gp-mediated drug–drug interaction involving P-gp inhibition at the BBB.

Alternative formulations need to be developed to improve the efficacy of treatments administered via the vaginal route. Mucoadhesive gels with disulfiram, a molecule that was originally approved as an antialcoholism drug, offer an attractive alternative to treat vaginal candidiasis. The aim of the current study was to develop and optimize a mucoadhesive drug delivery system for the local administration of disulfiram. Such formulations were composed of polyethylene glycol and carrageenan to improve the mucoadhesive and mechanical properties and to prolong the residence time in the vaginal cavity. Microdilution susceptibility testing showed that these gels had antifungal activity against Candida albicans, Candida parapsilosis, and Nakaseomyces glabratus. The physicochemical properties of the gels were characterized, and the in vitro release and permeation profiles were investigated with vertical diffusion Franz cells. After quantification, it was determined that the amount of the drug retained in the pig vaginal epithelium was sufficient to treat candidiasis infection. Together, our findings suggest that mucoadhesive disulfiram gels have the potential to be an effective alternative treatment for vaginal candidiasis.

Background The gut microbiota is in continuous interaction with the intestinal mucosa via metabolic, neuro-immunological, and neuroendocrine pathways. Disruption in levels of antimicrobial peptides produced by the enteroendocrine cells, such as catestatin, has been associated with changes in the gut microbiota and imbalance in intestinal homeostasis. However, whether the changes in the gut microbiota have a causational role in intestinal dyshomeostasis has remained elusive. Results To this end, we performed reciprocal fecal microbial transplantation in wild-type mice and mice with a knockout in the catestatin coding region of the chromogranin-A gene (CST-KO mice). Combined microbiota phylogenetic profiling, RNA sequencing, and transmission electron microscopy were employed. Fecal microbiota transplantation from mice deficient in catestatin (CST-KO) to microbiota-depleted wild-type mice induced transcriptional and physiological features characteristic of a distorted colon in the recipient animals, including impairment in tight junctions, as well as an increased collagen area fraction indicating colonic fibrosis. In contrast, fecal microbiota transplantation from wild-type mice to microbiota-depleted CST-KO mice reduced collagen fibrotic area, restored disrupted tight junction morphology, and altered fatty acid metabolism in recipient CST-KO mice. Conclusions This study provides a comprehensive overview of the murine metabolic- and immune-related cellular pathways and processes that are co-mediated by the fecal microbiota transplantation and supports a prominent role for the gut microbiota in the colonic distortion associated with the lack of catestatin in mice. Overall, the data show that the gut microbiota may play a causal role in the development of features of intestinal inflammation and metabolic disorders, known to be associated with altered levels of catestatin and may, thus, provide a tractable target in the treatment and prevention of these disorders.

[11C]metoclopramide PET imaging provides a sensitive and translational tool to explore P-glycoprotein (P-gp) function at the blood-brain barrier (BBB). Patients with neurological diseases are often treated with cytochrome (CYP) modulators which may impact the plasma and brain kinetics of [11C]metoclopramide. The impact of the CYP inducer carbamazepine or the CYP inhibitor ritonavir on the brain and plasma kinetics of [11C]metoclopramide was investigated in rats. Data obtained in a control group were compared with groups that were either orally pretreated with carbamazepine (45 mg/kg twice a day for 7 days before PET) or ritonavir (20 mg/kg, 3 h before PET) (n = 4 per condition). Kinetic modelling was performed to estimate the brain penetration (VT) of [11C]metoclopramide. CYP induction or inhibition had negligible impact on the plasma kinetics and metabolism of [11C]metoclopramide. Moreover, carbamazepine neither impacted the brain kinetics nor VT of [11C]metoclopramide (p > 0.05). However, ritonavir significantly increased VT (p < 0.001), apparently behaving as an inhibitor of P-gp at the BBB. Our data suggest that treatment with potent CYP inducers such as carbamazepine does not bias the estimation of P-gp function at the BBB with [11C]metoclopramide PET. This supports further use of [11C]metoclopramide for studies in animals and patients treated with CYP inducers.

Silver nanoparticles (AgNPs), due to their unique properties have been receiving immense attention in recent years. In addition to their antibacterial and antifungal activities, AgNPs also cause apoptosis, mitochondria disfunction, nucleic acid damage and show potent anticancer properties in both multidrug resistance (MDR) and sensitive tumors. The MDR phenomenon, caused by the presence of ATP-binding cassette (ABC) proteins, is responsible for the failure of chemotherapy. Thus, investigating the influence of widely used AgNPs on ABC transporters is crucial. In the present study, we have examined the cytotoxicity of silver nanoparticles of a nominal size of 20 nm (Ag20) on the cell lines of different tissue origins. In addition, we have checked the ATP-binding cassette transporters’ activity and expression under AgNP exposure. The results indicate that Ag20 shows a toxic effect on tested cells, as well as modulating the expression and transport activity of ABC proteins.

