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Scientific Reports Aug 2019The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which...
The lysosomal polypeptide transporter TAPL belongs to the superfamily of ATP-binding cassette transporters. TAPL forms a homodimeric transport complex, which translocates oligo- and polypeptides into the lumen of lysosomes driven by ATP hydrolysis. Although the structure and the function of ABC transporters were intensively studied in the past, details about the single steps of the transport cycle are still elusive. Therefore, we analyzed the coupling of peptide binding, transport and ATP hydrolysis for different substrate sizes. Although longer and shorter peptides bind with the same affinity and are transported with identical K values, they differ significantly in their transport rates. This difference can be attributed to a higher activation energy for the longer peptide. TAPL shows a basal ATPase activity, which is inhibited in the presence of longer peptides. Uncoupling between ATP hydrolysis and peptide transport increases with peptide length. Remarkably, also the type of nucleotide determines the uncoupling. While GTP is hydrolyzed as good as ATP, peptide transport is significantly reduced. In conclusion, TAPL does not differentiate between transport substrates in the binding process but during the following steps in the transport cycle, whereas, on the other hand, not only the coupling efficiency but also the activation energy varies depending on the size of peptide substrate.
Topics: ATP-Binding Cassette Transporters; Algorithms; Cell Membrane; Humans; Hydrolysis; Models, Biological; Peptides; Protein Binding; Protein Transport
PubMed: 31417173
DOI: 10.1038/s41598-019-48343-6 -
Journal of Pharmaceutical Sciences Jan 2012The sodium-coupled oligopeptide transporters 1 and 2 (SOPT1 and SOPT2) transport peptides consisting of at least five amino acids and show potential for the delivery of...
The sodium-coupled oligopeptide transporters 1 and 2 (SOPT1 and SOPT2) transport peptides consisting of at least five amino acids and show potential for the delivery of therapeutically relevant peptides/peptidomimetics. Here, we examined the expression of these two transporters in the retinal neuronal cell line RGC-5. These cells showed robust uptake activity for the synthetic pentapeptide DADLE ([D-Ala(2),D-Leu(5)]-Enkephalin). The uptake was Na(+) dependent and saturable (K(t), 6.2 ± 0.6 μM). A variety of oligopeptides inhibited DADLE uptake. The uptake of the competing oligopeptides was directly demonstrated with fluorescein isothiocyanate-labeled Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys in RGC-5 cells and primary mouse retinal ganglion cells. The characteristics of DADLE uptake matched those of SOPT2. We then examined the expression of SOPT1 in these cells with deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH(2)) as the substrate and found that RGC-5 cells also expressed SOPT1. As it is already known that SOPT1 is expressed in the neuronal cell line SK-N-SH, we investigated SOPT2 expression in these cells to determine whether the presence of both oligopeptide transporters is a common feature of neuronal cells. These studies showed that SK-N-SH cells also expressed SOPT2. This constitutes the first report on the functional characterization of SOPT1 and SOPT2 in retinal neuronal cells and on the expression of SOPT2 in nonretinal neuronal cells.
Topics: Amino Acids; Animals; Biological Transport; Cell Line; Enkephalin, Leucine-2-Alanine; Kinetics; Membrane Transport Proteins; Mice; Oligopeptides; Opioid Peptides; Retinal Ganglion Cells; Sodium; Substrate Specificity
PubMed: 21905028
DOI: 10.1002/jps.22733 -
Journal of Physics. Condensed Matter :... Nov 2010About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In...
About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40(SC) as well as a mutant Tom40(SC) (S(54 --> E) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40(SC) corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40(SC) S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with t(off) approximately = 1.1 ms for the wildtype, whereas the mutant Tom40(SC) S54E displayed a biphasic dwelltime distribution (t(off)(-1) approximately = 0.4 ms; t(off)(-2) approximately = 4.6 ms).
Topics: Computer Simulation; Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Mitochondrial Proteins; Models, Chemical; Peptides; Porosity; Protein Transport
PubMed: 21339590
DOI: 10.1088/0953-8984/22/45/454102 -
Amino Acids Aug 2022L-Carnosine (β-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of...
