Structure and Mechanism of Human ABC Transporters.

ABC transporters are essential for cellular physiology. Humans have 48 ABC genes organized into seven distinct families. Of these genes, 44 (in five distinct families) encode for membrane transporters, of which several are involved in drug resistance and disease pathways resulting from transporter dysfunction. Over the last decade, advances in structural biology have vastly expanded our mechanistic understanding of human ABC transporter function, revealing details of their molecular arrangement, regulation, and interactions, facilitated in large part by advances in cryo-EM that have rendered hitherto inaccessible targets amenable to high-resolution structural analysis. As a result, experimentally determined structures of multiple members of each of the five families of ABC transporters in humans are now available. Here we review this recent progress, highlighting the physiological relevance of human ABC transporters and mechanistic insights gleaned from their direct structure determination. We also discuss the impact and limitations of model systems and structure prediction methods in understanding human ABC transporters and discuss current challenges and future research directions.

Comparative Hepatic and Intestinal Efflux Transport of Statins.

Previous studies have shown that lipid-lowering statins are transported by various ATP-binding cassette (ABC) transporters. However, because of varying methods, it is difficult to compare the transport profiles of statins. Therefore, we investigated the transport of 10 statins or statin metabolites by six ABC transporters using human embryonic kidney cell-derived membrane vesicles. The transporter protein expression levels in the vesicles were quantified with liquid chromatography-tandem mass spectrometry and used to scale the measured clearances to tissue levels. In our study, apically expressed breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) transported atorvastatin, fluvastatin, pitavastatin, and rosuvastatin. Multidrug resistance-associated protein 3 (MRP3) transported atorvastatin, fluvastatin, pitavastatin, and, to a smaller extent, pravastatin. MRP4 transported fluvastatin and rosuvastatin. The scaled clearances suggest that BCRP contributes to 87%-91% and 84% of the total active efflux of rosuvastatin in the small intestine and the liver, respectively. For atorvastatin, the corresponding values for P-gp-mediated efflux were 43%-79% and 66%, respectively. MRP3, on the other hand, may contribute to 23%-26% and 25%-37% of total active efflux of atorvastatin, fluvastatin, and pitavastatin in jejunal enterocytes and liver hepatocytes, respectively. These data indicate that BCRP may play an important role in limiting the intestinal absorption and facilitating the biliary excretion of rosuvastatin and that P-gp may restrict the intestinal absorption and mediate the biliary excretion of atorvastatin. Moreover, the basolateral MRP3 may enhance the intestinal absorption and sinusoidal hepatic efflux of several statins. Taken together, the data show that statins differ considerably in their efflux transport profiles. SIGNIFICANCE STATEMENT: This study characterized and compared the transport of atorvastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin acid and four atorvastatin metabolites by six ABC transporters (BCRP, MRP2, MRP3, MRP4, MRP8, P-gp). Based on in vitro findings and protein abundance data, the study concludes that BCRP, MRP3, and P-gp have a major impact in the efflux of various statins. Together with in vitro metabolism, uptake transport, and clinical data, our findings are applicable for use in comparative systems pharmacology modeling of statins.

Pleiotropic Roles of ABC Transporters in Breast Cancer.

Chemotherapeutics are the mainstay treatment for metastatic breast cancers. However, the chemotherapeutic failure caused by multidrug resistance (MDR) remains a pivotal obstacle to effective chemotherapies of breast cancer. Although in vitro evidence suggests that the overexpression of ATP-Binding Cassette (ABC) transporters confers resistance to cytotoxic and molecularly targeted chemotherapies by reducing the intracellular accumulation of active moieties, the clinical trials that target ABCB1 to reverse drug resistance have been disappointing. Nevertheless, studies indicate that ABC transporters may contribute to breast cancer development and metastasis independent of their efflux function. A broader and more clarified understanding of the functions and roles of ABC transporters in breast cancer biology will potentially contribute to stratifying patients for precision regimens and promote the development of novel therapies. Herein, we summarise the current knowledge relating to the mechanisms, functions and regulations of ABC transporters, with a focus on the roles of ABC transporters in breast cancer chemoresistance, progression and metastasis.

