Proton-coupled Oligopeptide Transporter Research Articles

The mechanism of mammalian proton-coupled peptide transporters.

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

The mechanism of mammalian proton-coupled peptide transporters

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

Molecular Insights to the Structure-Interaction Relationships of Human Proton-Coupled Oligopeptide Transporters (PepTs).

Human proton-coupled oligopeptide transporters (PepTs) are important membrane influx transporters that facilitate the cellular uptake of many drugs including ACE inhibitors and antibiotics. PepTs mediate the absorption of di- and tri-peptides from dietary proteins or gastrointestinal secretions, facilitate the reabsorption of peptide-bound amino acids in the kidney, and regulate neuropeptide homeostasis in extracellular fluids. PepT1 and PepT2 have been the most intensively investigated of all PepT isoforms. Modulating the interactions of PepTs and their drug substrates could influence treatment outcomes and adverse effects with certain therapies. In recent studies, topology models and protein structures of PepTs have been developed. The aim of this review was to summarise the current knowledge regarding structure-interaction relationships (SIRs) of PepTs and their substrates as well as the potential applications of this information in therapeutic optimisation and drug development. Such information may provide insights into the efficacy of PepT drug substrates in patients, mechanisms of drug-drug/food interactions and the potential role of PepTs targeting in drug design and development strategies.

Proton-Coupled Oligopeptide Transport (Slc15) in the Brain: Past and Future Research.

This mini-review describes the role of the solute carrier (SLC)15 family of proton-coupled oligopeptide transporters (POTs) and particularly Pept2 (Slc15A2) and PhT1 (Slc15A4) in the brain. That family transports endogenous di- and tripeptides and peptidomimetics but also a number of drugs. The review focuses on the pioneering work of David E. Smith in the field in identifying the impact of PepT2 at the choroid plexus (the blood-CSF barrier) as well as PepT2 and PhT1 in brain parenchymal cells. It also discusses recent findings and future directions in relation to brain POTs including cellular and subcellular localization, regulatory pathways, transporter structure, species differences and disease states.

Mulberry leaf and konjac compound powder improves the metabolic capacity of old mice on a high-protein diet by regulating the structure of the intestinal microbiota.

The health of elderly individuals is closely linked to their protein intake and the abundance of intestinal microbiota. To investigate the impact of a compound powder made from mulberry leaf and konjac (hereinafter referred to as 'compound powder') on regulating the structure of intestinal microbiota in 15-month-old BALB/c mice that were fed a high-beef-protein diet, 16S rRNA high-throughput sequencing, reverse transcription quantitative polymerase reaction, western blot, and other biochemical methods were used to analyze the differences in intestinal microbiota, protein metabolism-related genes, short-chain fatty acid (SCFA) content, and serum cytokines. The results showed that the compound powder increased the content of SCFAs, reduced the inflammatory reaction of the body, adjusted the abundance of intestinal microbiota (Firmicutes and Bacteroidetes), and increased the ratio of Firmicutes to Bacteroidetes (F/B). Moreover, the compound powder could increase the abundance of Lactobacillus and some non-dominant bacteria that were related to amino acid metabolism and beneficial to human health, such as Eubacterium coprostanoligenes. These beneficial bacteria competitively reduced the abundance of harmful bacteria to protect the intestinal barrier and promote intestinal health, and upregulated the activities of aminopeptidase, proton-coupled oligopeptide transporter 1, and glutamate dehydrogenase at the transcription and translation levels. The compound powder could balance the abundance of intestinal microbiota, which may improve the metabolic capacity of old mice on a high-protein diet, and ultimately promoting the well-being of elderly individuals. © 2023 Society of Chemical Industry.

Functional Characterization of the Putative POT from Clostridium perfringens.

Proton-coupled oligopeptide transporters (POTs) are a fundamental part of the cellular transport machinery that provides plants, bacteria, and mammals with nutrition in the form of short peptides. However, POTs are not restricted to peptide transport; mammalian POTs have especially been in focus due to their ability to transport several peptidomimetics in the small intestine. Herein, we studied a POT from Clostridium perfringens (CPEPOT), which unexpectedly exhibited atypical characteristics. First, very little uptake of a fluorescently labelled peptide β-Ala-Lys-AMCA, an otherwise good substrate of several other bacterial POTs, was observed. Secondly, in the presence of a competitor peptide, enhanced uptake of β-Ala-Lys-AMCA was observed due to trans-stimulation. This effect was also observed even in the absence of a proton electrochemical gradient, suggesting that β-Ala-Lys-AMCA uptake mediated by CPEPOT is likely through the substrate-concentration-driving exchange mechanism, unlike any other functionally characterized bacterial POTs.

