Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1.

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Expression of peptide transporter 1 has a positive correlation in protoporphyrin IX accumulation induced by 5-aminolevulinic acid with photodynamic detection of non-small cell lung cancer and metastatic brain tumor specimens originating from non-small cell lung cancer.

BACKGROUND: Recently, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence was reported to be a useful tool during total surgical resection of high-grade gliomas. However, the labeling efficacy of protoporphyrin IX fluorescence is lower in metastatic brain tumors compared to that in high-grade gliomas, and the mechanism underlying protoporphyrin IX fluorescence in metastatic brain tumors remains unclear. Lung cancer, particularly non-small cell lung cancer (NSCLC), is the most common origin for metastatic brain tumor. Therefore, we investigated the mechanism of protoporphyrin IX fluorescence in NSCLC and associated metastatic brain tumors. METHODS: Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to evaluate the protein and mRNA levels of five transporters and enzymes involved in the porphyrin biosynthesis pathway: peptide transporter 1 (PEPT1), hydroxymethylbilane synthase (HMBS), ferrochelatase (FECH), ATP-binding cassette 2 (ABCG2), and heme oxygenase 1 (HO-1). The correlation between protein, mRNA, and protoporphyrin IX levels in NSCLC cells were evaluated in vitro. Immunohistochemistry was used to determine proteins that played a key role in intraoperative protoporphyrin IX fluorescence in clinical samples from patients with NSCLC and pathologically confirmed metastatic brain tumors. RESULTS: A significant correlation between PEPT1 expression and protoporphyrin IX accumulation in vitro was identified by western blotting (P = 0.003) and qRT-PCR (P = 0.04). Immunohistochemistry results indicated that there was a significant difference in PEPT1 between the intraoperative protoporphyrin IX fluorescence-positive and protoporphyrin IX fluorescence-negative groups (P = 0.009). CONCLUSION: Expression of PEPT1 was found to be positively correlated with 5-ALA-induced protoporphyrin IX accumulation detected by photodynamic reaction in metastatic brain tumors originating from NSCLC.

Resveratrol enhances the protective effects of JBP485 against indomethacin-induced rat intestinal damage in vivo and vitro through up-regulating oligopeptide transporter 1 (Pept1).

JBP485 is a hydrophilic dipeptide with protective effects on intestine, liver and kidney. However, oral bioavailability of JBP485 is limited due to the impaired absorptive function during intestinal injury. The purpose of this study was to investigate the effect of resveratrol (Res) on the protective effect of JBP485 against indomethacin-induced intestinal injury in rats and in IEC-6 cells. JBP485 treatment ameliorated indomethacin-induced enteropathy characterized by severe histopathological changes and a large number of TUNEL-positive cells in rat intestinal epithelium, elevated levels of MDA, MPO, TNF-α, IL-6, IL-1ß and increased expression of COX-2, and reduced levels of SOD, GSH, CAT, HDL-c, PGE2, expression of COX-1 and Bcl-2/Bax ratio in rat intestinal tissues. Co-treatment with Res enhanced activity of JBP485 against indomethacin-induced intestinal injury in rats and in IEC-6 cells. Furthermore, Res increased the plasma concentration of JBP485 in both indomethacin-treated and healthy rats, as well as the intracellular accumulation of JBP485 in IEC-6 cells, through upregulation of Pept1. In conclusion, JBP485 alleviated indomethacin-induced intestinal injury through restoring the redox balance, suppressing inflammation, reducing apoptosis, which was further enhanced by Res via upregulation of Pept1 and improvement of bioavailability of JBP485, at least in part.

Evaluating the intestinal and oral absorption of the prodrug valacyclovir in wildtype and huPepT1 transgenic mice.

The purpose of this work was to evaluate the intestinal permeability, oral absorption and disposition of the ester prodrug valacyclovir in wildtype mice and a huPepT1 transgenic mouse model. PepT1 (SLC15A1) is a transporter apically expressed along the lumen of the gastrointestinal tract and is responsible for the absorption of di-/tripeptides, ACE inhibitors, ß-lactam antibiotics and numerous prodrugs. Unfortunately, PepT1-mediated substrates that have been extensively studied were shown to exhibit species-dependent absorption and pharmacokinetics. Accordingly, in situ intestinal perfusion studies were conducted and valacyclovir uptake was shown to have a 30-fold lower Km and 100-fold lower Vmax in huPepT1 compared to wildtype mice. Moreover, inhibition studies demonstrated that the huPepT1 transporter alone was responsible for valacyclovir uptake, and segment-dependent studies reported significant reductions in permeability along the length of small intestine in huPepT1 mice. Subsequent oral administration studies revealed that the in vivo rate and extent of valacyclovir absorption were lower in huPepT1 mice, as indicated by 3-fold lower Cmax and 3-fold higher Tmax values, and an AUC0-180 that was 80% of that observed in wildtype mice. However, no significant changes in drug disposition were observed between genotypes after intravenous bolus administration of acyclovir. Lastly, mass balance studies established that the bioavailability of acyclovir, after oral dosing of valacyclovir, was 77.5% in wildtype mice and 52.8% in huPepT1 mice, which corroborated values of 51.3% in clinical studies. Thus, it appears the huPepT1 transgenic mice may be a better model to study prodrug absorption and disposition in humans than wildtype mice.