Background The expansion and metastasis of colorectal cancers are closely associated with the dynamic growth of cancer stem cells (CSCs). This study aimed to explore the possible effect of LXR (a regulator of glycolysis and lipid hemostasis) in the tumorgenicity of human colorectal CD133 cells. Methods Human HT-29 CD133+ cells were enriched by MACS and incubated with LXR agonist (T0901317) and antagonist (SR9243) for 72 h. Cell survival was evaluated using MTT assay and flow cytometric analysis of Annexin-V. The proliferation rate was measured by monitoring Ki-67 positive cells using IF imaging. The modulation of LXR was studied by monitoring the activity of all factors related to ABC transporters using real-time PCR assay and western blotting. Protein levels of metabolic enzymes such as PFKFB3, GSK3β, FASN, and SCD were also investigated upon treatment of CSCs with LXR modulators. The migration of CSCs was monitored after being exposed to LXR agonist using scratch and Transwell insert assays. The efflux capacity was measured using hypo-osmotic conditions. The intracellular content of reactive oxygen species was studied by DCFH-DA staining. Results Data showed incubation of CSCs with T0901317 and SR9243 reduced the viability of CD133 cells in a dose-dependent manner compared to the control group. The activation of LXR up-regulated the expression and protein levels of ABC transporters (ABCA1, ABCG5, and ABCG8) compared to the non-treated cells (p Calling attention, both T0901317 and SR9243 compounds induced apoptotic changes in cancer stem cells (p Conclusions The regulation of LXR activity can be considered as a selective targeting of survival, metabolism, and migration in CSCs to control the tumorigenesis and metastasis in patients with advanced colorectal cancers.

Structural data on ABCG5/G8 and ABCG2 reveal a unique molecular architecture for subfamily G ATP-binding cassette (ABCG) transporters and disclose putative substrate-binding sites. ABCG5/G8 and ABCG2 appear to use several unique structural motifs to execute transport, including the triple helical bundles, the membrane-embedded polar relay, the re-entry helices, and a hydrophobic valve. Interestingly, ABCG2 shows extreme substrate promiscuity, whereas ABCG5/G8 transports only sterol molecules. ABCG2 structures suggest a large internal cavity, serving as a binding region for substrates and inhibitors, while mutational and pharmacological analyses support the notion of multiple binding sites. By contrast, ABCG5/G8 shows a collapsed cavity of insufficient size to hold substrates. Indeed, mutational analyses indicate a sterol-binding site at the hydrophobic interface between the transporter and the lipid bilayer. In this review, we highlight key differences and similarities between ABCG2 and ABCG5/G8 structures. We further discuss the relevance of distinct and shared structural features in the context of their physiological functions. Finally, we elaborate on how ABCG2 and ABCG5/G8 could pave the way for studies on other ABCG transporters.

The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.

Identification of genetic markers of a human disease, which is generally sporadic, may become an essential tool for the investigation of its molecular mechanisms. The role of ABCA7 in Alzheimer’s disease (AD) was discovered less than ten years ago when meta-analyses provided evidence that rs3764650 is a new AD susceptibility locus. Recent research advances in this locus and new evidence regarding ABCA7 contribution to the AD pathogenesis brought a new understanding of the underlying mechanisms of this disorder. An interesting, up-to-date review article "ABCA7 and Pathogenic Pathways of Alzheimer’s Disease" by Aikawa et al. (2018), outlines the ABCA7 role in AD and summarizes new findings in this exciting area. ABC transporters or ATP-binding cassette transporters are a superfamily of proteins belonging to a cell transport system. Currently, members of the family are the focus of attention because of their central role in drug pharmacokinetics. Two recent findings are the reason why much attention is drawn to the ABCA7 family. First, is the biochemical data showing a role of ABCA7 in amyloid pathology. Second, genetic data identifying ABCA7 gene variants as loci responsible for the late-onset AD. These results point to the ABCA7 as a significant new contributor to the pathogenesis of AD.

