-
Molecular Pharmaceutics Dec 2017In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is...
In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target.
Topics: Biological Transport, Active; Drug Evaluation, Preclinical; Humans; Inhibitory Concentration 50; Intestinal Absorption; Kinetics; Ligands; Models, Chemical; Models, Molecular; Molecular Docking Simulation; Oligopeptides; Peptide Transporter 1; Prodrugs
PubMed: 29111754
DOI: 10.1021/acs.molpharmaceut.7b00775 -
Proceedings of the National Academy of... Mar 2023Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic...
Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood-brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.
Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Biological Transport; Cryoelectron Microscopy; Leukotriene C4; Multidrug Resistance-Associated Proteins; Peptides; Peptides, Cyclic
PubMed: 36893260
DOI: 10.1073/pnas.2220012120 -
Cellular and Molecular Life Sciences :... May 2011CD8(+) T lymphocytes screen the surface of all cells in the body to detect pathogen infection or oncogenic transformation. They recognize peptides derived from cellular... (Review)
Review
CD8(+) T lymphocytes screen the surface of all cells in the body to detect pathogen infection or oncogenic transformation. They recognize peptides derived from cellular proteins displayed at the plasma membrane by major histocompatibility complex (MHC) class I molecules. Peptides are mostly by-products of cytosolic proteolytic enzymes. Peptidic ligands of MHC class I molecules are also generated in the secretory and vesicular pathways. Features of protein substrates, of proteases and of available MHC class I molecules for loading peptides in these compartments shape a singular collection of ligands that also contain different, longer, and lower affinity peptides than ligands produced in the cytosol. Especially in individuals who lack the transporters associated with antigen processing, TAP, and in infected and tumor cells where TAP is blocked, which thus have no supply of peptides derived from the cytosol, MHC class I ligands generated in the secretory and vesicular pathways contribute to shaping the CD8(+) T lymphocyte response.
Topics: Animals; Antigen Presentation; Biological Transport; CD8-Positive T-Lymphocytes; Histocompatibility Antigens Class I; Humans; Ligands; Membrane Transport Proteins; Mice; Peptide Hydrolases; Peptides; Secretory Pathway; Virus Diseases
PubMed: 21387141
DOI: 10.1007/s00018-011-0661-2 -
International Journal of Molecular... Jan 2023The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and...
The glucosinolate transporters 1/2/3 (GTR1/2/3) from the Nitrate and Peptide transporter Family (NPF) play an essential role in the transport, accumulation, and distribution of the specialized plant metabolite glucosinolates. Due to representing both antinutritional and health-promoting compounds, there is increasing interest in characterizing GTRs from various plant species. We generated seven artificial glucosinolates (either aliphatic or benzenic) bearing different fluorophores (Fluorescein, BODIPY, Rhodamine, Dansylamide, and NBD) and investigated the ability of GTR1/2/3 from to import the fluorescent glucosinolates (F-GSLs) into oocytes from . Five out of the seven F-GSLs synthesized were imported by at least one of the GTRs. GTR1 and GTR2 were able to import three F-GSLs actively above external concentration, while GTR3 imported only one actively. Competition assays indicate that the F-GSLs are transported by the same mechanism as non-tagged natural glucosinolates. The GTR-mediated F-GSL uptake is detected via a rapid and sensitive assay only requiring simple fluorescence measurements on a standard plate reader. This is highly useful in investigations of glucosinolate transport function and provides a critical prerequisite for elucidating the relationship between structure and function through high-throughput screening of GTR mutant libraries. The F-GSL themselves may also be suitable for future studies on glucosinolate transport in vivo.
Topics: Arabidopsis Proteins; Glucosinolates; Arabidopsis; Biological Transport; Membrane Transport Proteins
PubMed: 36674437
DOI: 10.3390/ijms24020920 -
Structure (London, England : 1993) Mar 2018Proton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug...
Proton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepT from Streptococcus thermophilus, and structures were determined of PepT in complex with four physicochemically diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepT can adapt to different peptide side chains through movement of binding site residues and water molecules, and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also determined in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate molecules, which further underline the adaptability of the binding site.
Topics: Bacterial Proteins; Binding Sites; Biological Transport; Dipeptides; Membrane Transport Proteins; Models, Molecular; Protein Binding; Protein Conformation; Protein Stability; Protons; Streptococcus thermophilus
PubMed: 29429879
DOI: 10.1016/j.str.2018.01.005 -
The Journal of General and Applied... Dec 2023In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition...
