Substrate Transport Research Articles

Regulating Transport Preferences of Fucosylated Sugars: Revealing Transport Mechanisms via Sugar Efflux Transporter A Transformation Into a "Tight-in, Tight-out" Mode.

Sugar transporters play a crucial role in cellular metabolism across diverse organisms, regulating essential biological processes through efficient substrate transport. Despite extensive research efforts, the structures and mechanisms of transporters responsible for sugars have remained elusive. In this study, we investigated the transport efficiency of the Escherichia coli sugar efflux transporter A (SetA) for lactose and fucosylated lactose. By employing site and combinatorial mutations, we obtained a mutant exhibiting approximately sixfold enhanced transporter efficiency for fucosylated lactose while retaining its potency for lactose transport. In this mutant, the fundamental amino acids responsible for recognizing the galactosyl moiety remained unchanged, yet the introduction of two face-to-face aromatic ring residues facilitated the enhanced recognition of the fucosyl moiety. This indicated the transformation of SetA from a universal transporter into a specific "tight-in, tight-out" transporter. Utilizing SetA-based structural modeling, we mapped and investigated mutations associated with diseases. The structural and biochemical insights from SET in this study offer a valuable investigating framework for understanding substrate specificity mechanisms of fucosylated sugar transporters and, by extension, other transporters in broader contexts.

Dimerization and substrate recognition of human taurine transporter

Taurine is a conditionally essential nutrient and one of the most abundant amino acids in humans, with diverse physiological functions. The cellular uptake of taurine is primarily mediated by the taurine transporter (TauT), and its dysfunction leads to retinal regeneration, cardiomyopathy, neurological and aging-associated disorders. Here we determine structures of TauT in two states: the apo inward-facing open state and the occluded state bound with substrate taurine or γ-aminobutyric acid (GABA). In addition to monomer, the structures also reveal a TauT dimer, where two cholesterol molecules act as “molecular glue”, and close contacts of two TM5 from each protomer mediate the dimer interface. In combination with functional characterizations, our results elucidate the detailed mechanisms of substrate recognition, specificity and transport by TauT, providing a structural framework for understanding TauT function and exploring potential therapeutic strategies for taurine-deficiency-related disorders.

The structural characteristics of cellular phospholipid acyl chains required for ABCA1-mediated HDL formation.

The structural characteristics of cellular phospholipid acyl chains required for ABCA1-mediated HDL formation.

Strategies to Improve Pharmacokinetics of Hepatic Uptake Transporter Substrates.

For hepatic uptake transporter substrates, hepatic clearance depends not only on the rates of elimination (metabolism and biliary excretion) but also on the rates at which a drug moves between the liver and bloodstream. With representative extended clearance classification system Class 1B drugs, we performed mouse in vivo studies with chemical inhibitors of metabolism and hepatic uptake to demonstrate how permeability affects the relative contributions of hepatic uptake and metabolism to clearance. In addition, we show how half-life can be extended through reductions in metabolism but is largely unaffected by changes in hepatic uptake. Insights from the extended clearance model and pharmacokinetic modeling are used to supplement these studies. While most literature focuses on reducing hepatic uptake as the main strategy to reduce clearance for this class of drugs, we present a rationale to reduce clearance and extend half-life by increasing permeability and reducing metabolism.

Molecular basis for substrate recognition and transport of mammalian taurine transporters

The taurine transporter (TAUT) mediates cellular taurine uptake, playing a critical role in human health and longevity. In this study, we present cryogenic electron microscopy structures of both mouse and human TAUT in various conformational states. The taurine-bound, occluded forms of mouse and human TAUT reveal the substrate binding pocket and the ion binding sites. The amino group of taurine interacts with Glu406 at the binding site, constituting a key structural feature determining substrate preference. While both imidazole acetic acid and guanidinoethyl sulfonate (GES) inhibit TAUT by competing with taurine for the binding site, GES also functions as a substrate of TAUT. Moreover, mouse TAUT is captured in an inward-open apo conformation, where the tilted movement of transmembrane helix (TM) 1a opens the intracellular gate. Notably, TM6 exhibits two distinct conformational states: the canonical form consisting of two half-helices and a continuous straight helix. In the latter conformation, TM6 partially occupies the substrate binding site, likely promoting taurine release. Together, our findings provide critical insights into the molecular mechanisms by which TAUT recognizes and transports taurine.

