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Scientific Reports Jan 2019Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an...
Flavonoids have important developmental, physiological, and ecological roles in plants and are primarily stored in the large central vacuole. Here we show that both an ATP-binding cassette (ABC) transporter(s) and an H-antiporter(s) are involved in the uptake of cyanidin 3-O-glucoside (C3G) by Arabidopsis vacuolar membrane-enriched vesicles. We also demonstrate that vesicles isolated from yeast expressing the ABC protein AtABCC2 are capable of MgATP-dependent uptake of C3G and other anthocyanins. The uptake of C3G by AtABCC2 depended on the co-transport of glutathione (GSH). C3G was not altered during transport and a GSH conjugate was not formed. Vesicles from yeast expressing AtABCC2 also transported flavone and flavonol glucosides. We performed ligand docking studies to a homology model of AtABCC2 and probed the putative binding sites of C3G and GSH through site-directed mutagenesis and functional studies. These studies identified residues important for substrate recognition and transport activity in AtABCC2, and suggest that C3G and GSH bind closely, mutually enhancing each other's binding. In conclusion, we suggest that AtABCC2 along with possibly other ABCC proteins are involved in the vacuolar transport of anthocyanins and other flavonoids in the vegetative tissue of Arabidopsis.
Topics: ATP-Binding Cassette Transporters; Anthocyanins; Antiporters; Arabidopsis; Arabidopsis Proteins; Binding Sites; Biological Transport; Flavonoids; Glucosides; Glutathione; Mutagenesis, Site-Directed; Protein Binding; Saccharomyces cerevisiae; Transgenes; Vacuoles
PubMed: 30679715
DOI: 10.1038/s41598-018-37504-8 -
Pharmacological Research Oct 2023Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease...
Solute carrier (SLC) transport proteins are fundamental for the translocation of endogenous compounds and drugs across membranes, thus playing a critical role in disease susceptibility and drug response. Because only a limited number of transporter substrates are currently known, the function of a large number of SLC transporters is elusive. Here, we describe the proof-of-concept of a novel strategy to identify SLC transporter substrates exemplarily for the proton-coupled peptide transporter (PEPT) 2 (SLC15A2) and multidrug and toxin extrusion (MATE) 1 transporter (SLC47A1), which are important renal transporters of drug reabsorption and excretion, respectively. By combining metabolomic profiling of mice with genetically-disrupted transporters, in silico ligand screening and in vitro transport studies for experimental validation, we identified nucleobases and nucleoside-derived anticancer and antiviral agents (flucytosine, cytarabine, gemcitabine, capecitabine) as novel drug substrates of the MATE1 transporter. Our data confirms the successful applicability of this new approach for the identification of transporter substrates in general, which may prove particularly relevant in drug research.
Topics: Animals; Mice; Ligands; Membrane Transport Proteins; Solute Carrier Proteins; Biological Transport
PubMed: 37775020
DOI: 10.1016/j.phrs.2023.106941 -
Pharmacological Reviews Mar 2010Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier... (Review)
Review
Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology.
Topics: Animals; Bile Acids and Salts; Carrier Proteins; Cholesterol; Female; Gene Expression Regulation; Humans; Male; Membrane Glycoproteins; Membrane Transport Proteins; Organ Specificity; Phylogeny; Polymorphism, Genetic; Protein Isoforms; Protein Processing, Post-Translational; Protein Transport; Sex Characteristics; Substrate Specificity; Xenobiotics
PubMed: 20103563
DOI: 10.1124/pr.109.002014 -
Journal of Chemical Information and... Dec 2022Integration of statistical learning methods with structure-based modeling approaches is a contemporary strategy to identify novel lead compounds in drug discovery.... (Review)
Review
Integration of statistical learning methods with structure-based modeling approaches is a contemporary strategy to identify novel lead compounds in drug discovery. Hepatic organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are classical off-targets, and it is well recognized that their ability to interfere with a wide range of chemically unrelated drugs, environmental chemicals, or food additives can lead to unwanted adverse effects like liver toxicity and drug-drug or drug-food interactions. Therefore, the identification of novel (tool) compounds for hepatic OATPs by virtual screening approaches and subsequent experimental validation is a major asset for elucidating structure-function relationships of (related) transporters: they enhance our understanding about molecular determinants and structural aspects of hepatic OATPs driving ligand binding and selectivity. In the present study, we performed a consensus virtual screening approach by using different types of machine learning models (proteochemometric models, conformal prediction models, and XGBoost models for hepatic OATPs), followed by molecular docking of preselected hits using previously established structural models for hepatic OATPs. Screening the diverse drug-like set (Enamine) shows a comparable hit rate for OATP1B1 (36% actives) and OATP1B3 (32% actives), while the hit rate for OATP2B1 was even higher (66% actives). Percentage inhibition values for 44 selected compounds were determined using dedicated assays and guided the prioritization of several highly potent novel hepatic OATP inhibitors: six (strong) OATP2B1 inhibitors (IC values ranging from 0.04 to 6 μM), three OATP1B1 inhibitors (2.69 to 10 μM), and five OATP1B3 inhibitors (1.53 to 10 μM) were identified. Strikingly, two novel OATP2B1 inhibitors were uncovered ( and ) which show high affinity (IC values: 40 nM and 390 nM) comparable to the recently described estrone-based inhibitor (IC = 41 nM). A molecularly detailed explanation for the observed differences in ligand binding to the three transporters is given by means of structural comparison of the detected binding sites and docking poses.
