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Journal of Controlled Release :... Mar 2023Oral drug delivery increases patient compliance and is thus the preferred administration route for most drugs. However, for biologics the intestinal barrier greatly...
Oral drug delivery increases patient compliance and is thus the preferred administration route for most drugs. However, for biologics the intestinal barrier greatly limits the absorption and reduces their bioavailability. One strategy employed to improve on this is chemical modification of the biologic through the addition of lipid side chains. While it has been established that lipidation of peptides can increase transport, a mechanistic understanding of this effect remains largely unexplored. To pursue this mechanistic understanding, end-point detection of biopharmaceuticals transported through a monolayer of fully polarized epithelial cells is typically used. However, these methods are time-consuming and tedious. Furthermore, most established methods cannot be combined easily with high-resolution live-cell fluorescence imaging that could provide a mechanistic insight into cellular uptake and transport. Here we address this challenge by developing an axial PSF deconvolution scheme to quantify the transport of peptides through a monolayer of Caco-2 cells using single-cell analysis with live-cell confocal fluorescence microscopy. We then measure the known cross-barrier transport of several compounds in our model and compare the results with results obtained in an established microfluidic model finding similar transport phenotypes. This verifies that already after two days the Caco-2 cells in our model form a tight monolayer and constitute a functional barrier model. We then apply this assay to investigate the effects of side chain lipidation of the model peptide drug salmon calcitonin (sCT) modified with 4‑carbon and 8‑carbon-long fatty acid chains. Furthermore, we compare that with experiments performed at lower temperature and using inhibitors for some endocytotic pathways to pinpoint how lipidation length modifies the main avenues for the transport. We thus show that increasing the length of the lipid chain increases the transport of the drug significantly but also makes endocytosis the primary transport mechanism in a short-term cell culture model.
Topics: Humans; Caco-2 Cells; Biological Transport; Epithelial Cells; Peptides; Fatty Acids; Intestinal Absorption; Intestinal Mucosa
PubMed: 36724849
DOI: 10.1016/j.jconrel.2023.01.066 -
Nutrients Apr 2021The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible...
The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible beans. Our previous study demonstrated the anti-inflammatory potency of γ-EV against vascular inflammation at a concentration of 1mM, and that it can transport with the apparent permeability coefficient (P) of 1.56 × 10 ± 0.7 × 10 cm/s across the intestinal Caco-2 cells. The purpose of the current study was to explore whether the permeability of the peptide could be enhanced and to elucidate the mechanism of transport of γ-EV across Caco-2 cells. The initial results indicated that γ-EV was nontoxic to the Caco-2 cells up to 5 mM concentration and could be transported across the intestinal cells intact. During apical-to-basolateral transport, a higher peptide dose (5 mM) significantly ( < 0.01) enhanced the transport rate to 2.5 × 10 ± 0.6 × 10 cm/s. Cytochalasin-D disintegrated the tight-junction proteins of the Caco-2 monolayer and increased the P of γ-EV to 4.36 × 10 ± 0.16 × 10 cm/s ( < 0.001), while theaflavin 3'-gallate and Gly-Sar significantly decreased the P ( < 0.05), with wortmannin having no effects on the peptide transport, indicating that the transport route of γ-EV could be via both PepT1-mediated and paracellular.
Topics: Anti-Inflammatory Agents; Biological Transport; Caco-2 Cells; Cells, Cultured; Dipeptides; Humans; Intestinal Mucosa; Peptides
PubMed: 33923345
DOI: 10.3390/nu13051448 -
Microbiology (Reading, England) Jan 2020Bacteria offer resistance to a broad range of antibiotics by activating their export channels of ATP-binding cassette transporters. These transporters perform a central... (Review)
Review
Bacteria offer resistance to a broad range of antibiotics by activating their export channels of ATP-binding cassette transporters. These transporters perform a central role in vital processes of self-immunity, antibiotic transport and resistance. The majority of ATP-binding cassette transporters are capable of detecting the presence of antibiotics in an external vicinity and are tightly regulated by two-component systems. The presence of an extracellular loop and an adjacent location of both the transporter and two-component system offers serious assistance to induce a quick and specific response against antibiotics. Both systems have demonstrated their ability of sensing such agents, however, the exact mechanism is not yet fully established. This review highlighted the three key functions of antibiotic resistance, transport and self-immunity of ATP-binding cassette transporters and an adjacent two-component regulatory system.
