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14 pages, 3223 KiB

Open AccessArticle

Spns2 Transporter Contributes to the Accumulation of S1P in Cystic Fibrosis Human Bronchial Epithelial Cells

by Aida Zulueta, Michele Dei Cas, Francesco Luciano, Alessandra Mingione, Francesca Pivari, Ilaria Righi, Letizia Morlacchi, Lorenzo Rosso, Paola Signorelli, Riccardo Ghidoni, Rita Paroni and Anna Caretti

Biomedicines 2021, 9(9), 1121; https://doi.org/10.3390/biomedicines9091121 - 31 Aug 2021

Cited by 4 | Viewed by 1884

Abstract

The role of S1P in Cystic Fibrosis (CF) has been investigated since 2001, when it was first described that the CFTR channel regulates the inward transport of S1P. From then on, various studies have associated F508del CFTR, the most frequent mutation in CF [...] Read more.

The role of S1P in Cystic Fibrosis (CF) has been investigated since 2001, when it was first described that the CFTR channel regulates the inward transport of S1P. From then on, various studies have associated F508del CFTR, the most frequent mutation in CF patients, with altered S1P expression in tissue and plasma. We found that human bronchial epithelial immortalized and primary cells from CF patients express more S1P than the control cells, as evidenced by mass spectrometry analysis. S1P accumulation relies on two- to four-fold transcriptional up-regulation of SphK1 and simultaneous halving of SGPL1 in CF vs. control cells. The reduction of SGPL1 transcription protects S1P from irreversible degradation, but the excessive accumulation is partially prevented by the action of the two phosphatases that are up-regulated compared to control cells. For the first time in CF, we describe that Spns2, a non-ATP dependent transporter that normally extrudes S1P out of the cells, shows deficient transcriptional and protein expression, thus impairing S1P accrual dissipation. The in vitro data on CF human bronchial epithelia correlates with the impaired expression of Spns2 observed in CF human lung biopsies compared to healthy control. Full article

(This article belongs to the Special Issue Drug Transporters)

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13 pages, 2859 KiB

Open AccessArticle

Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

by Benedict-Uy Fabia, Joshua Bingwa, Jiyeon Park, Nguyen-Mihn Hieu and Jung-Hoon Ahn

Biomedicines 2021, 9(6), 679; https://doi.org/10.3390/biomedicines9060679 - 16 Jun 2021

Cited by 3 | Viewed by 2794

Abstract

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also [...] Read more.

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF. Full article

(This article belongs to the Special Issue Drug Transporters)

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15 pages, 3746 KiB

Open AccessArticle

Functional Characterization of the Dopaminergic Psychostimulant Sydnocarb as an Allosteric Modulator of the Human Dopamine Transporter

by Shaili Aggarwal, Mary Hongying Cheng, Joseph M. Salvino, Ivet Bahar and Ole Valente Mortensen

Biomedicines 2021, 9(6), 634; https://doi.org/10.3390/biomedicines9060634 - 02 Jun 2021

Cited by 6 | Viewed by 3299

Abstract

The dopamine transporter (DAT) serves a critical role in controlling dopamine (DA)-mediated neurotransmission by regulating the clearance of DA from the synapse and extrasynaptic regions and thereby modulating DA action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine [...] Read more.

The dopamine transporter (DAT) serves a critical role in controlling dopamine (DA)-mediated neurotransmission by regulating the clearance of DA from the synapse and extrasynaptic regions and thereby modulating DA action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DATs to alter their actions resulting in an enhancement in extracellular DA concentrations. We previously identified a novel allosteric site in the DAT and the related human serotonin transporter that lies outside the central orthosteric substrate- and cocaine-binding pocket. Here, we demonstrate that the dopaminergic psychostimulant sydnocarb is a ligand of this novel allosteric site. We identified the molecular determinants of the interaction between sydnocarb and DAT at the allosteric site using molecular dynamics simulations. Biochemical-substituted cysteine scanning accessibility experiments have supported the computational predictions by demonstrating the occurrence of specific interactions between sydnocarb and amino acids within the allosteric site. Functional dopamine uptake studies have further shown that sydnocarb is a noncompetitive inhibitor of DAT in accord with the involvement of a site different from the orthosteric site in binding this psychostimulant. Finally, DA uptake studies also demonstrate that sydnocarb affects the interaction of DAT with both cocaine and amphetamine. In summary, these studies further strengthen the prospect that allosteric modulation of DAT activity could have therapeutic potential. Full article

(This article belongs to the Special Issue Drug Transporters)

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12 pages, 484 KiB

Open AccessArticle

Natural Autoimmunity to the Thyroid Hormone Monocarboxylate Transporters MCT8 and MCT10

by Theresa Porst, Jörg Johannes, Hans Gluschke, Richard Köhler, Sebastian Mehl, Peter Kühnen, Kostja Renko, Waldemar B. Minich, Susanna Wiegand and Lutz Schomburg

Biomedicines 2021, 9(5), 496; https://doi.org/10.3390/biomedicines9050496 - 30 Apr 2021

Cited by 2 | Viewed by 2128

Abstract

The monocarboxylate transporters 8 (MCT8) and 10 (MCT10) are important for thyroid hormone (TH) uptake and signaling. Reduced TH activity is associated with impaired development, weight gain and discomfort. We hypothesized that autoantibodies (aAb) to MCT8 or MCT10 are prevalent in thyroid disease [...] Read more.