Topics: Organic Anion Transporters; Liver-Specific Organic Anion Transporter 1; Molecular Docking Simulation; Ligands; Solute Carrier Organic Anion Transporter Family Member 1B3; Biological Transport; Liver; Membrane Transport Proteins; Peptides; Drug Interactions
PubMed: 35274943
DOI: 10.1021/acs.jcim.1c01460 -
World Journal of Gastroenterology Dec 2012Glucose and other carbohydrates are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules. To date 14 members of... (Review)
Review
Glucose and other carbohydrates are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules. To date 14 members of this family, also called the solute carrier 2A proteins have been identified which are divided on the basis of transport characteristics and sequence similarities into several families (Classes 1 to 3). The expression of these different receptor subtypes varies between different species, tissues and cellular subtypes and each has differential sensitivities to stimuli such as insulin. The liver is a contributor to metabolic carbohydrate homeostasis and is a major site for synthesis, storage and redistribution of carbohydrates. Situations in which the balance of glucose homeostasis is upset such as diabetes or the metabolic syndrome can lead metabolic disturbances that drive chronic organ damage and failure, confirming the importance of understanding the molecular regulation of hepatic glucose homeostasis. There is a considerable literature describing the expression and function of receptors that regulate glucose uptake and release by hepatocytes, the most import cells in glucose regulation and glycogen storage. However there is less appreciation of the roles of GLUTs expressed by non parenchymal cell types within the liver, all of which require carbohydrate to function. A better understanding of the detailed cellular distribution of GLUTs in human liver tissue may shed light on mechanisms underlying disease pathogenesis. This review summarises the available literature on hepatocellular expression of GLUTs in health and disease and highlights areas where further investigation is required.
Topics: Animals; Biological Transport; Gene Expression Regulation; Glucose; Glucose Transport Proteins, Facilitative; Homeostasis; Humans; Insulin; Liver; Liver Neoplasms; Mice; Prognosis; Protein Isoforms; Tissue Distribution
PubMed: 23239915
DOI: 10.3748/wjg.v18.i46.6771 -
Biochimica Et Biophysica Acta Mar 2011The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible... (Review)
Review
The twin-arginine translocation (Tat) system operates in plant thylakoid membranes and the plasma membranes of most free-living bacteria. In bacteria, it is responsible for the export of a number of proteins to the periplasm, outer membrane or growth medium, selecting substrates by virtue of cleavable N-terminal signal peptides that contain a key twin-arginine motif together with other determinants. Its most notable attribute is its ability to transport large folded proteins (even oligomeric proteins) across the tightly sealed plasma membrane. In Gram-negative bacteria, TatABC subunits appear to carry out all of the essential translocation functions in the form of two distinct complexes at steady state: a TatABC substrate-binding complex and separate TatA complex. Several studies favour a model in which these complexes transiently coalesce to generate the full translocase. Most Gram-positive organisms possess an even simpler "minimalist" Tat system which lacks a TatB component and contains, instead, a bifunctional TatA component. These Tat systems may involve the operation of a TatAC complex together with a separate TatA complex, although a radically different model for TatAC-type systems has also been proposed. While bacterial Tat systems appear to require the presence of only a few proteins for the actual translocation event, there is increasing evidence for the operation of ancillary components that carry out sophisticated "proofreading" activities. These activities ensure that redox proteins are only exported after full assembly of the cofactor, thereby avoiding the futile export of apo-forms. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.
Topics: Amino Acid Sequence; Arginine; Biological Transport; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Molecular Sequence Data; Protein Sorting Signals; Protein Transport; Sequence Homology, Amino Acid
PubMed: 21126506
DOI: 10.1016/j.bbamem.2010.11.023 -
Cell Reports Aug 2023Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide...
Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders.
Topics: Molecular Docking Simulation; Models, Molecular; Membrane Transport Proteins; Peptides
PubMed: 37467108
DOI: 10.1016/j.celrep.2023.112831 -
Yakugaku Zasshi : Journal of the... 2021The blood-brain barrier (BBB) consists of brain capillary endothelial cells linked by tight junctions and serves to regulate the transfer of endogenous compounds and... (Review)
Review
The blood-brain barrier (BBB) consists of brain capillary endothelial cells linked by tight junctions and serves to regulate the transfer of endogenous compounds and xenobiotics between the circulating blood and brain interstitial fluid. We have developed a methodology to characterize brain-to-blood efflux transport in vivo, using the Brain Efflux Index and an in vitro culture model of the BBB, i.e., a conditionally immortalized cell line of the neurovascular unit. Employing these methods, we showed that the BBB plays an important role in protecting the brain by transporting neurotransmitters, neuromodulators, metabolites, uremic toxins, and xenobiotics together with atrial natriuretic peptide from the brain interstitial fluid to the circulating blood. We also developed a highly selective, sensitive LC-MS/MS method for simultaneous protein quantification. We found significant species differences in the expression amounts of various BBB transporter proteins among mice, rats, marmosets, cynomolgus monkeys, and humans. Among transporter proteins at the BBB, multidrug resistance protein 1 (Mdr1/Abcb1) is known to generate a concentration gradient of unbound substrate drugs between the blood and brain. Based on measurements of the intrinsic efflux transport rate of Mdr1 and the protein expression amounts of Mdr1 in mouse brain capillaries and Mdr1-expressing cell lines, we predicted the unbound drug concentration gradients of 7 drugs in the mouse brain in vivo. This was the first successful prediction of in vivo drug transport activity from in vitro experimental data and transporter protein concentration in tissues. This methodology and findings should greatly advance central nervous system barrier research.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Biological Transport; Blood-Brain Barrier; Brain; Cell Line; Chromatography, Liquid; Humans; Membrane Transport Proteins; Mice; Neurotransmitter Agents; Proteomics; Rats; Tandem Mass Spectrometry; Xenobiotics
PubMed: 33790111
DOI: 10.1248/yakushi.20-00232 -
Biochemistry Aug 2007Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in... (Review)
Review
Understanding the transport of hydrophilic proteins across biological membranes continues to be an important undertaking. The general secretory (Sec) pathway in Escherichia coli transports the majority of E. coli proteins from their point of synthesis in the cytoplasm to their sites of final localization, associating sequentially with a number of protein components of the transport machinery. The targeting signals for these substrates must be discriminated from those of proteins transported via other pathways. While targeting signals for each route have common overall characteristics, individual signal peptides vary greatly in their amino acid sequences. How do these diverse signals interact specifically with the proteins that comprise the appropriate transport machinery and, at the same time, avoid targeting to an alternate route? The recent publication of the crystal structures of components of the Sec transport machinery now allows a more thorough consideration of the interactions of signal sequences with these components.
Topics: Adenosine Triphosphatases; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Protein Transport; SEC Translocation Channels; SecA Proteins; Signal Recognition Particle; Signal Transduction
PubMed: 17676771
DOI: 10.1021/bi7010064 -
Structure (London, England : 1993) Jul 2022Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and...
Mammalian peptide transporters, PepT1 and PepT2, mediate uptake of small peptides and are essential for their absorption. PepT also mediates absorption of many drugs and prodrugs to enhance their bioavailability. PepT has twelve transmembrane (TM) helices that fold into an N-terminal domain (NTD, TM1-6) and a C-terminal domain (CTD, TM7-12) and has a large extracellular domain (ECD) between TM9-10. It is well recognized that peptide transport requires movements of the NTD and CTD, but the role of the ECD in PepT1 remains unclear. Here we report the structure of horse PepT1 encircled in lipid nanodiscs and captured in the inward-open apo conformation. The structure shows that the ECD bridges the NTD and CTD by interacting with TM1. Deletion of ECD or mutations to the ECD-TM1 interface impairs the transport activity. These results demonstrate an important role of ECD in PepT1 and enhance our understanding of the transport mechanism in PepT1.
Topics: Animals; Biological Transport; Horses; Mammals; Molecular Conformation; Peptide Transporter 1; Peptides; Symporters
PubMed: 35580608
DOI: 10.1016/j.str.2022.04.011