Insights into the effect of sertraline on anaerobic digestion: Stimulating CH4 and CO2 production.

Insights into the effect of sertraline on anaerobic digestion: Stimulating CH4 and CO2 production.

A data-mining analysis of host solute carrier family proteins in SARS-CoV-2 infection with reference to brain endothelial cells and the blood-brain barrier in COVID-19

BackgroundThe brain vasculature is a key player in neurological manifestations of COVID-19. Infection of brain endothelial cells with SARS-CoV-2 along with circulating cytokines may cause dysfunction of the blood-brain barrier (BBB). Solute carrier transporters (SLCs) in brain endothelial cells regulate substrate transport across the BBB. Here, it was hypothesized that transport functions of SLCs will be impaired by interactions with viral proteins, and subsequently, data-mining studies were performed.MethodsVirus-host protein-protein interaction data for SARS-CoV-2 infection were retrieved from the BioGRID database, filtered for SLCs, and then annotated for relevant expression in brain endothelial cells using a mouse brain transcriptomics database. Host SLCs expressed in brain endothelial cells were further explored using publicly available databases and information in the literature. Functional Annotation Clustering was performed using DAVID, and Enrichr served for pathway analysis. Substrates were retrieved from NCBI Gene. Links to monogenic disorders were retrieved from Online Mendelian Inheritance in Man™ and screened for disorders of the nervous system. Interactome data for viral proteins of SARS-CoV-2 were retrieved from BioGRID. Reports for host SLCs in viral receptor functions, viral entry mechanisms, and other major roles in the viral cycle were explored in databases (VThunter) and literature. ATP-binding cassette transporters (ABCs) were studied in parallel.ResultsN = 80 host SLCs showed relevant expression in brain endothelial cells whereby amino acid transporter stood out. N = 24/80 host SLCs were linked to monogenic disorders of the nervous system. N = 9/29 SARS-CoV-2 viral proteins had strong links to SLCs and key functions in viral infection (e.g., interferon response). SLCs serving as viral receptors and with closely associated functions were significantly enriched among all known listed viral receptors (chi-square test, p = 0.001). Literature searches for host SLCs revealed involvement of a subset of SLCs in infection mechanisms for SARS-CoV-2 and more broadly for other viruses. N = 17 host ABCs were found in brain endothelial cells where they may serve as efflux transporters.DiscussionThis hypothesis-generating work proposes a set of N = 80 host SLCs expressed in endothelial cells as contributors to BBB impairment after SARS-CoV-2 infection. Theoretically, persistent dysfunction of SLCs at the BBB, in particular insufficient transport of amino acids, could be one of many reasons for cognitive changes in long-COVID. Functions of SLCs in viral entry and associated roles deserve close attention.

Constructing Hydrazone-Linked Chiral Covalent Organic Frameworks with Different Pore Sizes for Asymmetric Catalysis

Chiral covalent organic frameworks (COFs) hold great promise in heterogeneous asymmetric catalysis due to their designable structures and well-defined chiral microenvironments. However, precise control over the pore size of chiral COFs to optimize asymmetric catalytic performance remains challenging. Herein, we designed a proline-derived dihydrazide chiral monomer (L-DBP-Boc), which was subjected to Schiff-base reactions with two aromatic aldehydes of different lengths, 1,3,5-triformyl phloroglucinol (BTA) and 4,4′,4″-(1,3,5-triazine-2,4,6-triyl)tribenzaldehyde (TZ), to construct two hydrazone-linked chiral COFs with distinct pore sizes (L-DBP-BTA COF and L-DBP-TZ COF). Interestingly, the Boc protecting groups were removed in situ during COF synthesis. We systematically investigated the catalytic performance of these two chiral COFs in asymmetric aldol reactions and found that their pore sizes significantly influenced both catalytic activity and enantioselectivity. The large-pore L-DBP-TZ COF (pore size: 3.5 nm) exhibited superior catalytic performance under aqueous conditions at room temperature, achieving a yield of 98% and an enantiomeric excess (ee) value of 78%. In contrast, the small-pore L-DBP-BTA COF (pore size: 2.0 nm) showed poor catalytic performance. Compared to L-DBP-BTA COF, L-DBP-TZ COF demonstrated a 1.69-fold increase in yield and a 1.56-fold enhancement in enantioselectivity, possibly attributed to the facilitated diffusion and transport of substrates and products within the larger pore, thus improving the accessibility of active sites. This study presents a facile synthesis of pyrrolidine-functionalized chiral COFs and establishes the possible structure–activity relationship in their asymmetric catalysis, offering new insights for the design of efficient chiral COF catalysts.

