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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 -
ACS Chemical Biology Mar 2021PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)K, targeting essential bacterial genes, such as P, can inhibit bacterial growth at...
PNA oligomers conjugated to bacteria penetrating peptides (BPPs), such as (KFF)K, targeting essential bacterial genes, such as P, can inhibit bacterial growth at one-digit micromolar concentrations. It has been found that the LPS of the outer membrane of Gram-negative bacteria is a barrier for cellular uptake of (KFF)K-eg-PNA and that the SbmA transporter protein is involved in the passage through the inner membrane. We now further elucidate the uptake mechanism of (KFF)K-eg-PNA by showing that the peptide part of (KFF)K-eg-PNA is unstable and is degraded by peptidases in the medium of a bacterial culture ( < 5 min) and inside the bacteria. Analysis of peptide-PNA conjugates present in the periplasmic space and the cytoplasm showed the presence of mainly PNA with only the FFK tripeptide and without a peptide, at a concentration 10-fold that added to the medium. Furthermore, the two main degradation products showed no antibacterial effect when added directly to a bacterial culture and the antibacterial effect decreased with peptide length, thereby demonstrating that an intact peptide is indeed crucial for uptake but not for intracellular antisense activity. Most surprisingly, it was found that although the corresponding series of the proteolytically stable D-form (kff)k-eg-PNAs exhibited an analogous reduction of activity with peptide length, the activity was dependent on the presence of SbmA for the shorter peptides (which is not the case with the full length peptide). Therefore, our results suggest that the BPP is necessary for crossing both the LPS/outer membrane as well as the inner membrane and that full length (KFF)K may spontaneously pass the inner membrane. Thus, SbmA dependence of (KFF)K-eg-PNA is ascribed to peptide degradation in the bacterial medium and in periplasmic space. Finally, the results show that stability and metabolism (by bacterial proteases/peptidases) should be taken into consideration upon design and activity/uptake analysis of BPPs (and antimicrobial peptides).
Topics: Anti-Bacterial Agents; Cell Culture Techniques; Cell Membrane Permeability; Drug Discovery; Escherichia coli; Escherichia coli Proteins; Kinetics; Membrane Transport Proteins; Microbial Sensitivity Tests; Molecular Structure; Oligonucleotides, Antisense; Peptide Hydrolases; Peptide Nucleic Acids; Peptides; Protein Stability
PubMed: 33684286
DOI: 10.1021/acschembio.0c00822 -
Scientific Reports May 2024Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins...
Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.
Topics: Klebsiella pneumoniae; Bacterial Proteins; Streptococcus pneumoniae; Transcriptome; Gene Expression Regulation, Bacterial; Humans; Ligands; Membrane Transport Proteins; Peptides
PubMed: 38816440
DOI: 10.1038/s41598-024-63217-2 -
British Journal of Pharmacology Jul 2017Organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs), encoded by a number of solute carrier (SLC)22A and SLC organic anion (SLCO) genes,... (Review)
Review
Trafficking and other regulatory mechanisms for organic anion transporting polypeptides and organic anion transporters that modulate cellular drug and xenobiotic influx and that are dysregulated in disease.
Organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs), encoded by a number of solute carrier (SLC)22A and SLC organic anion (SLCO) genes, mediate the absorption and distribution of drugs and other xenobiotics. The regulation of OATs and OATPs is complex, comprising both transcriptional and post-translational mechanisms. Plasma membrane expression is required for cellular substrate influx by OATs/OATPs. Thus, interest in post-translational regulatory processes, including membrane targeting, endocytosis, recycling and degradation of transporter proteins, is increasing because these are critical for plasma membrane expression. After being synthesized, transporters undergo N-glycosylation in the endoplasmic reticulum and Golgi apparatus and are delivered to the plasma membrane by vesicular transport. Their expression at the cell surface is maintained by de novo synthesis and recycling, which occurs after clathrin- and/or caveolin-dependent endocytosis of existing protein. Several studies have shown that phosphorylation by signalling kinases is important for the internalization and recycling processes, although the transporter protein does not appear to be directly phosphorylated. After internalization, transporters that are targeted for degradation undergo ubiquitination, most likely on intracellular loop residues. Epigenetic mechanisms, including methylation of gene regulatory regions and transcription from alternate promoters, are also significant in the regulation of certain SLC22A/SLCO genes. The membrane expression of OATs/OATPs is dysregulated in disease, which affects drug efficacy and detoxification. Several transporters are expressed in the cytoplasmic subcompartment in disease states, which suggests that membrane targeting/internalization/recycling may be impaired. This article focuses on recent developments in OAT and OATP regulation, their dysregulation in disease and the significance for drug therapy.