Topics: ATP-Binding Cassette Transporters; Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Drug Resistance, Bacterial; Gene Expression Regulation, Bacterial; Membrane Transport Proteins; Protein Domains; Signal Transduction; Substrate Specificity
PubMed: 31204967
DOI: 10.1099/mic.0.000823 -
Current Opinion in Immunology Jun 2021Major Histocompatibility Complex class I (MHC I) molecules are highly polymorphic, with allotypes differing in peptide binding preferences, and in their dependence upon... (Review)
Review
Major Histocompatibility Complex class I (MHC I) molecules are highly polymorphic, with allotypes differing in peptide binding preferences, and in their dependence upon tapasin for optimal peptide selection. The tapasin dependence of MHC allotypes is inversely correlated with their self-editing ability, and underpinned by conformational plasticity. Recently, TAPBPR has been shown to enhance MHC I assembly via a chaperone-like function, and by editing the peptide repertoire of some MHC I allotypes. Structural analysis has shown TAPBPR binding changes the conformation and dynamics of MHC I, with MHC protein dynamics likely to determine the prevailing TAPBPR function: generically enhancing MHC I assembly by stabilising highly dynamic peptide-empty MHC I; and by editing the peptide repertoire of highly dynamic MHC I allotypes.
Topics: Histocompatibility Antigens Class I; Humans; Immunoglobulins; Membrane Proteins; Membrane Transport Proteins; Peptides
PubMed: 34265495
DOI: 10.1016/j.coi.2021.06.016 -
Toxicology in Vitro : An International... Mar 2020Cinnabar, a mercury-containing mineral medicine, has been used as an ingredient in Traditional Chinese Medicines for treatment of various diseases for thousands of years...
Cinnabar, a mercury-containing mineral medicine, has been used as an ingredient in Traditional Chinese Medicines for treatment of various diseases for thousands of years and is still widely used today. The toxicity of cinnabar is much less than other mercury-containing compounds. This study aimed to evaluate the possible role of oligopeptide transporter1 (PEPT1) in intestinal uptake of cinnabar. Thus, the Caco-2 cell model was employed to investigate the differential transport levels and the probable transporter involved in the transport of cinnabar, mercury sulfide (HgS) and mercury chloride (HgCl). Cells were incubated with the same molar concentration of cinnabar, HgS or HgCl and then the inorganic mercury content of apical (AP), cellular and basolateral (BL) side of the cell was measured by ultra-high liquid chromatography-inductively coupled plasma mass spectrometry (UPLC-ICP/MS) after the treatment, respectively. Their transportation levels were also investigated when pH was changed to 5.5 in AP side to define the role of the H dependent transporter. Effects of cinnabar, HgS or HgCl on transporter mRNA and protein expression levels were assayed by RT-PCR and Western-blot method, respectively. The possible transporter involved in the transport was examined by siRNA silencing and chemical inhibition. The results showed that the levels of inorganic mercury in the BL side for cinnabar and HgS were 49.39% and 30.41% of that in HgCl group. The transport levels of cinnabar and HgCl were significantly increased when the pH was changed to 5.5 on the AP side as compared with the control group (pH 7.4). Cinnabar significantly decreased the mRNA and protein expression of PEPT1. Transport levels of cinnabar were significantly decreased by PEPT1-siRNA and chemical inhibition of PEPT1. The present study demonstrates that PEPT1 may be an important transporter in the entry of cinnabar into the intestinal epithelium, and intestinal transport levels of cinnabar and HgS was lower than that of HgCl.