Substrate space analysis of the bacterial proton-coupled oligopeptide transporter YdgR by cheminformatics.

Proton-dependent oligopeptide transporters (POTs) are recognized for their substrate promiscuity due to their ability to transport a wide range of substrates. POTs are conserved in all forms of life ranging from bacteria to humans. A dipeptide-fluorophore conjugate, H-(β-Ala)-Lys(AMCA)-OH, is a well-known substrate of the transporter YdgR that is commonly used as a fluorescent reporter. In order to understand the substrate space of YdgR, we used this dipeptide as a bait reference, when screening an ensemble of compounds (previously tested in PEPT/PTR/NPF space) via a cheminformatic analysis based on the Tanimoto similarity index. Eight compounds (sinalbin, abscisic acid, carnosine, jasmonic acid, N-acetyl-aspartate, N-acetyl-lysine, aspartame, and N-acetyl-aspartylglutamate), covering a wide range on the Tanimoto scale, were tested for YdgR-mediated transport. Carnosine was the only compound observed to be a YdgR substrate based on cell-based transport assays and molecular docking. The other compounds tested were neither inhibitors nor substrates. Thus, we found that neither the Tanimoto similarity index nor ADME (absorption, distribution, metabolism, and excretion) properties appear useful for the identification of substrates (e.g., dipeptides) in YdgR-mediated drug transport.

Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C.

Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa Pro-macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 Å. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

Dual-modified nanoparticles overcome sequential absorption barriers for oral insulin delivery

The efficacy of oral insulin drug delivery is seriously hampered by multiple gastrointestinal barriers, especially transepithelial barriers, including apical endocytosis, lysosomal degradation, cytosolic diffusion and basolateral exocytosis. In this study, a functional nanoparticle (PG-FAPEP) with dual-modification was constructed to sequentially address these important absorption obstacles for improved oral insulin delivery. The dual surface decorations folate and charge-convertible tripeptide endowed PG-FAPEP with the ability to target the apical and basolateral sides of enterocytes, respectively. After fast diffusion across the mucus layer, PG-FAPEP could be efficiently internalized into epithelial cells via a folate receptor-mediated pathway and subsequently became positively charged in acidic lysosomes due to the surface tripeptide, triggering the proton sponge effect to escape lysosomes. When entering the cytosolic medium, PG-FAPEP was converted to neutral charge again, attenuating intracellular adhesion, and gained improved motility toward the basolateral side. Finally, the tripeptide helped PG-FAPEP recognize the proton-coupled oligopeptide transporter (PHT1) in the basolateral membrane, boosting intact exocytosis across intestinal epithelial cells. The in vivo studies further verified that PG-FAPEP could traverse the intestinal epithelium by folate receptor-mediated endocytosis, lysosomal escape, and PHT1-mediated exocytosis, exhibiting a high oral insulin bioavailability of 14.3% and a prolonged hypoglycemic effect. This formulation addresses multiple absorption barriers on demand with a simple dual-modification strategy. Therefore, these features allow PG-FAPEP to unleash the potential of oral macromolecule delivery.

Intraluminal Behavior of Various Transporter Substrates in the Rat Gastrointestinal Tract.

The aim of this study was to evaluate the intraluminal behavior of various transporter substrates in different regions of the gastrointestinal (GI) tract. Drug solutions containing non-absorbable FITC-dextran 4000 (FD-4), were orally administered to rats. Residual water was sampled from the GI regions to measure the luminal drug concentration. Cephalexin (CEX), a substrate of the proton-coupled oligopeptide transporter, was absorbed rapidly, and no drug was detected in the lower small intestine. Saquinavir (SQV) was primarily absorbed in the upper region. However, unlike CEX, SQV was detected even in the lower segment probably due to the efflux of SQV via P-glycoprotein (P-gp). The concentration of methotrexate (MTX) showed a similar pattern to that of non-absorbable FD-4. The low absorption of MTX was probably due to efflux via several efflux transporters, and the limited expression of proton-coupled folate transporter, an absorptive transporter for MTX, in the upper region. This study revealed that the luminal concentration pattern of each drug differed considerably depending on the site because of the different absorption properties and luminal volumes. Although further investigation using a specific transporter inhibitor or transporter-knockout animals are necessary to clarify the actual contribution of each transporter to the drug absorption, this information will be valuable in evaluating transporter-mediated drug absorption in in vitro transport studies for ensuring optimal drug concentrations.