Topics: Animals; Disease; Humans; Organic Anion Transporters; Peptides; Pharmaceutical Preparations
PubMed: 28299773
DOI: 10.1111/bph.13785 -
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 -
Journal of Alzheimer's Disease : JAD 2021Alzheimer's disease (AD) is a chronic neurodegenerative disease that has been recognized as one of the most intractable medical problems with heavy social and economic... (Review)
Review
Alzheimer's disease (AD) is a chronic neurodegenerative disease that has been recognized as one of the most intractable medical problems with heavy social and economic costs. Amyloid-β (Aβ) has been identified as a major factor that participates in AD progression through its neurotoxic effects. The major mechanism of Aβ-induced neurotoxicity is by interacting with membrane receptors and subsequent triggering of aberrant cellular signaling. Besides, Aβ transporters also plays an important role by affecting Aβ homeostasis. Thus, these Aβ receptors and transporters are potential targets for the development of AD therapies. Here, we summarize the reported therapeutic strategies targeting Aβ receptors and transporters to provide a molecular basis for future rational design of anti-AD agents.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Membrane Transport Proteins; Neurons; Receptors, Cell Surface
PubMed: 33459712
DOI: 10.3233/JAD-200851 -
Journal of Agricultural and Food... Jan 2019The objective of this study was to characterize the expression profile, transport kinetics, and regulation of peptide transporters in bovine mammary epithelial cells...
The objective of this study was to characterize the expression profile, transport kinetics, and regulation of peptide transporters in bovine mammary epithelial cells (BMECs). Quantitative reverse-transcription real-time PCR, Western blotting, and immunofluorescence staining were used to investigate the expression of peptide transporters in bovine mammary tissues. The effects of time, pH, concentration, and specific inhibitors on β-alanyl-l-lysyl- Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA) uptake in BMECs were also studied. The results showed that the peptide transporters PepT2 and PhT1 are both expressed in bovine mammary glands. The optimal pH for the uptake of β-Ala-Lys-AMCA in BMECs was 6.5. The transport-kinetics study suggested that the uptake of β-Ala-Lys-AMCA in BMECs is saturable over the tested concentration, with a K value of 82 ± 18 μM and a V of 124 ± 11 pmol/min per milligram of protein. Other dipeptides, including Gly-Sar, Met-Gly, and Met-Met, competitively inhibited β-Ala-Lys-AMCA uptake in BMECs. However, histidine had no effect on β-Ala-Lys-AMCA uptake. Furthermore, knocking down PepT2 could significantly reduce β-Ala-Lys-AMCA uptake, but PhT1 interference had no effect on peptide uptake in BMECs. The inhibition of PI3K and Akt decreased the uptake of β-Ala-Lys-AMCA. The above results revealed functional characteristics of peptide transporters and demonstrated that PepT2 may play a major role in β-Ala-Lys-AMCA uptake in BMECs. Moreover, the PI3K-Akt signaling pathway may regulate the uptake of β-Ala-Lys-AMCA in BMECs.
Topics: Animals; Biological Transport; Cattle; Epithelial Cells; Female; Kinetics; Mammary Glands, Animal; Membrane Transport Proteins; Peptides
PubMed: 30525553
DOI: 10.1021/acs.jafc.8b05637 -
Journal of Visualized Experiments : JoVE May 2021Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for...
Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.
Topics: Cardiac Electrophysiology; Humans; Kinetics; Membrane Proteins; Membrane Transport Proteins; Single-Domain Antibodies
PubMed: 33999018
DOI: 10.3791/62578 -
Molecular Immunology Dec 2015Major histocompatibility complex class I (MHC I) proteins provide protection from intracellular pathogens and cancer via each of a cell's MHC I molecules binding and... (Review)
Review
Major histocompatibility complex class I (MHC I) proteins provide protection from intracellular pathogens and cancer via each of a cell's MHC I molecules binding and presenting a peptide to cytotoxic T lymphocytes. MHC I genes are highly polymorphic and can have significant diversity, with polymorphisms predominantly localised in the peptide-binding groove where they can change peptide-binding specificity. However, polymorphic residues may also determine other functional properties, such as how dependent MHC I alleles are on the peptide-loading complex for optimal acquisition of peptide cargo. We describe how differences in the peptide-binding properties of two MHC I alleles correlates with altered conformational flexibility in the peptide-empty state. We hypothesise that plasticity is an intrinsic property encoded by the protein sequence, and that co-ordinated movements of the membrane-proximal and membrane-distal domains collectively determines how dependent MHC I are on the peptide-loading complex for efficient assembly with high affinity peptides.
Topics: Alleles; Animals; Antigen Presentation; Antigen-Presenting Cells; Histocompatibility Antigens Class I; Humans; Membrane Transport Proteins; Molecular Dynamics Simulation; Peptides; Polymorphism, Genetic; Protein Binding; Protein Folding; Protein Structure, Tertiary; T-Lymphocytes, Cytotoxic
PubMed: 25818313
DOI: 10.1016/j.molimm.2015.03.010