The role of the degenerate nucleotide binding site in type I ABC exporters.

ATP-binding cassette (ABC) transporters are fascinating molecular machines that are capable of transporting a large variety of chemically diverse compounds. The energy required for translocation is derived from binding and hydrolysis of ATP. All ABC transporters share a basic architecture and are composed of two transmembrane domains and two nucleotide binding domains (NBDs). The latter harbor all conserved sequence motifs that hallmark the ABC transporter superfamily. The NBDs form the nucleotide binding sites (NBSs) in their interface. Transporters with two active NBSs are called canonical transporters, while ABC exporters from eukaryotic organisms, including humans, frequently have a degenerate NBS1 containing noncanonical residues that strongly impair ATP hydrolysis. Here, we summarize current knowledge on degenerate ABC transporters. By integrating structural information with biophysical and biochemical evidence of asymmetric function, we develop a model for the transport cycle of degenerate ABC transporters. We will elaborate on the unclear functional advantages of a degenerate NBS.

A twist in the ABC: regulation of ABC transporter trafficking and transport by FK506-binding proteins.

Post-transcriptional regulation of ATP-binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506-binding proteins (FKBPs), a type of peptidylprolyl cis-trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP-ABC modules in plants and mammals. We dissect a PPIase- and HSP90-dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis-trans isomerization of X-prolyl bonds.

Comprehensive classification of ABC ATPases and their functional radiation in nucleoprotein dynamics and biological conflict systems.

ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time we provide a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. We present a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism.

Structural and Mechanistic Principles of ABC Transporters.

ATP-binding cassette (ABC) transporters constitute one of the largest and most ancient protein superfamilies found in all living organisms. They function as molecular machines by coupling ATP binding, hydrolysis, and phosphate release to translocation of diverse substrates across membranes. The substrates range from vitamins, steroids, lipids, and ions to peptides, proteins, polysaccharides, and xenobiotics. ABC transporters undergo substantial conformational changes during substrate translocation. A comprehensive understanding of their inner workings thus requires linking these structural rearrangements to the different functional state transitions. Recent advances in single-particle cryogenic electron microscopy have not only delivered crucial information on the architecture of several medically relevant ABC transporters and their supramolecular assemblies, including the ATP-sensitive potassium channel and the peptide-loading complex, but also made it possible to explore the entire conformational space of these nanomachines under turnover conditions and thereby gain detailed mechanistic insights into their mode of action.

ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance: Structural and Functional Diversification across the Tree of Life.

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.

Towards Identification of the Substrates of ATP-Binding Cassette Transporters.

Most ATP-binding cassette (ABC) proteins function in transmembrane transport, and plant genomes encode a large number of ABC transporters compared with animal or fungal genomes. These transporters have been classified into eight subfamilies according to their topology and phylogenetic relationships. Transgenic plants and mutants with altered ABC transporter expression or function have contributed to deciphering the physiological roles of these proteins, such as in plant development, responses to biotic and abiotic stress, or detoxification activities within the cell. In agreement with the diversity of these functions, a large range of substrates (e.g. hormones and primary and secondary metabolites) have been identified. We review in detail transporters for which substrates have been unambiguously identified. However, some cases are far from clear, because some ABC transporters have the ability to transport several structurally unrelated substrates or because the identification of their substrates was performed indirectly without any flux measurement. Various heterologous or homologous expression systems have been used to better characterize the transport activity and other biochemical properties of ABC transporters, opening the way to addressing new issues such as the particular structural features of plant ABC transporters, the bidirectionality of transport, or the role of posttranslational modifications.

Cross-modality deep learning-based prediction of TAP binding and naturally processed peptide.