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.

Mechanistic Picture for Chemomechanical Coupling in a Bacterial Proton-Coupled Oligopeptide Transporter from Streptococcus Thermophilus.

Proton-coupled oligopeptide transporters (POTs) use the proton electrochemical gradient to transport peptides across the cell membrane. Despite the significant biological and biomedical relevance of these proteins, a detailed mechanistic picture for chemomechanical couplings involved in substrate/proton transport and protein structural changes is missing. Therefore, we performed microsecond-level molecular dynamics simulations of bacterial POT PepTSt, which shares ∼80% sequence identity with the human POT, PepT1, in the substrate-binding region. Three different conformational states of PepTSt were simulated, including (i) occluded, apo, (ii) inward-facing, apo, and (iii) inward-facingoccluded, Leu-Ala bound. We propose that the interaction of R33 with E299 and E300 acts as a conformational switch (i.e., to trigger the conformational change from an inward- to outward-facing state) in the substrate transport. Additionally, we propose that E299 and E400 disengage from interacting with the substrate either through protonation or through coordination with a cation for the substrate to get transported. This study provides clues to understand the chemomechanical couplings in POTs and paves the way to decipher the molecular-level underpinnings of the structure-function relationship in this important family of transporters.

Membrane Chemistry Tunes the Structure of a Peptide Transporter.

Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space.

Membrane Chemistry Tunes the Structure of a Peptide Transporter

AbstractMembrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space.

Recent advances in understanding prodrug transport through the SLC15 family of proton-coupled transporters

Solute carrier (SLC) transporters play important roles in regulating the movement of small molecules and ions across cellular membranes. In mammals, they play an important role in regulating the uptake of nutrients and vitamins from the diet, and in controlling the distribution of their metabolic intermediates within the cell. Several SLC families also play an important role in drug transport and strategies are being developed to hijack SLC transporters to control and regulate drug transport within the body. Through the addition of amino acid and peptide moieties several novel antiviral and anticancer agents have been developed that hijack the proton-coupled oligopeptide transporters, PepT1 (SCL15A1) and PepT2 (SLC15A2), for improved intestinal absorption and renal retention in the body. A major goal is to understand the rationale behind these successes and expand the library of prodrug molecules that utilise SLC transporters. Recent co-crystal structures of prokaryotic homologues of the human PepT1 and PepT2 transporters have shed important new insights into the mechanism of prodrug recognition. Here, I will review recent developments in our understanding of ligand recognition and binding promiscuity within the SLC15 family, and discuss current models for prodrug recognition.

Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide

Peptide transporters 1 and 2 (PEPT1 and PEPT2) are proton-coupled oligopeptide transporter members of the solute carrier 15 family and play a role in the cellular uptake of di/tri-peptides and peptidomimetics. Our previous work showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes. Here we show that PEPT2 expression decreases as keratinocyte differentiation progresses and that PEPT1 alternately is expressed at later stages. Absolute quantification using quantitative polymerase chain reaction revealed that the expression level of PEPT1 is about 17 times greater than that of PEPT2. Immunohistochemical study of human skin provided evidence of PEPT1 in the epidermis. The uptake of glycylsarcosine into keratinocytes was significantly blocked by PEPT inhibitors, including nateglinide and glibenclamide. Moreover, we found that PEPT1 knockdown in differentiated keratinocytes significantly suppressed the influence of a bacterial-derived peptide, muramyl dipeptide (MDP), on the production of proinflammatory cytokine interleukin-8, implying that bacteria-derived oligopeptides can be transported by PEPT1 in advanced differentiated keratinocytes. Taken together, PEPT1 and PEPT2 may concertedly play an important role in MDP-NOD2 signaling in the epidermis, which provides new insight into the mechanisms of skin homeostasis against microbial pathogens.