L-Carnosine (β-alanyl-L-histidine) is a well-known antioxidant and neuroprotector in various models on animals and cell cultures. However, while there is a plethora of data demonstrating its efficiency as a neuroprotector, there is a distinct lack of data regarding the mechanism of its take up by neurons. According to literature, cultures of rat astrocytes, SKPT cells and rat choroid plexus epithelial cells take up carnosine via the H-coupled PEPT2 membrane transporter. We've assessed the effectiveness and mechanism of carnosine transport, and its stability in primary rat cortical culture neurons. We demonstrated that neurons take up carnosine via active transport with Km = 119 μM and a maximum velocity of 0.289 nmol/mg (prot)/min. Passive transport speed constituted 0.21∙10 nmol/mg (prot)/min (with 119 μM concentration in the medium)-significantly less than active transport speed. However, carnosine concentrations over 12.5 mM led to passive transport speed becoming greater than active transport speed. Using PEPT2 inhibitor zofenopril, we demonstrated that PEPT2-dependent transport is one of the main modes of carnosine take up by neurons. Our experiments demonstrated that incubation with carnosine does not affect PEPT2 amount present in culture. At the same time, after removing carnosine from the medium, its elimination speed by culture cells reached 0.035 nmol/mg (prot)/min, which led to a decrease in carnosine quantity to control levels in culture within 1 h. Thus, carnosine is taken up by neurons with an effectiveness comparable to that of other PEPT2 substrates, but its elimination rate suggests that for effective use as a neuroprotector it's necessary to either maintain a high concentration in brain tissue, or increase the effectiveness of glial cell synthesis of endogenous carnosine and its shuttling into neurons, or use more stable chemical modifications of carnosine.
Topics: Animals; Biological Transport, Active; Carnosine; Choroid Plexus; Membrane Transport Proteins; Rats; Symporters
PubMed: 34694500
DOI: 10.1007/s00726-021-03094-5 -
Chembiochem : a European Journal of... May 2011In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of...
Transport of free and peptide-bound glycated amino acids: synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins.
In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.
Topics: Amino Acids; Biological Transport; Caco-2 Cells; Epithelium; Glycosylation; Humans; Intestinal Mucosa; Ligands; Magnetic Resonance Spectroscopy; Maillard Reaction; Membrane Transport Proteins; Models, Molecular; Peptides
PubMed: 21538757
DOI: 10.1002/cbic.201000759 -
Physiology (Bethesda, Md.) Apr 2006Uptake of nutrients into cells is essential to life and occurs in all organisms at the expense of energy. Whereas in most prokaryotic and simple eukaryotic cells... (Review)
Review
Uptake of nutrients into cells is essential to life and occurs in all organisms at the expense of energy. Whereas in most prokaryotic and simple eukaryotic cells electrochemical transmembrane proton gradients provide the central driving force for nutrient uptake, in higher eukaryotes it is more frequently coupled to sodium movement along the transmembrane sodium gradient, occurs via uniport mechanisms driven by the substrate gradient only, or is linked to the countertransport of a similar organic solute. With the cloning of a large number of mammalian nutrient transport proteins, it became obvious that a few "archaic'' transporters that utilize a transmembrane proton gradient for nutrient transport into cells can still be found in mammals. The present review focuses on the electrogenic peptide transporters as the best studied examples of proton-dependent nutrient transporters in mammals and summarizes the most recent findings on their physiological importance. Taking peptide transport as a general phenomenon found in nature, we also include peptide transport mechanisms in bacteria, yeast, invertebrates, and lower vertebrates, which are not that often addressed in physiology journals.
Topics: Amino Acid Sequence; Animals; Bacteria; Biological Transport; Eukaryotic Cells; Humans; Invertebrates; Membrane Transport Proteins; Molecular Sequence Data; Peptide Transporter 1; Phylogeny; Prokaryotic Cells; Protons; Substrate Specificity; Symporters; Vertebrates; Yeasts
PubMed: 16565475
DOI: 10.1152/physiol.00054.2005 -
The Journal of General Virology Feb 2004Equine herpesvirus-1 (EHV-1) downregulates surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. The objective of this study... (Comparative Study)
Comparative Study
Equine herpesvirus-1 (EHV-1) downregulates surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. The objective of this study was to investigate whether EHV-1 interferes with peptide translocation by the transporter associated with antigen processing (TAP) and to identify the proteins responsible. Using an in vitro transport assay, we showed that EHV-1 inhibited transport of peptides by TAP as early as 2 h post-infection (p.i). Complete shutdown of peptide transport was observed by 8 h p.i. Furthermore, pulse-chase experiments revealed that maturation of class I molecules in the endoplasmic reticulum (ER) was delayed in EHV-1-infected cells, which may be due to reduced availability of peptides in the ER as a result of TAP inhibition. Metabolic inhibition studies indicated that an early protein(s) of EHV-1 is responsible for this effect.