The barriers present in the interfaces between the blood and the central nervous system form a major hurdle for the pharmacological treatment of central nervous system injuries and diseases. The family of ATP-binding cassette (ABC) transporters has been widely studied regarding efflux of medications at blood-central nervous system barriers. These efflux transporters include P-glycoprotein (abcb1), ‘breast cancer resistance protein’ (abcg2) and the various ‘multidrug resistance-associated proteins’ (abccs). Understanding which efflux transporters are present at the blood-spinal cord, blood-cerebrospinal fluid and cerebrospinal fluid-spinal cord barriers is necessary to determine their involvement in limiting drug transfer from blood to the spinal cord tissue. Recent developments in the blood-brain barrier field have shown that barrier systems are dynamic and the profile of barrier defenses can alter due to conditions such as age, disease and environmental challenge. This means that a true understanding of ABC efflux transporter expression and localization should not be one static value but instead a range that represents the complex patient subpopulations that exist. In the present review, the blood-central nervous system barrier literature is discussed with a focus on the impact of ABC efflux transporters on: (i) protecting the spinal cord from adverse effects of systemically directed drugs, and (ii) limiting centrally directed drugs from accessing their active sites within the spinal cord.

Cancer multi-drug resistance (MDR) caused by P-glycoprotein (P-gp) efflux is a critical unresolved clinical concern. The present study analyzed the effect of cinnamophilin on P-gp inhibition and MDR reversion. The effect of cinnamophilin on P-gp was investigated through drug efflux assay, ATPase assay, MDR1 shift assay, and molecular docking. The cancer MDR-reversing ability and mechanisms were analyzed through cytotoxicity and combination index (CI), cell cycle, and apoptosis experiments. P-gp efflux function was significantly inhibited by cinnamophilin without influencing the drug’s expression or conformation. Cinnamophilin uncompetitively inhibited the efflux of doxorubicin and rhodamine 123 and exhibited a distinct binding behavior compared with verapamil, the P-gp standard inhibitor. The half maximal inhibitory concentration of cinnamophilin for doxorubicin and rhodamine 123 efflux was 12.47 and 11.59 μM, respectively. In regard to P-gp energy consumption, verapamil-stimulated ATPase activity was further enhanced by cinnamophilin at concentrations of 0.1, 1, 10, and 20 μM. In terms of MDR reversion, cinnamophilin demonstrated synergistic cytotoxic effects when combined with docetaxel, vincristine, or paclitaxel. The CI was

ATP-binding cassette (ABC) exporters transport substrates across biological membranes using ATP hydrolysis by a process that involves switching between inward- and outward-facing conformations. Most of the structural studies of ABC proteins have been performed with proteins in detergent micelles, locked in specific conformations and/or at low temperature. In this article, we present recent data from our laboratories where we studied the prototypical ABC exporter MsbA during ATP hydrolysis, at 37°C, reconstituted in a lipid bilayer. These studies were possible through the use of luminescence resonance energy transfer spectroscopy in MsbA reconstituted in nanodiscs. We found major differences between MsbA in these native-like conditions and in previous studies. These include a separation between the nucleotide-binding domains that was much smaller than previously thought, and a large fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions.

Plant hormones regulate a myriad of plant processes, from seed germination to reproduction, from complex organ development to microelement uptake. Much has been discovered on the factors regulating the activity of phytohormones, yet there are gaps in knowledge about their metabolism, signaling as well as transport. In this review we analyze the potential of the characterized phytohormonal transporters belonging to the ATP-Binding Cassette family (ABC proteins), thus to identify new candidate orthologs in model plants and species important for human health and food production. Previous attempts with phylogenetic analyses on transporters belonging to the ABC family suggested that sequence homology per se is not a powerful tool for functional characterization. However, we show here that sequence homology might indeed support functional conservation of characterized members of different classes of ABC proteins in several plant species, e.g., in the case of ABC class G transporters of strigolactones and ABC class B transporters of auxinic compounds. Also for the low-affinity, vacuolar abscisic acid (ABA) transporters belonging to the ABCC class we show that localization-, rather than functional-clustering occurs, possibly because of sequence conservation for targeting the tonoplast. The ABC proteins involved in pathogen defense are phylogenetically neighboring despite the different substrate identities, suggesting that sequence conservation might play a role in their activation/induction after pathogen attack. Last but not least, in case of the multiple lipid transporters belong to different ABC classes, we focused on ABC class D proteins, reported to transport/affect the synthesis of hormonal precursors. Based on these results, we propose that phylogenetic approaches followed by transport bioassays and in vivo investigations might accelerate the discovery of new hormonal transport routes and allow the designing of transgenic and genome editing approaches, aimed to improve our knowledge on plant development, plant–microbe symbioses, plant nutrient uptake and plant stress resistance.

Background P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein. Results In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8 Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer. Conclusion The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity. Electronic supplementary material The online version of this article (10.1186/s12900-018-0098-z) contains supplementary material, which is available to authorized users.