Exploring Conformational Transitions and Free-Energy Profiles of Proton-Coupled Oligopeptide Transporters.

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane α helices that come together to form a "V" shaped transporter with a central substrate binding site. Proton-coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF), and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding; however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands is still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free-energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both proton and ligand significantly change the conformational free-energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free-energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations became accessible. For the Glu300 protonated Holo-PepTst, the presence of a second free-energy minimum suggests that OF conformations are not only accessible, but also stable. The differences in the free-energy profiles demonstrate that transitions toward outward-facing conformation occur only after protonation, which is likely the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.

Carnosine Supplementation Enhances Post Ischemic Hind Limb Revascularization.

High (millimolar) concentrations of the histidine containing dipeptide – carnosine (β-alanine-L-histidine) are present in the skeletal muscle. The dipeptide has been shown to buffer intracellular pH, chelate transition metals, and scavenge lipid peroxidation products; however, its role in protecting against tissue injury remains unclear. In this study, we tested the hypothesis that carnosine protects against post ischemia by augmenting HIF-1α angiogenic signaling by Fe2+ chelation. We found that wild type (WT) C57BL/6 mice, subjected to hind limb ischemia (HLI) and supplemented with carnosine (1g/L) in drinking water, had improved blood flow recovery and limb function, enhanced revascularization and regeneration of myocytes compared with HLI mice placed on water alone. Carnosine supplementation enhanced the bioavailability of carnosine in the ischemic limb, which was accompanied by increased expression of proton-coupled oligopeptide transporters. Consistent with our hypothesis, carnosine supplementation augmented HIF-1α and VEGF expression in the ischemic limb and the mobilization of proangiogenic Flk-1+/Sca-1+ cells into circulation. Pretreatment of murine myoblast (C2C12) cells with octyl-D-carnosine or carnosine enhanced HIF-1α protein expression, VEGF mRNA levels and VEGF release under hypoxic conditions. Similarly pretreatment of WT C57/Bl6 mice with carnosine showed enhanced blood flow in the ischemic limb following HLI surgery. In contrast, pretreatment of hypoxic C2C12 cells with methylcarcinine, a carnosine analog, lacking Fe2+ chelating capacity, had no effect on HIF-1α levels and VEGF release. Collectively, these data suggest that carnosine promotes post ischemic revascularization via augmentation of pro-angiogenic HIF-1α/VEGF signaling, possibly by Fe2+ chelation.

Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells

PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, with Km values of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a Km value of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2.

The proton-coupled oligopeptide transporters PEPT2, PHT1 and PHT2 mediate the uptake of carnosine in glioblastoma cells.

The previous studies demonstrated that carnosine (β-alanyl-L-histidine) inhibits the growth of tumor cells in vitro and in vivo. Considering carnosine for the treatment of glioblastoma, we investigated which proton-coupled oligopeptide transporters (POTs) are present in glioblastoma cells and how they contribute to the uptake of carnosine. Therefore, mRNA expression of the four known POTs (PEPT1, PEPT2, PHT1, and PHT2) was examined in three glioblastoma cell lines, ten primary tumor cell cultures, in freshly isolated tumor tissue and in healthy brain. Using high-performance liquid chromatography coupled to mass spectrometry, the uptake of carnosine was investigated in the presence of competitive inhibitors and after siRNA-mediated knockdown of POTs. Whereas PEPT1 mRNA was not detected in any sample, expression of the three other transporters was significantly increased in tumor tissue compared to healthy brain. In cell culture, PHT1 expression was comparable to expression in tumor tissue, PHT2 exhibited a slightly reduced expression, and PEPT2 expression was reduced to normal brain tissue levels. In the cell line LN405, the competitive inhibitors β-alanyl-L-alanine (inhibits all transporters) and L-histidine (inhibitor of PHT1/2) both inhibited the uptake of carnosine. SiRNA-mediated knockdown of PHT1 and PHT2 revealed a significantly reduced uptake of carnosine. Interestingly, despite its low expression at the level of mRNA, knockdown of PEPT2 also resulted in decreased uptake. In conclusion, our results demonstrate that the transporters PEPT2, PHT1, and PHT2 are responsible for the uptake of carnosine into glioblastoma cells and full function of all three transporters is required for maximum uptake.