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BMC Biology Aug 2015Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral...
BACKGROUND
Peptide transporters are membrane proteins that mediate the cellular uptake of di- and tripeptides, and of peptidomimetic drugs such as β-lactam antibiotics, antiviral drugs and antineoplastic agents. In spite of their high physiological and pharmaceutical importance, the molecular recognition by these transporters of the amino acid side chains of short peptides and thus the mechanisms for substrate binding and specificity are far from being understood.
RESULTS
The X-ray crystal structure of the peptide transporter YePEPT from the bacterium Yersinia enterocolitica together with functional studies have unveiled the molecular bases for recognition, binding and specificity of dipeptides with a charged amino acid residue at the N-terminal position. In wild-type YePEPT, the significant specificity for the dipeptides Asp-Ala and Glu-Ala is defined by electrostatic interaction between the in the structure identified positively charged Lys314 and the negatively charged amino acid side chain of these dipeptides. Mutagenesis of Lys314 into the negatively charged residue Glu allowed tuning of the substrate specificity of YePEPT for the positively charged dipeptide Lys-Ala. Importantly, molecular insights acquired from the prokaryotic peptide transporter YePEPT combined with mutagenesis and functional uptake studies with human PEPT1 expressed in Xenopus oocytes also allowed tuning of human PEPT1's substrate specificity, thus improving our understanding of substrate recognition and specificity of this physiologically and pharmaceutically important peptide transporter.
CONCLUSION
This study provides the molecular bases for recognition, binding and specificity of peptide transporters for dipeptides with a charged amino acid residue at the N-terminal position.
Topics: Biological Transport; Carrier Proteins; Ligands; Membrane Transport Proteins; Static Electricity; Substrate Specificity; Yersinia enterocolitica
PubMed: 26246134
DOI: 10.1186/s12915-015-0167-8 -
Biomolecules Jun 2023ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein-protein interactions....
ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein-protein interactions. As peptides of molecular weights over 1000 Da are not usually evaluated, data on the drug-drug interaction (DDI) potential of stapled α-helical peptides remain scarce. Here, we evaluate the interaction of ATSP-7041 with hepatic cytochrome P450s (CYPs; CYP1A2, CYP2C9, CYP2C19, CYP3A4, and CYP2D6) and transporters (organic anion transporting polypeptides (OATPs; OATP1B1 and OATP1B3), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP)). ATSP-7041 demonstrated negligible metabolism in human liver S9 fraction and a limited inhibition of CYP activities in yeast microsomes or S9 fractions. On the contrary, a substantial uptake by OATPs in HEK 293 cells, a strong inhibition of OATP activities in the cells, and an inhibition of P-gp and BCRP activities in reversed membrane vesicles were observed for ATSP-7041. A recent report describes that ALRN-6924, an ATSP-7041 analog, inhibited OATP activities in vivo; therefore, we focused on the interaction between ATSP-7041 and OATP1B1 to demonstrate that ATSP-7041, as a higher molecular weight stapled peptide, is a substrate and strong inhibitor of OATP1B1 activity. Our findings demonstrated the possibility of transporter-mediated DDI potential by high molecular weight stapled peptides and the necessity of their evaluation for drug development.
Topics: Humans; Mice; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; HEK293 Cells; Neoplasm Proteins; Peptides; Membrane Transport Proteins; Organic Anion Transporters; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cytochrome P-450 Enzyme System
PubMed: 37371582
DOI: 10.3390/biom13061002 -
Scientific Reports Mar 2022Tripartite resistance-nodulation-division (RND) efflux pumps, such as AcrAB-TolC of Salmonella Typhimurium, contribute to antibiotic resistance and comprise an inner...
Tripartite resistance-nodulation-division (RND) efflux pumps, such as AcrAB-TolC of Salmonella Typhimurium, contribute to antibiotic resistance and comprise an inner membrane RND-transporter, an outer membrane factor, and a periplasmic adaptor protein (PAP). The role of the PAP in the assembly and active transport process remains poorly understood. Here, we identify the functionally critical residues involved in PAP-RND-transporter binding between AcrA and AcrB and show that the corresponding RND-binding residues in the closely related PAP AcrE, are also important for its interaction with AcrB. We also report a residue in the membrane-proximal domain of AcrA, that when mutated, differentially affects the transport of substrates utilising different AcrB efflux channels, namely channels 1 and 2. This supports a potential role for the PAP in sensing the substrate-occupied state of the proximal binding pocket of the transporter and substrate vetting. Understanding the PAP's role in the assembly and function of tripartite RND pumps can guide novel ways to inhibit their function to combat antibiotic resistance.
Topics: Adaptor Proteins, Signal Transducing; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Biological Transport; Escherichia coli Proteins; Membrane Transport Proteins; Multidrug Resistance-Associated Proteins; Periplasm; Salmonella typhimurium
PubMed: 35306531
DOI: 10.1038/s41598-022-08903-9 -
Proceedings of the National Academy of... Jan 2016The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface...
The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek's disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC).