Epitopes presented on MHC class I molecules pass multiple processing stages before their presentation on MHC molecules, the main ones being proteasomal cleavage and TAP binding. Transporter associated with antigen processing (TAP) binding is a necessary stage for most, but not all, MHC-I-binding peptides. The molecular determinants of TAP-binding peptides can be experimentally estimated from binding experiments and from the properties of peptides inducing a CD8 T cell response. We here propose novel optimization formalisms to combine binding and activation experimental results to produce a classifier for TAP binding using dual-output kernel and deep learning approaches. The application of these algorithms to the human and murine TAP binding leads to predictors that are much more precise than current state of the art methods. Moreover, the computed score is highly correlated with the observed binding energy. The new predictors show that TAP binding may be much more selective than previously assumed in humans and mice and sensitive to the properties of most positions of the peptides. Beyond the improved precision for TAP binding, we propose that the same approach holds in most molecular binding problems, where functional and binding measures are simultaneously available, and can be used to significantly improve the precision of binding prediction algorithms in general and immune system molecules specifically.

Mechanistic diversity in ATP-binding cassette (ABC) transporters.

ABC transporters catalyze transport reactions, such as the high-affinity uptake of micronutrients into bacteria and the export of cytotoxic compounds from mammalian cells. Crystal structures of ABC domains and full transporters have provided a framework for formulating reaction mechanisms of ATP-driven substrate transport, but recent studies have suggested remarkable mechanistic diversity within this protein family. This review evaluates the differing mechanistic proposals and outlines future directions for the exploration of ABC-transporter-catalyzed reactions.

Structural basis for the mechanism of ABC transporters.

The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

Structure based investigation on the binding interaction of transport proteins in leishmaniasis: insights from molecular simulation.

Leishmania major is the causative agent of cutaneous leishmaniasis which affects over 1 million people in 88 different countries. The incidence of this disease is on the rise due to the current problems associated with the present chemotherapeutics. In addition, Leishmania confronts resistance to the traditional drugs like sodium stibogluconate and newer repurposed drugs like miltefosine. ABC transporters are involved in the development of drug resistance. Miltefosine, the drug used for the treatment of leishmaniasis, is effluxed by P4 ATPase and ABC transporter, which is the prime focus of our study in this paper. P4 ATPase (MDR1) along with an unnamed protein (cdc50) translocates miltefosine from the outer to the inner leaflet by the process of flipping which is ATP driven. In contrast, miltefosine also escapes from the cells by an energy dependent mechanism that involves the ABC transporter protein (ABC). It is known that certain genes in the parasite amplify the portions of a gene which encodes ABC transporter and P4 ATPase involved in translocating phospholipids and hence resistance to miltefosine. We observed the ABC and P4 ATPase genes, 39 T-box elements were observed in the ABC transporter protein and three elements were observed in the P4 ATPase gene suggesting its role in transcription regulation. To the best of our knowledge, there are no structural and regulatory reports on these two proteins in L. major. Computational structural biology tools may aid in understanding the interaction of miltefosine with the P4-ATPase-cdc50 complex and the ABC transporter. This can be achieved by modeling the target protein structures, studying the dynamics associated with the different domains of the protein and later using activators and inhibitors to alter the functioning of the protein. Molecular dynamics simulation with a lipid bilayer is performed to investigate the conformational changes and structure-activity relationship. As transporters are difficult to model, the relevant structural motifs and domains may help to understand the allosteric relation with the substrate and the cofactors. The dynamics of a protein molecule ultimately defines the functional mechanism involving excursions of multiple conformational states. To understand these functional mechanisms of transporter proteins, computational modeling and simulations will be carried out with the goal of elucidating the atomistic details of allosteric conformational transitions and propagations during the transport processes. In particular these studies are designed to investigate the critical structural and dynamic elements that determine individual and combined ligand-binding specificities, the interactions among transporters, their coupled-proteins and the associations of transporters within the lipid bilayer. The nature of results from such studies also makes it possible to rationally optimize existing ligands for these proteins and develop some new compounds that can shift the conformational equilibrium of transporters which may aid in functional studies leading to drug discovery.