Background: Proteins that belong to the ATP-binding cassette superfamily include transporters that mediate the efflux of substrates from cells. Among these exporters, P-glycoprotein and MRP1 are involved in cancer multidrug resistance, protection from endo and xenobiotics, determination of drug pharmacokinetics, and the pathophysiology of a variety of disorders. Objective: To review the information available on ATP-binding cassette exporters, with a focus on Pglycoprotein, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Methods: Evaluation of selected publications on the structure and function of ATP-binding cassette proteins. Conclusions: Conformational changes on the nucleotide-binding domains side of the exporters switch the accessibility of the substrate-binding pocket between the inside and outside, which is coupled to substrate efflux. However, there is no agreement on the magnitude and nature of the changes at the nucleotide- binding domains side that drive the alternate-accessibility. Comparison of the structures of Pglycoprotein and MRP1 helps explain differences in substrate selectivity and the bases for polyspecificity. P-glycoprotein substrates are hydrophobic and/or weak bases, and polyspecificity is explained by a flexible hydrophobic multi-binding site that has a few acidic patches. MRP1 substrates are mostly organic acids, and its polyspecificity is due to a single bipartite binding site that is flexible and displays positive charge. Fil: Arana, Maite Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Altenberg, Guillermo Alejandro. Texas Tech University Health Sciences Center; Estados Unidos

Transport of a wide variety of substrates, including xenobiotics, is one of the main functions attributed to human ATP-binding cassette (ABC) proteins. Overexpression of ABC genes is considered to be an important mechanism facilitating the development of chemoresistance. Relationships between the expression levels of ABC genes in tumor tissues and established clinicopathological features were extensively studied previously. The current study tested our hypothesis that the expression levels of ABC genes in non-neoplastic (control) tissues also provide important information in relation to the relevant tumor progression. Expression levels of all human ABC genes (48 protein coding and one pseudogene), measured by qRT-PCR, were bioinformatically analyzed. The data originated from four independently collected cohorts covering three types of tumors - breast, colorectal and pancreatic carcinomas. ABC gene expression profiles (signatures) in non-neoplastic tissues (matched to tumor samples from three different tumor types) were characteristically clustered into three main types - those with the vast majority of the genes downregulated, upregulated or heterogeneously regulated. The clusters with mostly downregulated and upregulated genes were shown to possess significant relations to good and poor prognostic markers, respectively, in pancreatic and colorectal cancers. The present findings support the theory that the expression of ABC genes in non-neoplastic tissues can significantly contribute to tumor pathogenesis. Suggested multi-gene panels, consisting of the reduced number of ABC genes, have the potential to be implemented as new prognostic markers, which are especially urgent in pancreatic cancer. The results can also stimulate further primary research in carcinogenesis.

Graphical abstract, Highlights • Ligand recognition by antibacterial Siderocalin controls the competition for iron during infection. • We determined nine crystal structures of Siderocalin mutants with ligands. • We determined three candidate ligands did not bind. • We determined the crystal structure of SBP YfiY. • Multiplexed specificity of Siderocalin was determined., Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration – including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism.

ATP-binding cassette (ABC) transporters are involved in a large number of processes and contribute to various human genetic diseases. Among other functions, ABC proteins are involved in the transport of multiple drugs through cells. Most of the genes coding for these transporters are highly polymorphic and DNA variants in these genes can affect the normal functioning of these proteins, affecting the way drugs are transported, increasing or decreasing drug levels. These changes in the intracellular and extracellular drug levels may be associated with altered drug effectiveness or severe drug-induced adverse events. This review presents a state-of-art of the most pharmacogenetics clinically relevant ABC transporters closed to the clinical implementation.

Multi Drug Resistance (MDR) is one of the major causes of chemotherapy failure in human malignancies. Curcumin, the active constituent of Curcuma longa is a proven anticancer agent potentially modulating the expression and function of these MDR proteins. In this study, we attempted to test curcumin for its potential to inhibit the expression and function of multidrug resistance associated protein 1 (MRP1) in retinoblastoma (RB) cell lines through western blot, RT-PCR and functional assays. In silico analysis were also performed to understand the molecular interactions conferred by curucmin on MRP1 in RB cells. Western blot and RTPCR analysis did not show any correlation of MRP1 expression with increase in concentration of curcumin. However, inhibitory effect of curcumin on MRP1 function was observed as a decrease in the efflux of fluorescent substrate. Moreover, Curcumin did not affect 8-azido-ATP-biotin binding to MRP1 and it also showed inhibition of ATP-hydrolysis stimulated by quercetin, which is indicative of curcumin's interaction with the substrate binding site of MRP1. Furthermore, homology modelling and docking simulation studies of MRP1 also provided deeper insights into the molecular interactions, thereby inferring the potential binding mode of curcumin into the substrate binding site of MRP1.

