No description available.

Absorption of dietary proteins can be met through the uptake of free amino acids or as small peptides. A peptide transport protein, PepT1, is responsible for the absorption of intact peptides arising from digestion of dietary proteins. PepT1 is driven by a H+-coupled transport system that allows for the absorption of small peptides through the intestinal brush border membrane. Screening of a porcine intestinal cDNA library with a sheep PepT1 cDNA probe resulted in the identification of three porcine PepT1 (pPepT1) cDNAs of varying sizes and sequences. Each variant cDNA isolated was cloned into a mammalian expression vector, sequenced, and expressed in Chinese hamster ovary (CHO) cells. Peptide transport was assessed by uptake studies using the radiolabeled dipeptide [3H]-Gly-Sar. Only one of the three cDNAs encoding for a protein of 708 amino acids induced H+-dependent peptide transport activity. Through computer analysis, a putative protein structure for pPepT1 was developed. The transporter has an unusual 13 transmembrane structure with the N-terminus located extracellularly and the C-terminus located intracellularly. Seven glycosylation sites and three protein kinase C phosphorylation sites are located throughout the protein. Expression of pPepT1 activity in CHO cells had a optimal peptide uptake at 18-24 hours. The transporter showed optimal uptake at a pH of 5.5-6.0. Eighteen different unlabeled dipeptides and tripeptides were found to inhibit the uptake of [3H] -Gly-Sar in competition studies. The IC50 of 13 of the dipeptides and two tripeptides ranged between 0.015 to 0.4 mmol/L. The exceptions were Lys-Lys, Arg-Lys, and Lys-Trp-Lys, which showed IC50 values greater than 1.37 mmol/L and appear to be poor substrates for pPepT1. All three of the tetrapeptides examined showed very high IC50 values and inhibition of the uptake of Gly-Sar was too small to measure even at a 10mM concentration. Dipeptides and tripeptides appear to be substrates for the porcine intestinal peptide transporter while tetrapeptides do not appear to be transported. / Master of Science

No description available.

No description available.

No description available.

PepT1 and PepT2 are integral membrane proteins which couple the uptake of di- and tri-peptides to the proton electro-chemical gradient. PepT1 is predominantly expressed in the small intestine and is the main route through which dietary protein is absorbed. PepT2 shares 46% sequence identity with PepT1 and is expressed in the kidneys, lung and central nervous system. Many commonly prescribed drugs, such as penicillin are peptide mimetics, and PepT1 and PepT2 play a direct role in their transport and pharmacokinetic properties. Recent X-ray crystal structures and functional studies of the bacterial peptide transporters have provided significant insight into the likely mechanism by which such drugs are recognised by PepT1 and PepT2. The bacterial peptide transporters share approximately 30% sequence identity within their trans-membrane domains to the mammalian PepT1 and PepT2 transporters. However, a key structural difference exists; an additional 20 kDa extra-cellular loop is inserted between trans-membrane helices 9 and 10 in the animal peptide transporters, and this loop is absent in the bacterial homologues. It was not known, prior to this thesis, if this extra-cellular loop was structured and/or integral to the transport cycle, or whether it served an additional function to assist or regulate peptide transport. To investigate the role of this domain, the crystal structures of the Mus musculus PepT1 and Rattus norvegicus PepT2 'loops' were determined to 2.10 and 2.06 Å resolution respectively. The structures indicated that the loop region in both PepT1 and PepT2 forms a bi-lobal, all β-sheet, self-contained extra-cellular domain (ECD). Despite low sequence similarity, the ECDs from PepT1 and PepT2 share a common architecture; two transthyretin-like folds connected by a flexible linker. Sequence and structural analyses have indicated that the lobe interface of MmPepT^ECD is maintained by two highly conserved salt bridges, whereas in RnPepT2^ECD only one salt bridge is observed. Small-angle X-ray diffraction experiments indicated that the extra-cellular domains form a compact structural arrangement, although the lobe conformation of PepT2^ECD was more dynamic than PepT1. Using the X-ray crystal structures of the M. musculus and R. norvegicus ECDs, and the trans-membrane domain of PepT_so from Shewanella oneidensis, the first structure-based homology models of H. sapiens PepT1 and PepT2 were constructed. The hybrid models indicated that the ECD sits on top of the transporters. Two-electrode voltage clamp studies then revealed that the ECDs do not play a part in the transport mechanism of the transporter, although PepT1^ECD may play a role in transporter stability. Surface plasmon resonance binding assays were performed between the ECDs and the intestinal proteases trypsin and α-chymotrypsin. An interaction between both ECDs and α-chymotrypsin was observed, although this interaction could not be saturated using this assay. Trypsin binding however, could be saturated for both MmPepT1^ECD and RnPepT2^ECD giving K_ds of 90 ± 20 and 170 ± 30 μM. Physiologically the interaction would give trypsin a predisposition for the peptide transporter ECD; locating the protease in the vicinity of the transporter and aiding the presentation of substrate. The interaction between trypsin and PepT1^ECD was explored further with a mutational analysis of potential binding residues. The results could not locate the binding site definitively, however, the work did highlight the probable binding face which is perhaps conserved between PepT1 and PepT2 ECD.