Phylogenetic analysis and DNA-based species confirmation in Anopheles (Nyssorhynchus).

Specimens of neotropical Anopheles (Nyssorhynchus) were collected and identified morphologically. We amplified three genes for phylogenetic analysis-the single copy nuclear white and CAD genes, and the COI barcode region. Since we had multiple specimens for most species we were able to test how well the single or combined genes were able to corroborate morphologically defined species by placing the species into exclusive groups. We found that single genes, including the COI barcode region, were poor at confirming species, but that the three genes combined were able to do so much better. This has implications for species identification, species delimitation, and species discovery, and we caution that single genes are not enough. Higher level groupings were partially resolved with some well-supported groupings, whereas others were found to be either polyphyletic or paraphyletic. There were examples of known groups, such as the Myzorhynchella Section, which were poorly supported with single genes but were well supported with combined genes. From this we can infer that more sequence data will be needed in order to show more higher-level groupings with good support. We got unambiguously good support (0.94-1.0 Bayesian posterior probability) from all DNA-based analyses for a grouping of An. dunhami with An. nuneztovari and An. goeldii, and because of this and because of morphological similarities we propose that An. dunhami be included in the Nuneztovari Complex. We obtained phylogenetic corroboration for new species which had been recognised by morphological differences; these will need to be formally described and named.

Use of shape similarities for the classification of P-glycoprotein substrates and nonsubstrates.

BACKGROUND: The multidrug transporter P-glycoprotein (P-gp) ATP-binding cassette B1 (ABCB1) is one of the key proteins influencing bioavailability and uptake of drugs in the brain. In addition, it is one of the main factors contributing to multidrug resistance in tumor therapy. Due to its promiscuous substrate recognition, prediction of substrate properties for the multidrug transporter P-gp represents a challenging task. RESULTS: Here, we present data on three classification methods of ABCB1 substrates and nonsubstrates based on 2D and 3D shape similarity calculations with special emphasis on the use of the similarity-based relationship approach. The results indicate that a reference set structurally similar to the data set performs superiorly to those selected on the basis of maximum diversity and suggests Random Forest as the most suitable classification method for this data set. CONCLUSION: This study suggests 2D descriptors representing 3D features best suited to the classification of P-gp substrates and nonsubstrates.

ABC proteins protect the human body and maintain optimal health.

Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

ABC multidrug transporters: target for modulation of drug pharmacokinetics and drug-drug interactions.

Nine proteins of the ABC superfamily (P-glycoprotein, 7 MRPs and BCRP) are involved in multidrug transport. Being localised at the surface of endothelial or epithelial cells, they expel drugs back to the external medium (if located at the apical side [P-glycoprotein, BCRP, MRP2, MRP4 in the kidney]) or to the blood (if located at the basolateral side [MRP1, MRP3, MRP4, MRP5]), modulating thereby their absorption, distribution, and elimination. In the CNS, most transporters are oriented to expel drugs to the blood. Transporters also cooperate with Phase I/Phase II metabolism enzymes by eliminating drug metabolites. Their major features are (i) their capacity to recognize drugs belonging to unrelated pharmacological classes, and (ii) their redundancy, a single molecule being possibly substrate for different transporters. This ensures an efficient protection of the body against invasion by xenobiotics. Competition for transport is now characterized as a mechanism of interaction between co-administered drugs, one molecule limiting the transport of the other, potentially affecting bioavailability, distribution, and/or elimination. Again, this mechanism reinforces drug interactions mediated by cytochrome P450 inhibition, as many substrates of P-glycoprotein and CYP3A4 are common. Induction of the expression of genes coding for MDR transporters is another mechanism of drug interaction, which could affect all drug substrates of the up-regulated transporter. Overexpression of MDR transporters confers resistance to anticancer agents and other therapies. All together, these data justify why studying drug active transport should be part of the evaluation of new drugs, as recently recommended by the FDA.