Resistance to cytotoxic drugs is a significant problem of systemic treatment of cancers. Apart from drug inactivation, changes in target enzymes and proteins, increased DNA repair and suppression of apoptosis, an important mechanism of resistance is an active drug efflux from cancer cells. Drug efflux across the cell membrane is caused by transport proteins such as ABC proteins (ATP-binding cassette). This review focuses on the ABCC protein subfamily, whose members are responsible for multidrug cross-resistance of cancer cells to cytotoxic agents. The authors discuss the structure of ABCC proteins, their physiological function and diseases provoked by mutations of respective genes, their expression in many different malignancies and its connection with resistance to anticancer drugs, as well as methods of reversion of such resistance.

Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.

Vertebrate evolution has been largely driven by the duplication of genes that allow for the acquisition of new functions. The ATP-binding cassette (ABC) proteins constitute a large and functionally diverse family of membrane transporters. The members of this multigene family are found in all cellular organisms, most often engaged in the translocation of a wide variety of substrates across lipid membranes. Because of the diverse function of these genes, their large size, and the large number of orthologs, ABC genes represent an excellent tool to study gene family evolution. We have identified ABC proteins from the sea squirt (Ciona intestinalis), zebrafish (Danio rerio), and chicken (Gallus gallus) and, using phylogenetic analysis, identified those genes with a one-to-one orthologous relationship to human ABC proteins. All ABC protein subfamilies found in Ciona and zebrafish correspond to the human subfamilies, with the exception of a single ABCH subfamily gene found only in zebrafish. Multiple gene duplication and deletion events were identified in different lineages, indicating an ongoing process of gene evolution. As many ABC genes are involved in human genetic diseases, and important drug transport phenotypes, the understanding of ABC gene evolution is important to the development of animal models and functional studies.

We present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The accessory domains include the substrate-binding proteins, that function as high affinity receptors in ABC type uptake systems, and regulatory or catalytic domains that can be fused to either the TMDs or NBDs. The regulatory domains add unique functions to the transporters allowing the systems to act as channel conductance regulators, osmosensors regulators, and assemble into macromolecular complexes with specific properties. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

We have studied the role of five ABC transporter genes ( MgAtr to MgAtr5) from the wheat pathogen Mycosphaerella graminicola in multidrug resistance (MDR). Complementation of Saccharomyces cerevisiae mutants with the ABC transporter genes from M. graminicola showed that all the genes tested encode proteins that provide protection against chemically unrelated compounds, indicating that their products function as multidrug transporters with distinct but overlapping substrate specificities. Their substrate range in yeast includes fungicides, plant metabolites, antibiotics, and a mycotoxin derived from Fusarium graminearum (diacetoxyscirpenol). Transformants of M. graminicola in which individual ABC transporter genes were deleted or disrupted did not exhibit clear-cut phenotypes, probably due to the functional redundancy of transporters with overlapping substrate specificity. Independently generated MgAtr5 deletion mutants of M. graminicola showed an increase in sensitivity to the putative wheat defence compound resorcinol and to the grape phytoalexin resveratrol, suggesting a role for this transporter in protecting the fungus against plant defence compounds. Bioassays with antagonistic bacteria indicated that MgAtr2 provides protection against metabolites produced by Pseudomonas fluorescens and Burkholderia cepacia. In summary, our results show that ABC transporters from M. graminicola play a role in protection against toxic compounds of natural and artificial origin.

Mutations in the ATP-binding cassette transporter 1 (ABCA1) gene have been recently identified as the molecular defect in Tangier disease (TD) and familial high density lipoprotein deficiency (FHA). We here report novel mutations in the ABCA1 gene in two sisters from a Japanese family with TD who have been described previously (S. Ohtaki, H. Nakagawa, N. Kida, H. Nakamura, K. Tsuda, S. Yokoyama, T. Yamamura, S. Tajima, A. Yamamoto, Atherosclerosis 49 (1983)) and a family with FHA. Both probands of TD and FHA developed coronary heart disease. Sequence analysis of the ABCA1 gene from the patients with TD revealed a homozygous G to A transition at nucleotide 3805 of the cDNA resulting in the substitution of Asp 1229 with Asn in exon 27, and a C to T at nucleotide 6181 resulting in the substitution of Arg 2021 with Trp in exon 47. Sequence analysis of the ABCA1 gene from the FHA patient revealed a homozygous 4 bp CGCC deletion from nucleotide 3787 to 3790 resulting in premature termination by frameshift at codon 1224. These mutations were confirmed by restriction digestion analysis, and were not found in 141 control subjects. Our findings indicate that mutations in the ABCA1 gene are associated with TD as well as FHA.