Effect of Hepatic Impairment on the Pharmacokinetics of Baicalin in Rats: Critical Roles of Gut Microbiota and Hepatic Transporters

Background: Baicalin (BG) has been used in the treatment of many diseases. However, the effect of hepatic insufficiency on its pharmacokinetics has not been reported, and there is a lack of clinical guidance for the use of BG in patients with hepatic impairment. Methods: Carbon tetrachloride (CCl4)-induced rat models were used to simulate hepatic failure patients to assess the effect of hepatic impairment on the pharmacokinetics and distribution of BG. In vitro metabolism and transporter studies were employed to elucidate the potential mechanisms. Results: After intragastric administration of 10 mg/kg of BG, the peak plasma concentration and exposure (AUC0–t) of BG decreased by 64.6% and 52.6%, respectively, in CCl4-induced rats. After intravenous administration, the AUC0–t decreased by 73.6%, and unlike in the control group, the second absorption peak of BG was not obvious in the concentration–time curve of CCl4-induced rats. The cumulative excretion of BG in the feces increased, but that in the bile decreased. In vivo data indicated that the absorption and enterohepatic circulation of BG were affected. In vitro studies found that the hydrolysis of BG to the aglycone baicalein decreased significantly in the intestinal tissues and contents of the CCl4-induced rats. And BG was identified as a substrate for multiple efflux and uptake transporters, such as breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs), organic anion transporting polypeptides (OATP1B1, 1B3, 2B1), and organic anion transporters (OATs). The bile acids accumulated by liver injury inhibited the uptake of BG by OATPs, especially that by OATP2B1. Conclusions: Hepatic impairment reduced BG hydrolysis by intestinal microflora and inhibited its transporter-mediated biliary excretion, which synergistically led to the attenuation of the enterohepatic circulation of BG, which altered its pharmacokinetics.

Substrate Specificity of the Organic Cation Transporters MATE1 and MATE2K and Functional Overlap with OCT1 and OCT2.

The multidrug and toxin extrusion proteins MATE1 and MATE2K may determine the pharmacokinetics and drug-drug interactions of many drugs. However, their substrate spectrum and synergy with organic cation transporters OCT1 and OCT2 remain incompletely understood. Therefore, we screened 590 predominantly positively charged, low molecular weight compounds for transport via these four transporters in HEK293 cells using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). MATE1 and MATE2K transported 164 and 114 compounds, respectively, with significant overlap. High-affinity substrates included berberine, pentamidine, and amisulpride, while epinephrine and atenolol had the highest Vmax. Despite less than 16% sequence homology, there was high overlap among MATE1/-2K and OCT1/-2 substrates. Neither isolated physicochemical properties nor their linear combinations predicted the substrates of these organic cation transporters. However, machine learning classifiers using 15 parameters allowed 69 to 87% correct prediction. The large number of substrates indicates a possibly broad role of multidrug and toxin extrusion (MATE) transporters in pharmacokinetics and drug interactions.

Sex-Specific Differences in the Transport and Availability of Energy Substrates in the Hippocampus of Rats During Neurodevelopment.