The monocarboxylate transporters 8 (MCT8) and 10 (MCT10) are important for thyroid hormone (TH) uptake and signaling. Reduced TH activity is associated with impaired development, weight gain and discomfort. We hypothesized that autoantibodies (aAb) to MCT8 or MCT10 are prevalent in thyroid disease and obesity. Analytical tests for MCT8-aAb and MCT10-aAb were developed and characterized with commercial antiserum. Serum samples from healthy controls, thyroid patients and young overweight subjects were analyzed, and prevalence of the aAb was compared. MCT8-aAb were additionally tested for biological effects on thyroid hormone uptake in cell culture. Positive MCT8-aAb and MCT10-aAb were detected in all three clinical cohorts analyzed. MCT8-aAb were most prevalent in thyroid patients (11.9%) as compared to healthy controls (3.8%) and overweight adolescents (4.2%). MCT8-aAb positive serum reduced T4 uptake in cell culture in comparison to MCT8-aAb negative control serum. Prevalence of MCT10-aAb was highest in the group of thyroid patients as compared to healthy subjects or overweight adolescents (9.0% versus 4.5% and 6.3%, respectively). We conclude that MCT8 and MCT10 represent autoantigens in humans, and that MCT8-aAb may interfere with regular TH uptake and signaling. The increased prevalence of MCT8-aAb and MCT10-aAb in thyroid disease suggests that their presence may be of pathophysiological relevance. This hypothesis deserves an analysis in large prospective studies. Full article

(This article belongs to the Special Issue Drug Transporters)

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16 pages, 4972 KiB

Open AccessArticle

Reduced Thiamine Availability and Hyperglycemia Impair Thiamine Transport in Renal Glomerular Cells through Modulation of Thiamine Transporter 2

by Aurora Mazzeo, Federica Barutta, Linda Bellucci, Marina Trento, Gabriella Gruden, Massimo Porta and Elena Beltramo

Biomedicines 2021, 9(4), 385; https://doi.org/10.3390/biomedicines9040385 - 05 Apr 2021

Cited by 6 | Viewed by 2648

Abstract

Thiamine helps transketolase in removing toxic metabolites, counteracting high glucose-induced damage in microvascular cells, and progression of diabetic retinopathy/nephropathy in diabetic animals. Diabetic subjects show reduced thiamine levels. Hyperglycemia and reduced thiamine availability concur in impairing thiamine transport inside the blood-retinal barrier, with [...] Read more.

Thiamine helps transketolase in removing toxic metabolites, counteracting high glucose-induced damage in microvascular cells, and progression of diabetic retinopathy/nephropathy in diabetic animals. Diabetic subjects show reduced thiamine levels. Hyperglycemia and reduced thiamine availability concur in impairing thiamine transport inside the blood-retinal barrier, with thiamine transporter-2 (THTR2) primarily involved. Here, we examined the behavior of thiamine transporter-1 (THTR1), THTR2, and their transcription factor Sp1 in response to high glucose and altered thiamine availability in renal cells involved in diabetic nephropathy. Human proximal tubule epithelial cells, podocytes, glomerular endothelial, and mesangial cells were exposed to high glucose and/or thiamine deficiency/oversupplementation. Localization and modulation of THTR1, THTR2, and Sp1; intracellular thiamine; transketolase activity; and permeability to thiamine were examined. Reduced thiamine availability and hyperglycemia impaired thiamine transport and THTR2/Sp1 expression. Intracellular thiamine, transketolase activity, and permeability were strongly dependent on thiamine concentrations and, partly, excess glucose. Glomerular endothelial cells were the most affected by the microenvironmental conditions. Our results confirmed the primary role of THTR2 in altered thiamine transport in cells involved in diabetic microvascular complications. Lack of thiamine concurs with hyperglycemia in impairing thiamine transport. Thiamine supplementation could represent a therapeutic option to prevent or slow the progression of these complications. Full article

(This article belongs to the Special Issue Drug Transporters)

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22 pages, 2947 KiB

Open AccessArticle

Screening of Natural Compounds as P-Glycoprotein Inhibitors against Multidrug Resistance

by Sérgio M. Marques, Lucie Šupolíková, Lenka Molčanová, Karel Šmejkal, David Bednar and Iva Slaninová

Biomedicines 2021, 9(4), 357; https://doi.org/10.3390/biomedicines9040357 - 30 Mar 2021

Cited by 29 | Viewed by 5771

Abstract

Multidrug resistance (MDR) is a common problem when fighting cancer with chemotherapy. P-glycoprotein (P-gp, or MDR1) is an active pump responsible for the efflux of xenobiotics out of the cell, including anti-cancer drugs. It is a validated target against MDR. No crystal structure [...] Read more.