Tangier disease (TD) is an autosomal co-dominant disorder in which homozygotes have a marked deficiency of high density lipoprotein (HDL) cholesterol and, in some cases, peripheral neuropathy and premature coronary heart disease (CHD). Homozygotes are further characterized by cholesteryl ester deposition in various tissues throughout the body, most notably in those of the reticuloendothelial system. Several studies have demonstrated that the excess lipid deposition in TD is due to defective apolipoprotein-mediated efflux of cellular cholesterol and phospholipids. Although much progress has been made in our understanding of the metabolic basis of TD, the precise molecular defect had remained elusive until very recently. By positional cloning methods, we: 1) confirm the assignment of TD to chromosome 9q31, 2) provide evidence that human ATP-binding cassette-1 (hABC-1) maps to a 250 kb region on 9q31, and 3) describe novel deletion, insertion, and missense mutations in the gene encoding hABC-1 in four unrelated TD kindreds. These results establish a causal role for mutations in hABC-1 in TD and indicate that this transporter has a critical function in the regulation of intracellular lipid trafficking that dramatically affects plasma HDL cholesterol levels. —Brousseau, M. E., E. J. Schaefer, J. Dupuis, B. Eustace, P. Van Eerdewegh, A. L. Goldkamp, L. M. Thurston, M. G. FitzGerald, D. Yasek-McKenna, G. O'Neill, G. P. Eberhart, B. Weiffenbach, J. M. Ordovas, M. W. Freeman, R. H. Brown, Jr., and J. Z. Gu. Novel mutations in the gene encoding ATP-binding cassette 1 in four Tangier disease kindreds. J. Lipid Res. 2000. 41: 433–441.

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.

Micromolar concentrations of tolbutamide will inhibit (SUR1/K(IR)6. 2)(4) channels in pancreatic beta-cells, but not (SUR2A/K(IR)6.2)(4) channels in cardiomyocytes. Inhibition does not require Mg(2+) or nucleotides and is enhanced by intracellular nucleotides. Using chimeras between SUR1 and SUR2A, we show that transmembrane domains 12-17 (TMD12-17) are required for high-affinity tolbutamide inhibition of K(ATP) channels. Deletions demonstrate involvement of the cytoplasmic N-terminus of K(IR)6.2 in coupling sulfonylurea-binding with SUR1 to the stabilization of an interburst closed configuration of the channel. The increased efficacy of tolbutamide by nucleotides results from an impairment of their stimulatory action on SUR1 which unmasks their inhibitory effects. The mechanism of inhibition of beta-cell K(ATP) channels by sulfonylureas during treatment of non-insulin-dependent diabetes mellitus thus involves two components, drug-binding and conformational changes within SUR1 which are coupled to the pore subunit through its N-terminus and the disruption of nucleotide-dependent stimulatory effects of the regulatory subunit on the pore. These findings uncover a molecular basis for an inhibitory influence of SUR1, an ATP-binding cassette (ABC) protein, on K(IR)6.2, a ion channel subunit.

Pathogenic bacteria acquire transition metals for cell viability and persistence of infection in competition with host nutritional defenses. The human host employs a variety of mechanisms to stress the invading pathogen with both cytotoxic metal ions and oxidative and nitrosative insults while withholding essential transition metals from the bacterium. For example, the S100 family protein calprotectin (CP) found in neutrophils is a calcium-activated chelator of extracellular Mn and Zn and is found in tissue abscesses at sites of infection by Staphylococcus aureus. In an adaptive response, bacteria have evolved systems to acquire the metals in the face of this competition while effluxing excess or toxic metals to maintain a bioavailability of transition metals that is consistent with a particular inorganic fingerprint under the prevailing conditions. This review highlights recent biological, chemical and structural studies focused on manganese (Mn) acquisition and homeostasis and connects this process to oxidative stress resistance and iron (Fe) availability that operates at the human host-pathogen interface.

