Coupled Oligopeptide Transporter Research Articles

Conformational Transition Pathway of GkPOT

GkPOT, a Proton-Coupled coupled Oligopeptide Transporter transporter (POTs) from Geobacillus Kaustophilus, is among the symporter members of the Major Facilitator Superfamily (MFS) of transporters. Functional and structural analysis of various POTs suggest that the uptake of small peptides and peptide- like molecules to the cell across the cell membrane, via the alternating access mechanism, utilizes an inward directed proton electrochemical gradient the source of energy for their active symport function. The transition between the inward-facing (IF) and outward-facing (OF) conformations is important in understanding the structural relationship between large-scale protein conformational changes and substrate translocation that has often times remained elusive due to the short time scales of molecular dynamics simulations (MD) limitations of computational and experimental effort techniques. Using an extensive set of enhanced sampling techniques, we have characterized the conformational transition pathway of GkPOT. We have aimed at describing the large-scale conformational changes between the IF and OF states while in the presence and absence of substrate (dialanine peptide). Nonequlibrium pulling simulations were initiated from the known inward facing IF conformation and steered the system toward the unknown outward facing OF conformation using orientation based collective variables defined on different transmembrane helices. The transition pathways are far from equilibrium and are smoothed with string method with swarms of trajectories (SMwST), thus generating microseconds of biased and unbiased simlulations. Free energy profiles of the conformational transition pathways were obtained from a novel maximum-likelihood estimation method. Our results indicate that the presence of ligand significantly changes the conformational landscape with respect to the apo system which contains a greater degree of conformational variability.

Transport Characteristics of 5-Aminosalicylic Acid Derivatives Conjugated with Amino Acids via Human H+-Coupled Oligopeptide Transporter PEPT1.

5-Aminosalicylic acid (5-ASA) is used as first line therapy for symptom remission and maintenance of inflammatory bowel disease (IBD). Because 5-ASA is well absorbed from the small intestine when orally administered, several 5-ASA formulations for selective delivery to the colon have been developed and used in clinical practice. However, its delivery efficiency to local inflamed colonic sites remains low. Intestinal H+-coupled oligopeptide transporter 1 (PEPT1) expression in the colon is low, whereas its expression is induced in the colon under chronic inflammation conditions, such as IBD. Therefore, we considered that PEPT1 would be a target transporter to improve 5-ASA delivery efficiency to local colonic lesions. We evaluated the transport characteristics of dipeptide-like 5-ASA derivatives, which were coupling glycine (Gly), lysine, glutamic acid (Glu), valine (Val) and tyrosine to amino or carboxyl group of 5-ASA, in Caco-2 cells. [3H]Glycylsarcosine (Gly-Sar) uptake into Caco-2 cells was inhibited by all 5-ASA derivatives. In addition, 5-ASA derivatives (Gly-ASA, Glu-ASA and Val-ASA), which were coupled by glycine, glutamic acid and valine to amino group of 5-ASA, were taken up in a pH- and concentration-dependent manner and their uptake was inhibited by excess Gly-Sar. Two-electrode voltage-clamp experiment using human PEPT1 expressing Xenopus oocytes showed that Gly-ASA, Glu-ASA and Val-ASA induced marked currents at pH 6.0. Taken together, these results showed that these 5-ASA derivatives are transportable substrates for PEPT1.

Establishment of a novel microscale thermophoresis ligand-binding assay for characterization of SLC solute carriers using oligopeptide transporter PepT1 (SLC15 family) as a model system

IntroductionMembrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purification tasks even more challenging compared to soluble proteins. This issue also holds true for conventional ligand-binding assays (LBAs) which usually require high-quality, pure and concentrated protein samples. Herein, we report a novel binding assay strategy to overcome these issues, taking advantage of a unique combination of yeast expression and microscale thermophoresis (MST). Following yeast overexpression of SLC15A1/PepT1 ortholog from moss Physcomitrella patens, PepTPp, which exhibits remarkable similarity to human PepT1, the approach was validated using dipeptide glycylsarcosine (Gly-Sar) and antiviral prodrug valacyclovir as test substrates. MethodThe originality of our approach is based on the comparative analysis of solubilized total membrane preparations with or without expression of the SLC target of interest, using a yeast strain (S. cerevisiae), in which the corresponding endogenous SLC homolog is depleted. MST is a recently developed technique that takes advantage of the properties of biomolecules in solution to migrate along a temperature gradient. Importantly, this migration is affected by substrate binding. It is being monitored by fluorescence using labelled SLC molecules in the presence of different ligand concentrations. ResultsWe herein report a novel MST/yeast-based method to characterize binding of ligands to SLCs without the need for a prior SLC-purification step. For validation purposes, we used a close eukaryotic homolog of the human H+-coupled oligopeptide transporter PepT1 (SLC15A1) that mediates uptake of di-tripeptides and peptide-like drugs as a test model. This approach allowed the successful confirmation of the binding of Gly-Sar at the mM range and revealed for the first time the KD of the antiviral prodrug valacyclovir to the PepT1 homolog at around 50 μM. DiscussionThis novel LBA approach is independent of protein purification. It is suitable for drug discovery as it is upscalable to high throughput compound screening. It works well for SLC transporters which are underrepresented targets due to their difficulties to study them. Moreover, this approach could make a significant contribution toward “deorphanization” of SLCs, revealing their transport substrates.