Phenotype prediction of non-synonymous single-nucleotide polymorphisms in human ATP-binding cassette transporter genes.

A large number of non-synonymous single-nucleotide polymorphisms (nsSNPs) have been found in human genome, but there is poor knowledge on the relationship between the genotype and phenotype of these nsSNPs. Human ATP-binding cassette (ABC) transporters are able to transport a number of important substrates including endogenous and exogenous compounds. This study aimed to predict the phenotypical impact of nsSNPs of human ABC transporter genes, and the predicted results were further validated by reported phenotypical data from site-directed mutagenesis and clinical genetic studies. One thousand and six hundred thirty-two nsSNPs were found from 49 human ABC transporter genes. Using the PolyPhen and SIFT algorithms, 41.8-53.6% of nsSNPs in ABC transporter genes were predicted to have an impact on protein function. The prediction accuracy was up to 63-85% when compared with known phenotypical data from in vivo and in vitro studies. There was a significant concordance between the prediction results using SIFT and PolyPhen. Of nsSNPs predicted as deleterious, the prediction scores by SIFT and PolyPhen were significantly related to the number of nsSNPs with known phenotypes confirmed by experimental and human studies. The amino acid substitution variants are supposed to be the pathogenetic basis of increased susceptibility to certain diseases with Mendelian or complex inheritance, altered drug resistance and altered drug clearance and response. Predicting the phenotypic consequence of nsSNPs using computational algorithms may provide a better understanding of genetic differences in susceptibility to diseases and drug response. The prediction of nsSNPs in human ABC transporter genes would be useful hints for further genotype-phenotype studies.

Membrane porters of ATP-binding cassette transport systems are polyphyletic.

The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents, which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. Using five distinct computer programs, we here provide convincing statistical data suggesting that the transmembrane domains of ABC exporters are polyphyletic, having arisen at least three times independently. ABC1 porters arose by intragenic triplication of a primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins. ABC2 porters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, nonhomologous to the ABC1 proteins. ABC3 porters arose by duplication of a primordial four-TMS-encoding genetic element, yielding either eight- or 10-TMS proteins. We assign each of 48 of the 50 currently recognized families of ABC exporters to one of the three evolutionarily distinct ABC types. Currently available high-resolution structural data for ABC porters are fully consistent with our findings. These results provide guides for future structural and mechanistic studies of these important transport systems.

Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro.

ATP-driven efflux transport proteins at the blood-brain barrier protect the healthy brain but impede pharmacotherapy of the disordered CNS. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated during inflammation or infection we analysed the effects of the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on the expression of brain multidrug resistance proteins in primary cultures of porcine brain capillary endothelial cells. We found that TNF-alpha and IL-1beta rapidly decrease Abcg2 (BMDP/BCRP) mRNA expression within 6 h. After 24 and 48 h the mRNA level came back to control values. The mRNA reduction at 6 h was counter-regulated by the anti-inflammatory glucocorticoid hydrocortisone. Abcg2 protein levels were suppressed at prolonged stimulations but not after 6 h of stimulation which correlates with Abcg2 specific substrate uptake measurements. Abcb1 (p-glycoprotein) protein expression was transiently increased after TNF-alpha addition within 6 h of incubation followed by a reduction after 24 and 48 h whereas the Abcb1 mRNA levels were not changed. IL-1beta caused a continuous decrease in protein expression of both ABC-transporters. Long-term treatment with an assumed TNF-alpha-downstream agent, the vasoconstrictor endothelin-1, induced Abcg2 protein expression but suppressed Abcb1. Other efflux pumps like multidrug resistance-associated proteins/Abcc were rarely affected. The present results imply a complex regulation of the two most abundant ABC-transporters at the blood-brain barrier during early inflammation stages suggesting that Abcb1 (p-glycoprotein) is an early target of TNF-alpha-signalling counterbalanced by Abcg2.