In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition to intracellular metabolic modification, is important from a productivity perspective. However, there has been little progress in analyses of the functions of microbial membrane transporters, and because of the difficulty in finding transporters that transport target compounds, only a few transporters have been put to practical use. Here, we constructed a Corynebacterium glutamicum-derived transporter expression library (CgTP-Express library) with the fusion partner gene mstX and used a peptide-feeding method with the dipeptide L-Ala-L-Ala to search for alanine exporters in the library. Among 39 genes in the library, five candidate alanine exporters (NCgl2533, NCgl2683, NCgl0986, NCgl0453, and NCgl0929) were found; expression of NCgl2533 increased the alanine concentration in cell culture. The CgTP-Express library was thus effective for finding a new transporter candidate.
Topics: Fermentation; Membrane Transport Proteins; Corynebacterium glutamicum; Alanine; Biological Transport; Metabolic Engineering
PubMed: 36567121
DOI: 10.2323/jgam.2022.12.002 -
Research in Microbiology 2019The transport of peptides in microorganisms plays an important role in their physiology and behavior, both as a nutrient source and as a proxy to sense their... (Review)
Review
The transport of peptides in microorganisms plays an important role in their physiology and behavior, both as a nutrient source and as a proxy to sense their environment. This latter function is evidenced in Gram-positive bacteria where cell-cell communication is mediated by small peptides. Here, we highlight the importance of the oligopeptide permease (Opp) systems in the various major processes controlled by signaling peptides, such as sporulation, virulence and conjugation. We underline that the functioning of these communication systems is tightly linked to the developmental status of the bacteria via the regulation of opp gene expression by transition phase regulators.
Topics: ATP-Binding Cassette Transporters; Bacterial Proteins; Biological Transport; Gene Expression Regulation, Bacterial; Gram-Positive Bacteria; Membrane Transport Proteins; Peptide Termination Factors; Quorum Sensing; Signal Transduction
PubMed: 31376485
DOI: 10.1016/j.resmic.2019.07.004 -
The Journal of Biological Chemistry Jun 1997Human cytotoxic T lymphocytes (CTL) recognize specific complexes of HLA class I molecules and peptides, which assemble when nascent class I molecules bind peptides...
Human cytotoxic T lymphocytes (CTL) recognize specific complexes of HLA class I molecules and peptides, which assemble when nascent class I molecules bind peptides transported from the cytoplasm into the endoplasmic reticulum by the heterodimeric transporter associated with antigen processing (TAP). Increased class I molecule expression on the cell surface increases the efficiency of CTL lysis. The kinetics of interferon (IFN)-gamma induction of TAP, peptide transport capacity, and HLA class I molecule expression was determined in endothelial cells, which are targets of CTL following transplantation or viral infection. TAP mRNAs are induced rapidly, increasing 20-fold (TAP1) or 10-fold (TAP2) by 12 h, whereas HLA class I mRNA is induced more slowly, increasing 10-fold in 24 h. TAP1 and TAP2 proteins are also induced rapidly, increasing 10-fold in 24 h, whereas HLA class I heavy chain proteins and surface expression increase more slowly. Peptide transport capacity in endothelial and HeLa cells increases within 6 h of IFN-gamma treatment, suggesting that the IFN-gamma-induced TAP heterodimers are functional. Therefore, the IFN-gamma-induced increase in TAP proteins is accompanied by an increased peptide transport capacity, which may be important in supporting the subsequent rise in HLA class I protein expression.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP Binding Cassette Transporter, Subfamily B, Member 3; ATP-Binding Cassette Transporters; Biological Transport; Endothelium, Vascular; Genes, MHC Class I; HeLa Cells; Histocompatibility Antigens Class I; Humans; Interferon-gamma; Peptides; RNA, Messenger
PubMed: 9195970
DOI: 10.1074/jbc.272.26.16585 -
Biopolymers 2008The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting... (Review)
Review
The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.
Topics: Adenosine Triphosphatases; Amino Acid Sequence; Bacterial Proteins; Binding Sites; Cross-Linking Reagents; Membrane Transport Proteins; Models, Biological; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Sorting Signals; Protein Transport; SEC Translocation Channels; SecA Proteins; Signal Recognition Particle
PubMed: 17918185
DOI: 10.1002/bip.20856 -
Cellular and Molecular Life Sciences :... May 2000The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in... (Review)
Review
The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.
Topics: Amino Acid Sequence; Animals; Binding Sites; Biological Transport, Active; Caenorhabditis elegans; Carrier Proteins; Female; Genome; Hormones; Humans; Molecular Sequence Data; Peptide Transporter 1; Peptides; Pregnancy; Second Messenger Systems; Sequence Homology, Amino Acid; Symporters; Tissue Distribution
PubMed: 10892342
DOI: 10.1007/s000180050040