Multidrug resistance (MDR) is a common problem when fighting cancer with chemotherapy. P-glycoprotein (P-gp, or MDR1) is an active pump responsible for the efflux of xenobiotics out of the cell, including anti-cancer drugs. It is a validated target against MDR. No crystal structure of the human P-gp is available to date, and only recently several cryo-EM structures have been solved. In this paper, we present a comprehensive computational approach that includes constructing the full-length three-dimensional structure of the human P-gp and its refinement using molecular dynamics. We assessed its flexibility and conformational diversity, compiling a dynamical ensemble that was used to dock a set of lignan compounds, previously reported as active P-gp inhibitors, and disclose their binding modes. Based on the statistical analysis of the docking results, we selected a system for performing the structure-based virtual screening of new potential P-gp inhibitors. We tested the method on a library of 87 natural flavonoids described in the literature, and 10 of those were experimentally assayed. The results reproduced the theoretical predictions only partially due to various possible factors. However, at least two of the predicted natural flavonoids were demonstrated to be effective P-gp inhibitors. They were able to increase the accumulation of doxorubicin inside the human promyelocytic leukemia HL60/MDR cells overexpressing P-gp and potentiate the antiproliferative activity of this anti-cancer drug. Full article

(This article belongs to the Special Issue Drug Transporters)

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12 pages, 1242 KiB

Open AccessArticle

Organic Cation Transporters (OCTs) in EpiAirway™, a Cellular Model of Normal Human Bronchial Epithelium

by Amelia Barilli, Rossana Visigalli, Francesca Ferrari, Maria Di Lascia, Benedetta Riccardi, Paola Puccini, Valeria Dall’Asta and Bianca Maria Rotoli

Biomedicines 2020, 8(5), 127; https://doi.org/10.3390/biomedicines8050127 - 19 May 2020

Cited by 7 | Viewed by 3099

Abstract

Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney; in the lung, OCTs mediate inhaled drugs’ transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human [...] Read more.

Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney; in the lung, OCTs mediate inhaled drugs’ transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human airways were mostly performed in immortal or transformed cell lines; here, we studied OCTs in EpiAirway™, a recently developed in vitro model of normal bronchial epithelium. Calu-3 monolayers were used for comparison. The activity of OCTs was evaluated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP⁺) at the apical and basolateral side of monolayers and protein expression through Western Blot analysis. OCTs and OCTNs expression, along with that of Amino acid Transporter B^0,+ (ATB^0,+)transporter, was determined by measuring the number of mRNA molecules through quantitative Polymerase Chain Reaction (qPCR). The interaction of the transporters with bronchodilators was also assessed. Results highlight significant differences between Calu-3 cells and EpiAirway™, since, in the latter, OCTs are active only on the basolateral membrane where they interact with the bronchodilator ipratropium. No activity of OCTs is detectable at the apical side; there, the most abundant carrier is, instead, SLC6A14/ATB^0,+, that can thus be potentially listed among organic cation transporters responsible for drug delivery in the lung. Full article

(This article belongs to the Special Issue Drug Transporters)

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Review

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16 pages, 576 KiB

Open AccessReview

Membrane Carriers and Transporters in Kidney Physiology and Disease

by Marek Drozdzik, Maria Drozdzik and Stefan Oswald

Biomedicines 2021, 9(4), 426; https://doi.org/10.3390/biomedicines9040426 - 14 Apr 2021

Cited by 13 | Viewed by 3934

Abstract

The growing information suggests that chronic kidney disease may affect expression and function of membrane carriers and transporters in the kidney. The dysfunction of carriers and transporters entails deficient elimination of uremic solutes as well as xenobiotics (drugs and toxins) with subsequent clinical [...] Read more.

The growing information suggests that chronic kidney disease may affect expression and function of membrane carriers and transporters in the kidney. The dysfunction of carriers and transporters entails deficient elimination of uremic solutes as well as xenobiotics (drugs and toxins) with subsequent clinical consequences. The renal carriers and transporters are also targets of drugs used in clinical practice, and intentional drug–drug interactions in the kidney are produced to increase therapeutic efficacy. The understanding of membrane carriers and transporters function in chronic kidney disease is important not only to better characterize drug pharmacokinetics, drug actions in the kidney, or drug–drug interactions but also to define the organ pathophysiology. Full article

(This article belongs to the Special Issue Drug Transporters)

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