Topics: Biological Transport; Caco-2 Cells; Humans; Ibuprofen; Intestinal Mucosa; Mercury Compounds; Peptide Transporter 1; RNA, Messenger; RNA, Small Interfering
PubMed: 31838184
DOI: 10.1016/j.tiv.2019.104747 -
Nature Structural & Molecular Biology Dec 2023Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex...
Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. The mechanisms through which proteins are translocated via the TOM and TIM23 complexes remain unclear. Here we report the assembly of the active TOM-TIM23 supercomplex of Saccharomyces cerevisiae with translocating polypeptide substrates. Electron cryo-microscopy analyses reveal that the polypeptide substrates pass the TOM complex through the center of a Tom40 subunit, interacting with a glutamine-rich region. Structural and biochemical analyses show that the TIM23 complex contains a heterotrimer of the subunits Tim23, Tim17 and Mgr2. The polypeptide substrates are shielded from lipids by Mgr2 and Tim17, which creates a translocation pathway characterized by a negatively charged entrance and a central hydrophobic region. These findings reveal an unexpected pre-sequence pathway through the TOM-TIM23 supercomplex spanning the double membranes of mitochondria.
Topics: Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Carrier Proteins; Mitochondrial Membrane Transport Proteins; Saccharomyces cerevisiae Proteins; Protein Transport; Mitochondria; Saccharomyces cerevisiae; Mitochondrial Proteins; Peptides; Membrane Proteins
PubMed: 37696957
DOI: 10.1038/s41594-023-01103-7 -
Journal of Molecular Biology Oct 2023ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance...
ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.
Topics: Humans; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cryoelectron Microscopy; Hydrolysis; Membrane Transport Proteins; Neoplasm Proteins; Single-Domain Antibodies
PubMed: 37597690
DOI: 10.1016/j.jmb.2023.168234 -
Amino Acids Feb 2021Some dipeptides have been implicated in myocardial protection, but little is known about their membrane transporter PEPT2. The aim of this study was to determine whether...
Some dipeptides have been implicated in myocardial protection, but little is known about their membrane transporter PEPT2. The aim of this study was to determine whether the expression and activity of the cardiac-type PEPT2 cotransporter could be affected by ageing and/or hypertension. Sarcolemmal vesicles (SV) were isolated from the hearts of all rat groups using a standard procedure to investigate the transport activity and protein abundance by fluorescence spectroscopy and Western blot, respectively. SLC15A2 "PEPT2" gene expression was relatively quantified by RT-qPCR. In the Wistar rat groups, the protein and gene expression of PEPT2 were upregulated with ageing. These changes were accompanied by corresponding increases in the competitive inhibition and the transport rate (V) of β-Ala-Lys (AMCA) into SV isolated from middle-aged hearts. Although, the transport rate of β-Ala-Lys (AMCA) into SV isolated from old hearts was significantly the lowest compared to middle-aged and young adult hearts, the inhibition percentage of β-Ala-Lys (AMCA) transport by Gly-Gln was the highest. In the WKY and SHR rat groups, Y-SHR hypertrophied hearts showed an increase in PEPT2 gene expression accompanied by a significant decrease in protein expression and activity. With advanced age, however, M-SHR hypertrophied hearts revealed significantly lower gene expression, but higher protein expression and activity than Y-SHR hearts. These findings suggest that increased expression of PEPT2 cotransporter in all types of middle-aged hearts could be exploited to facilitate di-and tripeptide transport by PEPT2 in these hearts, which subsequently could result in improved myocardial protection in these populations.
Topics: Aging; Animals; Biological Transport; Cardiomegaly; Dipeptides; Hypertension; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Symporters
PubMed: 33404911
DOI: 10.1007/s00726-020-02936-y -
Parasites & Vectors Oct 2021The roundworm Ascaris suum is one of the parasites with the greatest economic impact on pig farming. In this context, lower weight gain is hypothesized to be due to...
BACKGROUND
The roundworm Ascaris suum is one of the parasites with the greatest economic impact on pig farming. In this context, lower weight gain is hypothesized to be due to decreased nutrient absorption. This study aims at characterizing the effects of A. suum infection on intestinal nutrient transport processes and potential molecular mechanisms.