Topics: Amino Acid Sequence; Animals; Biological Transport; Cell Membrane; Chickens; Epitopes; Erythrocytes; Haplotypes; Heterozygote; Histocompatibility Antigens Class I; Homozygote; Membrane Transport Proteins; Molecular Sequence Data; Peptides; Protein Stability; Substrate Specificity; Temperature; beta 2-Microglobulin
PubMed: 26699458
DOI: 10.1073/pnas.1511859113 -
Yakugaku Zasshi : Journal of the... 2016Membrane proteins allow a cell to communicate with its environment by relaying a signal or transporting a molecule through the cell membrane. Elucidation of the... (Review)
Review
Membrane proteins allow a cell to communicate with its environment by relaying a signal or transporting a molecule through the cell membrane. Elucidation of the three-dimensional structure of a membrane protein provides a greater understanding of its function and mechanisms. Ultimately, this knowledge will enlighten researchers on how these proteins can be regulated to elicit a desired cellular response, which could lead to novel therapeutic medicine. Unfortunately, the determination of the high-resolution crystal structures of transmembrane proteins remains a challenge due to their poor solubility and high conformational flexibility. Additives and cocrystallization ligands are being used to address these problems. In vitro selected macrocyclic peptides have recently been successfully employed as cocrystallization ligands. Although originally intended as inhibitors and drug lead molecules, in vitro selected macrocyclic peptides are now showing that their pharmacodynamic properties also allow them to serve as excellent cocrystallization ligands. Structures for macrocyclic peptide-bound transporters, the multidrug and toxic compound extrusion family transporter from Pyrococcus furiosus (PfMATE) and the ABC transporter subfamily B member 1 from Cyanidioschyzon meraloe (CmABCB1), have been elucidated using X-ray crystallography. The cocrystal structures reveal that the macrocyclic peptides improve crystallization by binding in a similar manner as a small molecule or a biologic. The PfMATE-binding macrocyclic peptides MaD3S and MaD5 bind to the surfaces buried in the center channel of the transporters. Although both transporters possess a center channel and substrate-binding pocket, the CmABCB1-binding macrocyclic peptide, aCAP, binds to the outer surface of the transporter in a similar manner to a biologic.
Topics: ATP-Binding Cassette Transporters; Cell Communication; Cell Membrane; Crystallization; Crystallography, X-Ray; In Vitro Techniques; Ligands; Membrane Transport Proteins; Multiprotein Complexes; Peptides; Protein Binding; Protein Conformation; Signal Transduction
PubMed: 26831792
DOI: 10.1248/yakushi.15-00229-4 -
Scientific Reports Sep 2019In γ-proteobacterial species, such as Escherichia coli, the Arc (anoxic redox control) two-component system plays a major role in mediating the metabolic transition...
In γ-proteobacterial species, such as Escherichia coli, the Arc (anoxic redox control) two-component system plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis, and thus is crucial for anaerobic growth but dispensable for aerobic growth. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, Arc (SoArc) primarily affects aerobic growth. To date, how this occurs has remained largely unknown although the growth defect resulting from the loss of DNA-binding response regulator SoArcA is tryptone-dependent. In this study, we demonstrated that the growth defect is in part linked to utilization of oligopeptides and di-tripeptides, and peptide uptake but not peptide degradation is significantly affected by the SoArcA loss. A systematic characterization of major small peptide uptake systems manifests that ABC peptide transporter Sap and four proton-dependent oligopeptide transporters (POTs) are responsible for transport of oligopeptides and di-tripeptides respectively. Among them, Sap and DtpA (one of POTs) are responsive to the SoarcA mutation but only dtpA is under the direct control of SoArcA. We further showed that both Sap and DtpA, when overproduced, improve growth of the SoarcA mutant. While the data firmly establish a link between transport of oligopeptides and di-tripeptides and the SoarcA mutation, other yet-unidentified factors are implicated in the growth defect resulting from the SoArcA loss.
Topics: Anaerobiosis; Bacterial Outer Membrane Proteins; Bacterial Proteins; Biological Transport; Gene Expression Regulation, Bacterial; Membrane Transport Proteins; Mutation; Oligopeptides; Shewanella
PubMed: 31554843
DOI: 10.1038/s41598-019-50201-4 -
Biochemical and Biophysical Research... Feb 2011Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25...
Retinal pigment epithelial cells (RPE) express two transport systems (SOPT1 and SOPT2) for oligopeptides. Hepcidin is an iron-regulatory peptide hormone consisting of 25 amino acids. This hormone binds to ferroportin, an iron exporter expressed on the cell surface, and facilitates its degradation. Here we investigated if hepcidin is a substrate for SOPT1 and SOPT2 and if the hormone has any intracellular function in RPE. Hepcidin inhibited competitively the uptake of deltorphin II (a synthetic oligopeptide substrate for SOPT1) and DADLE (a synthetic oligopeptide substrate for SOPT2) with IC50 values in the range of 0.4-1.7 μM. FITC-hepcidin was taken up into RPE, and this uptake was inhibited by deltorphin II and DADLE. The entry of FITC-hepcidin into cells was confirmed by flow cytometry. Incubation of RPE with hepcidin decreased the levels of ferroportin mRNA. This effect was not a consequence of hepcidin-induced ferroportin degradation because excessive iron accumulation in RPE, which is expected to occur in these cells as a result of ferroportin degradation, did not decrease but instead increased the levels of ferroportin mRNA. This study reveals for the first time a novel intracellular function for hepcidin other than its established cell surface action on ferroportin.