Background The ATP-Binding Cassette (ABC) functional superfamily includes integral transmembrane exporters that have evolved three times independently, forming three families termed ABC1, ABC2 and ABC3, upon which monophyletic ATPases have been superimposed for energy-coupling purposes [e.g., J Membr Biol 231(1):1-10, 2009]. The goal of the work reported in this communication was to understand how the integral membrane constituents of ABC uptake transporters with different numbers of predicted or established transmembrane segments (TMSs) evolved. In a few cases, high resolution 3-dimensional structures were available, and in these cases, their structures plus primary sequence analyses allowed us to predict evolutionary pathways of origin. Results All of the 35 currently recognized families of ABC uptake proteins except for one (family 21) were shown to be homologous using quantitative statistical methods. These methods involved using established programs that compare native protein sequences with each other, after having compared each sequence with thousands of its own shuffled sequences, to gain evidence for homology. Topological analyses suggested that these porters contain numbers of TMSs ranging from four or five to twenty. Intragenic duplication events occurred multiple times during the evolution of these porters. They originated from a simple primordial protein containing 3 TMSs which duplicated to 6 TMSs, and then produced porters of the various topologies via insertions, deletions and further duplications. Except for family 21 which proved to be related to ABC1 exporters, they are all related to members of the previously identified ABC2 exporter family. Duplications that occurred in addition to the primordial 3 → 6 duplication included 5 → 10, 6 → 12 and 10 → 20 TMSs. In one case, protein topologies were uncertain as different programs gave discrepant predictions. It could not be concluded with certainty whether a 4 TMS ancestral protein or a 5 TMS ancestral protein duplicated to give an 8 or a 10 TMS protein. Evidence is presented suggesting but not proving that the 2TMS repeat unit in ABC1 porters derived from the two central TMSs of ABC2 porters. These results provide structural information and plausible evolutionary pathways for the appearance of most integral membrane constituents of ABC uptake transport systems. Conclusions Almost all integral membrane uptake porters of the ABC superfamily belong to the ABC2 family, previously established for exporters. Most of these proteins can have 5, 6, 10, 12 or 20 TMSs per polypeptide chain. Evolutionary pathways for their appearance are proposed.

The requirement to cross a biological membrane can be a complex process especially if multidrug transporters such as P-gp must be considered. Drug partitioning into the lipid membrane and efflux by P-gp are tightly coupled processes wherein H-bonding interactions play a key role. All H-bond donors and acceptors are not equal in terms of the strength of the H-bonds that they form, hence it is important to consider their relative strength. Using various examples from literature, we illustrate the benefits of considering the relative strengths of individual H-bonds and introducing intramolecular H-bonds to increase membrane permeability and/or decrease P-gp efflux.

The ABC transporter-encoding gene MgAtr7 from the wheat pathogen Mycosphaerella graminicola was cloned based upon its high homology to ABC transporters involved in azole-fungicide sensitivity. Genomic and cDNA sequences indicated that the N-terminus of this ABC transporter contains a motif characteristic for a dityrosine/pyoverdine biosynthesis protein. This makes MgAtr7 the first member of a new class of fungal ABC transporters harboring both a transporter and a biosynthetic moiety. A homologue of MgAtr7 containing the same biosynthetic moiety was only found in the Fusarium graminearum genome and not in any other fungal genome examined so far. The gene structure of both orthologous transporters is highly conserved and the genomic area surrounding the ABC transporter exhibits micro-synteny between M. graminicola and F. graminearum. Functional analyses revealed that MgAtr7 is neither required for virulence nor involved in fungicide sensitivity but indicated a role in maintenance of iron homeostasis.

ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.ATP-binding cassette (ABC) transporters have often been refractory to over-expression. Using the C41(DE3) E. coli as a host strain, membrane vesicles highly enriched (>50%) in YvcC, a previously uncharacterized ABC transporter from Bacillus subtilis homologous to P-glycoprotein multidrug transporters, were obtained. The functionality of YvcC was assessed by its high vanadate-sensitive ATPase activity and its ability to transport a fluorescent drug, the Hoechst 33342.

Mammalian physiological processes can distinguish between dietary cholesterol and non-cholesterol, retaining very little of the non-cholesterol in their bodies. We have recently identified two genes, ABCG5 and ABCG8, encoding sterolin-1 and -2 respectively, mutations of which cause the human disease sitosterolemia. We report here the mouse cDNAs and genomic organization of Abcg5 and Abcg8. Both genes are arranged in an unusual head-to-head configuration, and only 140 bases separate their two respective start-transcription sites. A single TATA motif was identified, with no canonical CCAT box present between the two genes. The genes are located on mouse chromosome 17 and this complex spans no more than 40 kb. Expression of both genes is confined to the liver and intestine. For both genes, two different sizes of transcripts were identified which differ in the lengths of their 3′ UTRs. Additionally, alternatively spliced forms for Abcg8 were identified, resulting from a CAG repeat at the intron 1 splice-acceptor site, causing a deletion of a glutamine. We screened 20 different mouse strains for polymorphic variants. Although a large number of polymorphic variants were identified, strains reported to show significant differences in cholesterol absorption rates did not show significant genomic variations in Abcg5 or Abcg8.—Lu, K., M-H. Lee, H. Yu, Y. Zhou, S. A. Sandell, G. Salen, and S. B. Patel. Molecular cloning, genomic organization, genetic variations, and characterization of murine sterolin genes Abcg5 and Abcg8.