METHODS
Three groups of six piglets each were infected orally (10,000 embryonated A. suum eggs) in a single dose ("single infection"). Another three groups were infected orally (1000 embryonated eggs) for 10 consecutive days ("trickle infection"). Animals were necropsied 21, 35 and 49 days post-infection (dpi). Three groups served as respective controls. The Ussing chamber technique was applied for the functional characterization of small intestinal tissues [short-circuit currents (I) as induced by glucose, alanine and peptides; H-glucose net flux rates; tissue conductance (G)]. Transcription and expression levels of relevant cytokines and nutrient transporters were evaluated (qPCR/western blot).
RESULTS
Peptide- and alanine-induced changes in I were significantly decreased in the jejunum and ileum of the trickle-infected group at 49 dpi and in the ileum of the single-infected group at 49 dpi. No significant differences regarding glucose transport were observed between the Ascaris-infected groups and the control group in Ussing chamber experiments. Transcription levels of the glucose and peptide transporters as well as of selected transcription factors (transcription of signal transducer and activator of transcription 6 [STAT6] and hypoxia-inducible factor 1-alpha [Hif-1α]) were significantly increased in response to both infection types after some periods. The transcription of interleukins 4 and 13 varied between decrease and increase regarding the respective time points, as did the protein expression of glucose transporters. The expression of the peptide transporter PepT1 was significantly decreased in the ileal single-infected group at 35 dpi. Hif-1α was significantly increased in the ileal tissue from the single-infected group at 21 dpi and in the trickle-infected group at 35 dpi. The expression levels of Na/K-ATPase and ASCT1 remained unaffected.
CONCLUSIONS
In contrast to the current hypothesis, these results indicate that the nutrient deprivation induced by A. suum cannot be explained by transcriptional or expression changes alone and requires further studies.
Topics: Alanine; Animals; Ascariasis; Ascaris suum; Biological Transport; Cytokines; Glucose; Intestines; Nutrients; Peptides; Swine; Swine Diseases
PubMed: 34649607
DOI: 10.1186/s13071-021-05029-1 -
Cell-Penetrating Peptides: Correlation between Peptide-Lipid Interaction and Penetration Efficiency.Chemphyschem : a European Journal of... Mar 2021Cell-penetrating peptides are used in the delivery of peptides and biologics, with some cell-penetrating peptides found to be more efficient than others. The exact...
Cell-penetrating peptides are used in the delivery of peptides and biologics, with some cell-penetrating peptides found to be more efficient than others. The exact mechanism of how they interact with the cell membrane and penetrate it, however, remains unclear. This study attempts to investigate the difference in free energy profiles of three cell-penetrating peptides (TAT, CPP1 and CPP9) with a model lipid bilayer (DOPC) using molecular dynamics pulling simulations with umbrella sampling. Potential mean force (PMF) and free energy barrier between the peptides and DOPC are determined using WHAM analysis and MM-PBSA analysis, respectively. CPP9 is found to have the smallest PMF value, followed by CPP1 and TAT, consistent with the experimental data. YDEGE peptide, however, does not give the highest PMF value, although it is a non-cell-permeable peptide. YDEGE is also found to form water pores, alongside with TAT and CPP9, suggesting that it is difficult to distinguish true water pore formation from artefacts arising from pulling simulations. On the contrary, free energy analysis of the peptide-DOPC complex at the lipid-water interface with MM-PBSA provides results consistent with experimental data with CPP9 having the least interaction with DOPC and lowest free energy barrier, followed by CPP1, TAT and YDEGE. These findings suggest that peptide-lipid interaction at the lipid-water interface has a direct correlation with the penetration efficiency of peptides across the lipid bilayer.
Topics: Biological Transport; Cell Membrane; Lipids; Models, Biological; Molecular Dynamics Simulation; Peptides; Thermodynamics; Water
PubMed: 33377300
DOI: 10.1002/cphc.202000873