Topics: Animals; Antimicrobial Cationic Peptides; Cation Transport Proteins; Cell Line; Down-Regulation; Hepcidins; Homeostasis; Humans; Iron; Iron-Regulatory Proteins; Membrane Transport Proteins; Mice; Oligopeptides; Protein Transport; RNA, Messenger; Retinal Pigment Epithelium
PubMed: 21219868
DOI: 10.1016/j.bbrc.2011.01.018 -
Biochimica Et Biophysica Acta Mar 1997The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the renal peptide transporter (PEPT...
The present study was undertaken to investigate the interaction of anionic cephalosporins (cefixime, ceftibuten, and cefdinir) with the renal peptide transporter (PEPT 2) and the intestinal peptide transporter (PEPT 1) using four different experimental model systems. In the first approach, the human colon carcinoma cell line Caco-2 which expresses PEPT 1 and the SHR rat kidney cell line SKPT which expresses PEPT 2 were used. The uptake of the dipeptide Gly-Sar mediated by PEPT 1 or PEPT 2 in these cells was inhibited significantly by the anionic cephalosporins, with the following order of potency: ceftibuten > cefixime > cefdinir. The inhibition was competitive in nature. Even though the order of potency was the same for PEPT 1 and PEPT 2, PEPT 1 exhibited much lesser sensitivity to inhibition than PEPT 2. In the second approach, the cloned human PEPT 1 and PEPT 2 were functionally expressed in HeLa cells following which the cells were used to study the interaction of anionic cephalosporins with PEPT 1 and PEPT 2. Again, Gly-Sar uptake mediated by the human PEPT 1 and PEPT 2 in HeLa cells was found to be inhibited by the anionic cephalosporins with the same order potency as in Caco-2 and SKPT cells. In the third approach, brush border membrane vesicles isolated from rat kidneys were employed. In this approach also it was found that PEPT 2-mediated Gly-Sar uptake was inhibited by cefixime and ceftibuten. In the fourth approach, the human PEPT 1 was expressed in Xenopus laevis oocytes and PEPT 1-mediated transport of ceftibuten was investigated directly by electrophysiological methods. Ceftibuten evoked inward currents in PEPT 1-expressing oocytes but not in water-injected oocytes, showing that the transport of the anionic cephalosporin via PEPT 1 is associated with transfer of positive charge. The ceftibuten-evoked currents were saturable with respect to ceftibuten concentration and were markedly dependent on membrane potential. It is concluded that anionic cephalosporins interact with the peptide transporters expressed in the intestine (PEPT 1) as well as in the kidney (PEPT 2).
Topics: Animals; Biological Transport; Caco-2 Cells; Carrier Proteins; Cell Line; Cephalosporins; Colon; Dipeptides; Evoked Potentials; HeLa Cells; Humans; Kidney; Microvilli; Oocytes; Peptide Transporter 1; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Symporters; Transfection; Xenopus laevis
PubMed: 9092716
DOI: 10.1016/s0005-2736(96)00234-9 -
Applied and Environmental Microbiology Jul 2018The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of is an ABC-type...
The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA. characterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides. Fundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.
Topics: Amides; Bacterial Proteins; Biological Transport; Dipeptides; Escherichia coli; Escherichia coli Proteins; Glutamic Acid; Glutathione; Kinetics; Membrane Transport Proteins; Metabolic Networks and Pathways; Mutation; Peptides; Periplasmic Binding Proteins; Substrate Specificity; gamma-Glutamyltransferase
PubMed: 29728377
DOI: 10.1128/AEM.00340-18 -
Microbiological Research 2003The twin arginine translocation (Tat) system is a machinery which can translocate folded proteins across energy transducing membranes. Currently it is supposed that Tat... (Review)
Review
The twin arginine translocation (Tat) system is a machinery which can translocate folded proteins across energy transducing membranes. Currently it is supposed that Tat substrates bind directly to Tat translocon components before a ApH-driven translocation occurs. In this review, an alternative model is presented which proposes that membrane integration could precede Tat-dependent translocation. This idea is mainly supported by the recent observations of Tat-independent membrane insertion of Tat substrates in vivo and in vitro. Membrane insertion may allow i) a quality control of the folded state by membrane bound proteases like FtsH, ii) the recognition of the membrane spanning signal peptide by Tat system components, and iii) a pulling mechanism of translocation. In some cases of folded Tat substrates, the membrane targeting process may require ATP-dependent N-terminal unfolding-steps.
Topics: Bacterial Proteins; Biological Transport; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Models, Biological; Mutation; Protein Conformation; Protein Folding; Protein Sorting Signals; Substrate Specificity
PubMed: 12608575
DOI: 10.1078/0944-5013-00176