To study peptide absorption in chickens, an intestinal peptide transporter cDNA (cPepT1) was isolated from a chicken cDNA library. The cDNA was 2,914-bp and encoded a protein of 714 amino acid residues. Twenty-three di-, tri-, and tetra-peptides were used for functional analysis of cPepT1 in Xenopus oocytes and Chinese hamster ovary (CHO) cells. For most di- and tripeptides tested, the Kt was in the micromolar range, except Lys-Lys and Lys-Trp-Lys. Northern analysis demonstrated that cPepT1 is expressed strongly in the small intestine, and at lower levels in kidney and cecum. These results demonstrated the presence and functions of a peptide transporter in chickens. cPepT1 mRNA abundance was evaluated in response to developmental and dietary regulations. In Experiment 1, eggs at incubation day 18 (E18) and Cobb chicks after hatch (d 0) were sampled before treatments. Three groups of chicks were fed diets containing 12, 18, or 24% crude protein (CP). Feed intake of chicks fed the 18 or 24% CP diets was restricted to that of chicks fed the 12% CP diet. In Experiment 2, a fourth group with free access to the 24% CP diet was added. cPepT1 mRNA abundance was quantified from northern blots. By d 0, there was a 50-fold increase in cPepT1 mRNA abundance compared with E 18. In chicks fed the 12% CP diet, cPepT1 mRNA abundance decreased throughout the 35 d. Chicks fed 18 or 24% CP diets showed an increase in cPepT1 mRNA abundance with time. In chicks with free access to the 24% CP diet, cPepT1 mRNA decreased until d 14 but returned to an intermediate level at d 35. Our results indicate that cPepT1 mRNA is regulated by both dietary protein and developmental stage. To investigate the kinetics of an ovine peptide transporter (oPepT1), CHO cells were transfected with oPepT1 cDNA. Uptake of Gly-Sar by transfected cells was pH-dependent, concentration-dependent, and saturable. Competition studies showed that all di-, tri-, and tetra-peptides inhibited uptake of Gly-Sar. Pretreatment of the cells with staurosporine resulted in an increase in peptide transport. This increase was blocked by pretreatment with PMA. The results indicate that protein kinase plays a role in oPepT1 function. / Ph. D.

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.

Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepT_So and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepT_So reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.

S. thermophilus est une bactérie largement employée dans la fabrication des produits laitiers. La capacité des souches de S. thermophilus à générer des peptides bioactifs à partir des caséines bovines a été étudiée. Dix souches exprimant différemment la protéase de surface, PrtS, ont été incubées en présence de la caséine [alpha]s1, [alpha]s2 ou [bêta]. Le nombre et le type de peptides libérés dépendent de la souche utilisée. Des peptides connus comme des peptides bioactifs ont été détectés : 13 peptides ont été générés à partir de la caséine [bêta], 5 peptides à partir de la caséine [alpha]s2 et 2 peptides à partir de la caséine [alpha]s1. L'utilisation de cette bactérie pour la production de tels peptides dans l'aliment requiert qu'elle en internalise le moins possible. Nous nous sommes intéressés aux systèmes de transport de peptides présents au sein de l'espèce S. thermophilus. Une collection de 22 souches de S. thermophilus a été choisie pour étudier la variabilité des systèmes de transport des peptides présents au sein de l'espèce. Toute d'abord, la proximité phylogénétique entre les souches a été évaluée par MLST, puis la variabilité génétique du système de transport des oligopeptides Ami et du transporteur de di- et tripeptides DtpT a été étudiée au sein de cette collection. Un cluster, composé de 4 gènes, annoté en tant que transporteur ABC de peptides et de nickel a été détecté au sein du génome de la souche LMD-9, et appelé Ots. Il est présent chez 9 sur 22 souches de S. thermophilus et est transcrit tout au long de la croissance en milieu M17. La caractérisation du système Ots suggère qu'il est impliqué dans l'internalisation de peptides de petites tailles / S. thermophilus is a widely used bacterium in the manufacture of dairy products. The capacity of S. thermophilus to generate bioactive peptides from bovine caseins was studied. Ten strains expressing different levels of the cell envelope protease, PrtS, were incubated with [alpha]s1-, [alpha]s2- or [beta]-casein. Number and type of peptides released were strain-dependent. Peptides known as bioactive peptides were detected: 13 peptides were generated from [beta]-casein, 5 peptides from [alpha]s2-casein and 2 peptides from [alpha]s1-casein. The use of this bacterium for the production of such peptides in the food products requires the least internalization of these peptides by this bacterium. We were interested in knowing the peptide transport system present in the species S. thermophilus. A collection of 22 strains of S. thermophilus was chosen to study the genetic variability of peptide transport systems present within the species. First of all, we evaluated the phylogenetic proximity between selected strains by MLST, and then the genetic variability of the transport system of oligopeptides Ami and di- and tripeptides transporter, DtpT were studied in this collection. A cluster consisting of four genes, annotated as ABC transporter of peptides and nickel was detected in the genome of strain LMD-9, and called Ots. It is present in 9 of 22 strains of S. thermophilus and is transcribed throughout the growth in M17 medium. The Ots system seems to be involved in the internalization of smaller sized peptides