The mammalian brain uses several energy substrates during development, such as lactate, ketone bodies, and glucose, to avoid energy imbalances that may lead to cellular dysfunction. Sex differences in brain function are well-documented, with males being more susceptible to neurodevelopmental disturbances than females; however, the underlying reason for this vulnerability is not fully elucidated. Present study aimed to investigate sex differences in energy metabolism at different postnatal ages. Wistar rats of both sexes were evaluated on postnatal days (PND) 1, 7, 14, 21, and 60. Biochemical parameters included the immunocontent of ketone bodies (KB) and glucose transporters, glucose uptake in the hippocampus, and the levels of lactate, glucose, and beta-hydroxybutyrate (BOHB) in serum and cerebrospinal fluid. Moreover, beta-hydroxybutyrate dehydrogenase (BHD) activity was evaluated in the liver and the hippocampus. Immunofluorescence staining was performed to label the distribution of monocarboxylate transporters (MCTs) in the hippocampus and the corpus callosum. Serum lactate and BOHB levels were higher at PND1 than at other ages. Animals at PND14 predominantly utilize BOHB as a substrate, followed by a transition phase to glucose during weaning. Female rats exhibited an increase in the immunocontent of MCT2, MCT1, MCT4, and GLUT1 throughout postnatal development, suggesting a metabolic advantage over males due to greater uptake efficiency of energy substrates. Present findings reveal sex-specific differences in the expression of glucose and MCTs monocarboxylate transporters during postnatal brain development. A deeper understanding of these metabolic differences among healthy individuals may elucidate crucial sex-specific features of in neurological disorders, such as hypoxia-ischemia. This understanding could facilitate the advancement of more precise and targeted therapeutic interventions.

EFFECT OF SEX HORMONES ON THE EXPRESSION OF GENES ENCODING ORGANIC ANION-TRANSPORTING POLYPEPTIDES OATP1B1/OATP1B3 IN VITRO

Introduction. OATP1B1 and OATP1B3 are influx proteins belonging to the SLC transporter family and providing substrate transport into hepatocytes. The mechanisms of their regulation are currently being actively studied. Aim. To study the effect of sex hormones on the expression of the SLCO1B1 and SLCO1B3 genes encoding OATP1B1 and OATP1B3, respectively. Material and methods. The study was performed on the HepG2 cell line. Estradiol, progesterone, and testosterone were added to the cells at concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and incubated for 24 h. After incubation, total RNA was isolated and gene expression was tested by real-time polymerase chain reaction. Results. Estradiol at concentrations of 10 and 100 μM increased the expression of the SLCO1B1 gene encoding OATP1B1 by 50.9% (p=0.027) and 60.2% (p=0.009), respectively. Testosterone at a concentration of 1 μM increased the expression of the SLCO1B1 gene by 53.2% (p=0.014), at a concentration of 10 μM – by 62.7% (p=0.004), at a concentration of 100 μM – by 66.7% (p=0.0026). Estradiol at concentrations of 1, 10 and 100 μM increased the expression of the SLCO1B3 gene by 80.3% (p=0.0021)

ConformationalLandscape of the Di- and TripeptidePermease A Transport Cycle

Dipeptide and tripeptidepermease A (DtpA) transporteris a bacterialhomologue of the human PepT that is responsible for the uptake ofdi- and tripeptides from the small intestine and transports them acrossthe cell membrane utilizing an inward-directed proton electrochemicalgradient. Despite its importance, the structural dynamics governingthe conformational transitions of DtpA remain poorly understood. Inthis study, we employed Adaptive Bandit enhanced sampling moleculardynamics simulations to investigate the five major conformationalstates of DtpA adopted during the transport cycle. We identified keymetastable states and transitions underlying the transport cycle usingMarkov State Models (MSMs). Our findings reveal that intra- and interhelicalinteractions drive conformational changes by inducing bending androtation of helices lining the pore, resulting in its opening andclosure. This study explains the substrate transport mechanism inDtpA, enhancing our understanding of bacterial proton-dependent oligopeptidetransporters (POTs) and opening new drug design and development opportunities.

Decrease in In Vivo Efflux Transport via P-glycoprotein at the Rat Inner Blood-Retinal Barrier by Peripheral Administration of Lipopolysaccharide.