Topics: ATP-Binding Cassette Transporters; Animals; Biological Transport, Active; Cell Line; Down-Regulation; Endoplasmic Reticulum; Herpesvirus 1, Equid; Histocompatibility Antigens Class I; Immediate-Early Proteins; Membrane Transport Modulators; Membrane Transport Proteins
PubMed: 14769892
DOI: 10.1099/vir.0.19563-0 -
Current Opinion in Cell Biology Aug 1991This review focuses primarily on the progress made in the last couple of years in the understanding of the intestinal peptide transporter, a prototype for H(+)-coupled... (Review)
Review
This review focuses primarily on the progress made in the last couple of years in the understanding of the intestinal peptide transporter, a prototype for H(+)-coupled solute transport systems in the animal cell plasma membrane. The impressive number of transport systems currently known to be energized by the components of the proton-motive force indicates that the role of H+ as the coupling ion for active transport has not been lost during evolution.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Animal Population Groups; Animals; Biological Transport, Active; Cadherins; Carrier Proteins; Cell Membrane; Dipeptides; Energy Metabolism; Hydrogen; Intestinal Absorption; Intestine, Small; Kidney; Membrane Transport Proteins; Microvilli; Molecular Sequence Data; Peptides; Protons; Rabbits; Rats; Rats, Inbred F344; Species Specificity
PubMed: 1663375
DOI: 10.1016/0955-0674(91)90043-x -
Biochimica Et Biophysica Acta May 2013Glutathione (GSH) is synthesized in the cytoplasm but there is a requirement for glutathione not only in the cytoplasm, but in the other organelles and the extracellular... (Review)
Review
BACKGROUND
Glutathione (GSH) is synthesized in the cytoplasm but there is a requirement for glutathione not only in the cytoplasm, but in the other organelles and the extracellular milieu. GSH is also imported into the cytoplasm. The transports of glutathione across these different membranes in different systems have been biochemically demonstrated. However the molecular identity of the transporters has been established only in a few cases.
SCOPE OF REVIEW
An attempt has been made to present the current state of knowledge of glutathione transporters from different organisms as well as different organelles. These include the most well characterized transporters, the yeast high-affinity, high-specificity glutathione transporters involved in import into the cytoplasm, and the mammalian MRP proteins involved in low affinity glutathione efflux from the cytoplasm. Other glutathione transporters that have been described either with direct or indirect evidences are also discussed.
MAJOR CONCLUSIONS
The molecular identity of a few glutathione transporters has been unambiguously established but there is a need to identify the transporters of other systems and organelles. There is a lack of direct evidence establishing transport by suggested transporters in many cases. Studies with the high affinity transporters have led to important structure-function insights.
GENERAL SIGNIFICANCE
An understanding of glutathione transporters is critical to our understanding of redox homeostasis in living cells. By presenting our current state of understanding and the gaps in our knowledge the review hopes to stimulate research in these fields. This article is part of a Special Issue entitled Cellular functions of glutathione.
Topics: Animals; Biological Transport; Glutathione; Humans; Membrane Transport Proteins; Oxidation-Reduction
PubMed: 23206830
DOI: 10.1016/j.bbagen.2012.11.018 -
Molecular Nutrition & Food Research Oct 2010Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine...
Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β-casein (β-CN, 193-209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β-CN (193-209) peptide across Caco-2 cell monolayer. In addition, we evaluated the possible routes of the β-CN (193-209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β-CN (193-209) peptide resisted the action of brush-border membrane peptidases, and that it was transported through the Caco-2 cell monolayer. The main route involved in transepithelial transport of the β-CN (193-209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. Our results demonstrated the transport of an intact long-chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.
Topics: Animals; Caco-2 Cells; Caseins; Cattle; Chymosin; Humans; Hydrophobic and Hydrophilic Interactions; Immunologic Factors; Intestinal Absorption; Membrane Transport Modulators; Membrane Transport Proteins; Microvilli; Osmolar Concentration; Peptide Fragments; Peptide Transporter 1; Protein Isoforms; Symporters; Tight Junctions; Time Factors; Transcytosis
PubMed: 20397193
DOI: 10.1002/mnfr.200900443