Symmetry and Structure in the POT Family of Proton Coupled Peptide Transporters

The POT family of proton coupled oligopeptide transporters belong to the Major Facilitator Superfamily of secondary active transporters and are found widely distributed in bacterial, plant, fungal and animal genomes. POT transporters use the inwardly directed proton electrochemical gradient to drive the concentrative uptake of di- and tri-peptides across the cell membrane for metabolic assimilation. Mammalian members of the family, PepT1 and PepT2, are responsible for the uptake and retention of dietary protein in the human body, and due to their promiscuity in ligand recognition, play important roles in the pharmacokinetics of drug transport. Recent crystal structures of bacterial and plant members have revealed the overall architecture for this protein family and provided a framework for understanding proton coupled transport within the POT family. An interesting outcome from these studies has been the discovery of symmetrically equivalent structural and functional sites. This review will highlight both the symmetry and asymmetry in structure and function within the POT family and discuss the implications of these considerations in understanding transport and regulation.

Targeting PEPT1: a novel strategy to improve the antitumor efficacy of doxorubicin in human hepatocellular carcinoma therapy.

Proton coupled oligopeptide transporter 1 (PEPT1) is a member of the peptide transporter superfamily and plays important role in the absorption of oligopeptide and peptidomimetic drugs. Our previous research verified that PEPT1 expressed specifically in human Hepatocellular carcinoma (HCC) tissue and cell lines and showed potential transport activity to be a new candidate of the tumor therapeutic target. In this study, we aim to explore the feasibility of a novel tumor target therapeutic strategy: Targeting PEPT1 to improve the antitumor efficacy of Doxorubicin in human HCC therapy. First, Doxorubicin was conjugated with Glycylglycylglycine (Gly-Gly-Gly) − a tripeptide which was known as the substrate of PEPT1 and characterized by HPLC and MS successfully. Doxorubicin-tripeptide conjugate was then observed to clarify the target delivery by PEPT1 and the antitumor effect on human hepatocarcinoma in vivo and in vitro. Furthermore, the improvement of the toxic and side effect of Doxorubicin after conjugation was also evaluated by some biochemical tests. Our results reveal that targeting PEPT1 may contribute to the efficient delivery of Doxorubicin to hepatocarcinoma cells and the reduction of drug toxicity. PEPT1 has the prospect to be a novel target of HCC therapy.

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.

Investigation of the Route of Absorption of Lipid and Sugar Modified Leu- Enkephalin Analogues and Their Enzymatic Stability Using the Caco-2 Cell Monolayer System

It has been demonstrated that conjugation of lipoamino acids or glucose units to the endogenous opioid peptide, Leu-enkephalin can significantly improve the peptide's metabolic stability and absorption across biological barriers. The purpose of this study was to investigate the possible involvement of specific carrier proteins in the absorption of these peptide conjugates. A series of lipo- glycol- and liposaccharide peptide conjugates were synthesised and examined using the Caco-2 monolayer assay for evidence of interaction with the human H(+)-coupled oligopeptide transporter (hPepT1), glucose transporters and the multidrug resistance efflux pump, p-glycoprotein. The investigation involved determining the apparent permeability of each compound in the absence of any inhibitors and comparing this to the apparent permeabilities of each compound in the presence of glycylsarcosine, glucose or vinblastine, respective inhibitors of the above mentioned transporters. None of the peptide conjugates were found to be substrates for p-glycoprotein. Of the six peptide conjugates examined, only the C-terminus glucose conjugate of Leu-enkephalin (Enk-glu) showed evidence of transport by both glucose transporters and hPepT1. In contrast, N-terminus conjugation of both lipids and sugars appeared to provide the greatest protection against enzymatic degradation.

A Novel H+-coupled Oligopeptide Transporter (OPT3) from Caenorhabditis elegans with a Predominant Function as a H+ Channel and an Exclusive Expression in Neurons

We have cloned and functionally characterized a novel, neuron-specific, H(+)-coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans that functions predominantly as a H(+) channel. The opt3 gene is approximately 4.4 kilobases long and consists of 13 exons. The cDNA codes for a protein of 701 amino acids with 11 putative transmembrane domains. When expressed in mammalian cells and in Xenopus laevis oocytes, OPT3 cDNA induces H(+)-coupled transport of the dipeptide glycylsarcosine. Electrophysiological studies of the transport function of OPT3 in Xenopus oocytes show that this transporter, although capable of mediating H(+)-coupled peptide transport, functions predominantly as a H(+) channel. The H(+) channel activity of OPT3 is approximately 3-4-fold greater than the H(+)/peptide cotransport activity as determined by measurements of H(+) gradient-induced inward currents in the absence and presence of the dipeptide using the two-microelectrode voltage clamp technique. A downhill influx of H(+) was accompanied by a large intracellular acidification as evidenced from the changes in intracellular pH using an ion-selective microelectrode. The H(+) channel activity exhibits a K(0.5)(H) of 1.0 microM at a membrane potential of -50 mV. At the level of primary structure, OPT3 has moderate homology with OPT1 and OPT2, two other H(+)-coupled oligopeptide transporters previously cloned from C. elegans. Expression studies using the opt3::gfp fusion constructs in transgenic C. elegans demonstrate that opt3 gene is exclusively expressed in neurons. OPT3 may play an important physiological role as a pH balancer in the maintenance of H(+) homeostasis in C. elegans.

O.36 Combined growth hormone (GH) and epidermalgrowth factor (EGF) administration upregulates H +-coupled oligopeptide transporter PEPT1 gene expression in rabbit jejunum after small bowel resection

O.36 Combined growth hormone (GH) and epidermalgrowth factor (EGF) administration upregulates H +-coupled oligopeptide transporter PEPT1 gene expression in rabbit jejunum after small bowel resection

Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of β-lactam antibiotics

A polyclonal antibody (anti-PepT1/C) was raised against the rabbit intestinal H +-coupled oligopeptide transporter, PepT1. Anti-PepT1/C detected 70–80-kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush-border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5′-coding region of rabbit PepT1 cDNA.

Mammalian ion‐coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.