This study aimed to determine in vivo alterations in the rat retinal distribution of a substrate for P-glycoprotein (P-gp), which restricts drug transport to the retina at the inner blood-retinal barrier (BRB), by peripheral administration of lipopolysaccharide (LPS), an inflammatory agent. Using retinal capillaries isolated from rats 24h after peripheral 5mg/kg LPS administration, transport analyses with a fluorescent substrate of P-gp were performed. In vivo retinal distribution of [3H]digoxin, a P-gp substrate, in the LPS-administered rats was evaluated after intravenous or intracarotid artery injection. The mRNA and protein expression levels of P-gp in the retinal capillaries were evaluated. P-gp-mediated luminal transport of the fluorescent substrate was significantly attenuated in retinal capillaries of the LPS-administered rats. Moreover, in vivo retinal [3H]digoxin distribution in LPS-injected rats was significantly greater than that in saline-injected rats. Since the retinal distribution of [3H]D-mannitol, a paracellular transport marker, was not significantly altered in LPS-treated rats, it is suggested that in vivo elevation of retinal [3H]digoxin distribution is caused by P-gp downregulation at the inner BRB, but not a change in paracellular transport in the barrier. In retinal capillaries isolated from LPS-administered rats, expression analyses of P-gp mRNAs and protein indicated a reduction in its expression on the luminal membrane of the inner BRB. Our study demonstrated that in vivo retinal distribution of P-gp substrates was elevated in LPS-administered rats via a decrease in the function and expression of P-gp at the inner BRB.

Helicobacter pylori CagT4SS proteins CagN and CagM bind DNA and CagN is involved in heptose-independent pro-inflammatory substrate translocation by the T4SS.

Helicobacter pylori CagT4SS proteins CagN and CagM bind DNA and CagN is involved in heptose-independent pro-inflammatory substrate translocation by the T4SS.

Transcriptome and lipidome integration unveils key mechanisms constraining bivalve larval sensitivity in an acidifying sea.

Transcriptome and lipidome integration unveils key mechanisms constraining bivalve larval sensitivity in an acidifying sea.

In vitro identification of decreased function genetic variants of ABCB1.

In vitro identification of decreased function genetic variants of ABCB1.

Multisite synergistic evolution of oleate hydratase via DCCM and the orthogonal location of distal sites.

Multisite synergistic evolution of oleate hydratase via DCCM and the orthogonal location of distal sites.

Plant steroids on the move: mechanisms of brassinosteroid export.

Plant steroids on the move: mechanisms of brassinosteroid export.

Proof of principle concept for the analysis and functional prediction of rare genetic variants in the CYP2C19 and CYP2D6 genes

BackgroundVariations in pharmacogenes that regulate drug absorption, distribution, metabolism, and excretion (ADME) contribute to approximately 20–30% of interindividual differences in drug response. While many common variants are successfully utilized in clinical settings to predict individual drug responses, a significant portion of the genetic basis underlying this variability remains unidentified. This includes rare variants, which are estimated to account for 4–6% of drug response variability.ResultsTo comprehensively elucidate the functional consequences and molecular mechanisms of rare variants, we conducted in vitro enzyme expression studies combined with in silico structure–function analyses. We selected 11 rare variants in the CYP2C19 and CYP2D6 genes identified among participants within the Estonian Biobank. Variant cDNAs were heterologously expressed in HEK-293 cells, and detailed enzyme activity analyses were performed. The experimental results were further validated against average scores from five optimized in silico prediction models: LRT, Mutation Assessor, PROVEAN, VEST3, and CADD. To explore structure–activity relationships, we performed in silico docking of substrates into available 3D enzyme structures. Our findings reveal that most of the rare genetic variants caused significant functional alterations, including: (i) Likely impairments in substrate transport to the active site due to narrowing of access channels; (ii) Changes in catalytic rates; and (iii) Potential effects on substrate extrusion rates from the active site. The in silico prediction tools accurately anticipated the functional impact of 6 out of the 11 variants (54%). ConclusionsEvaluating the functionality of rare variants will become increasingly essential as rapid and cost-effective whole-genome sequencing technologies continue to advance. Our results highlight the need for further refinement of in silico prediction models, particularly those leveraging 3D crystal enzyme structures, to enhance the accuracy of functional predictions for rare genetic variants.