A cDNA encoding a putative ATP-binding cassette (ABC) transporter from Arabidopsis was cloned and sequenced based on an EST clone homologous to ABC sequences in other species. The cDNA is 5.5 kb long and contains an ORF encoding a 1623 amino acids protein. This sequence is the first MRP-like protein found in plants.

BACKGROUND: Protein distribution profiles along the human intestinal tract of transporters involved in the absorption of cholesterol and long-chain fatty acids (LCFA) have been scarcely evaluated. METHODOLOGY/PRINCIPAL FINDINGS: In post-mortem samples from 11 subjects, intestinal transporter distribution profiles were determined via Western Blot. Differences in transporter protein levels were statistically tested using ANOVA and Tukey's Post Hoc comparisons. Levels in all segments were expressed relative to those in duodenum. Except for ABCG5 and FATP4, levels (mean+/-SEM) were the highest in the ileum. For ABCA1, ileal levels (1.80+/-0.26) differed significantly from those in duodenum (P = 0.049) and proximal colon (0.92+/-0.14; P = 0.029). ABCG8 levels in ileum (1.91+/-0.30) differed from those in duodenum (P = 0.041) and distal colon (0.84+/-0.22; P = 0.010) and jejunum (1.64+/-0.26) tended to be higher than distal colon (0.84+/-0.22; P = 0.087). Ileal NPC1L1 levels (2.56+/-0.51) differed from duodenum levels (P = 0.019) and from distal colon (1.09+/-0.22; P = 0.030). There was also a trend (P = 0.098) for higher jejunal (2.23+/-0.37) than duodenal NPC1L1 levels. The levels of ABCG5 did not correlate with those of ABCG8. FAT/CD36 levels in ileum (2.03+/-0.42) differed from those in duodenum (P = 0.017), and proximal and distal colon (0.89+/-0.13 and 0.97+/-0.15 respectively; P = 0.011 and P = 0.014). FABPpm levels in ileum (1.04+/-0.13) differed from proximal (0.64+/-0.07; P = 0.026) and distal colon (0.66+/-0.09; P = 0.037). CONCLUSIONS/SIGNIFICANCE: The distribution profiles showed a bell-shape pattern along the GI-tract with the highest levels in ileum for ABCA1, ABCG8, NPC1L1, FATCD36 and FABPm, suggesting a prominent role for ileum in transporter-mediated uptake of cholesterol and LCFAs.

Overexpression of P-glycoprotein (PGP), a multidrug-related (MDR) protein, is one of the most important factors responsible for reduced drug sensitivity in acute myeloid leukemia (AML). Recently, we demonstrated that the presence of CD56 antigen, an isoform of the neural adhesion molecule, in AML cells is a negative independent prognostic factor for the achievement of complete remission (CR) and correlates with shorter survival. Since in our previous report we observed a more frequent PGP expression in CD56+ patients, we hypothesized that the reduced response to chemotherapy in this group of patients was due to increased PGP-mediated drug efflux. To confirm this hypothesis in this study PGP and CD56 expression on AML cells was correlated with other clinical and biological features and treatment response.Immunophenotypic analysis, including evaluation of CD56 and PGP expression, was performed using multiparameter flow cytometry on fresh and/or cryopreserved blast cells, obtained after informed consent, from bone marrow and/or peripheral blood of 143 consecutive newly diagnosed AML cases at the time of diagnosis. Samples expressing CD56 in at least 15% or more cells were considered as positive (CD56+). PGP expression was expressed as a mean fluorescence index (MFI) i.e. as the ratio of sample mean fluorescence channel and the isotypic control mean fluorescence channel.Overall results showed that 67/143 cases were PGP-/CD56-, 23/143 were PGP+ /CD56+, 40/143 were PGP+/CD56- and the remaining 13/143 were PGP-/CD56+. CD56+ and PGP+ on AML cells significantly reduced the CR rate (83% in the PGP-/CD56- group vs 60% in the PGP-/CD56+ group, 46% in the PGP+/CD56- group and 58% in the PGP+/CD56+ group, p = 0.002). In addition we observed a significantly higher proportion of total failures in patients expressing PGP or CD56 compared to in the group not expressing either (73% vs 27%, respectively; p = 0.0001). CD56 and PGP overexpression influenced the overall survival: in fact, the median survival of CD56+ and PGP+ patients ranged from 10 to 23 months, while the actuarial survival of CD56-/PGP- patients at 5 years is 52% (p = 0.023).Our data underline the independent negative prognostic role of PGP and CD56 expression in acute myeloid leukemia. Since the mechanism by which CD56 reduces drug sensitivity is still unknown, further investigations are required.