Citrate Uptake Research Articles

Structure-Guided Discovery of a Potent Inhibitor of the Ferric Citrate Binding Protein FecB in Vibrio Bacteria.

Infections caused by Gram-negative bacteria present a significant risk to human health worldwide. Novel strategies are needed to deal with the challenge caused by drug-resistant bacteria. Here, we report a new approach to combat infections by targeting iron-binding proteins to suppress bacterial growth. We investigated the function of the conserved periplasmic binding protein FecB from Vibrio alginolyticus. FecB was known to play a crucial role in the bacterial growth and to relate with biofilm formation. We then solved the crystal structures and elucidated the binding mechanism of FecB with ferric ion chelated by citrate. The results indicated that FecB binds weakly to one citrate molecule and strongly to the Fe3+-(citrate)2 complex. Based on these results, a structure-based virtual screening approach was conducted against FecB to identify small molecules that block ferric citrate uptake. Further evaluations in vivo and in vitro demonstrated that salvianolic acid C significantly suppressed bacterial growth, indicating that targeting bacterial nutrient absorption is a promising strategy for identifying potential antibacterial drugs.

Structure‐Guided Discovery of a Potent Inhibitor of the Ferric Citrate Binding Protein FecB in Vibrio Bacteria

AbstractInfections caused by Gram‐negative bacteria present a significant risk to human health worldwide. Novel strategies are needed to deal with the challenge caused by drug‐resistant bacteria. Here, we report a new approach to combat infections by targeting iron‐binding proteins to suppress bacterial growth. We investigated the function of the conserved periplasmic binding protein FecB from Vibrio alginolyticus. FecB was known to play a crucial role in the bacterial growth and to relate with biofilm formation. We then solved the crystal structures and elucidated the binding mechanism of FecB with ferric ion chelated by citrate. The results indicated that FecB binds weakly to one citrate molecule and strongly to the Fe3+‐(citrate)2 complex. Based on these results, a structure‐based virtual screening approach was conducted against FecB to identify small molecules that block ferric citrate uptake. Further evaluations in vivo and in vitro demonstrated that salvianolic acid C significantly suppressed bacterial growth, indicating that targeting bacterial nutrient absorption is a promising strategy for identifying potential antibacterial drugs.

Molecular Phenotypes Segregate Missense Mutations in SLC13A5 Epilepsy

The sodium-coupled citrate transporter (NaCT, SLC13A5) mediates citrate uptake across the plasma membrane via an inward Na+ gradient. Mutations in SLC13A5 cause early infantile epileptic encephalopathy type-25 (EIEE25, SLC13A5 Epilepsy) due to impaired citrate uptake in neurons and astrocytes. Despite clinical identification of disease-causing mutations, underlying mechanisms and cures remain elusive. Here we mechanistically classify six frequent SLC13A5 mutations by phenotyping their protein cell surface expression and citrate transport functions. Mutants C50R, T142M, and T227M exhibit impaired citrate transport despite normal expression at the cell surface. In contrast, mutations G219R, S427L, and L488P show low total protein expression levels, absence of mature, glycosylated proteins at the cell surface, retention of the proteins in the endoplasmic reticulum, and diminished transport activity. This mechanistic classification divides SLC13A5 mutants into two groups, Class I (C50R, T142M, and T227M) and Class II (G219R, S427L, and L488P). Importantly, mutants’ mRNA levels resemble wildtype, suggesting post-translational defects. Class II mutations display immature core-glycosylation and shortened half-lives, indicating protein folding defects. Together, these experiments provide a comprehensive understanding of the disease-causing mutation’s defects in SLC13A5 Epilepsy at the biochemical and molecular level and shed light into the trafficking pathway(s) of NaCT. The two classes of mutations will require fundamentally different approaches for treatment to either restore transport function of the mutant protein that is capable of reaching the cell surface (Class I), or therapies that enable the correction of protein folding defects to enable escape to the cell surface where it may restore transport function (Class II).

Anticancer effects of high-dose extracellular citrate treatment in pancreatic cancer cells under different glucose concentrations

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive solid tumor. Recently, the uptake of extracellular citrate by the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, has been demonstrated to exert profound effects on cancer cell metabolism. However, research on the function of extracellular citrate in PDAC pathogenesis and the relationship between NaCT expression and the tumor metabolic microenvironment is limited. Therefore, we aimed to evaluate the expression of citrate transporters across a spectrum of glucose concentrations in pancreatic cancer and systematically explore the effects of sodium citrate treatment on pancreatic cancer cells at different glucose concentrations. We observed a positive correlation between glucose concentration and NaCT expression in PDAC cell lines. Extracellular sodium citrate significantly reduced cell viability partially due to reduction in intracellular Ca2+ levels and decreased the migration of human PDAC cells. Furthermore, we observed a decrease in the levels of the stem cell marker prominin I (CD133) following sodium citrate treatment. Notably, the combination treatment of gemcitabine and extracellular sodium citrate exhibited a synergistic anticancer effect in both two-dimensional (2D) and three-dimensional (3D) culture systems. Additionally, we confirmed that pH slightly increased upon administration of sodium citrate, indicating that this could potentially augment the efficacy of gemcitabine. Altogether, these findings suggest that exogenous sodium citrate treatment, particularly in combination with gemcitabine, may represent a novel therapeutic strategy for treating PDAC. This approach holds promise for disrupting PDAC cell metabolism and inhibiting tumor progression.

Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness.

Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR- genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR- strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR- strains. Citrate uptake by LasR- strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR- strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR- strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities.IMPORTANCECross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.

Targeting Longevity Gene SLC13A5: A Novel Approach to Prevent Age-Related Bone Fragility and Osteoporosis.

Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1-5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts' specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.

3D human foreskin model for testing topical formulations of sildenafil citrate

Sildenafil citrate is an approved drug used for the treatment of erectile dysfunction and premature ejaculation. Despite a widespread application, sildenafil citrate shows numerous adverse cardiovascular effects in high-risk patients. Local transdermal drug delivery of this drug is therefore being explored as an interesting and noninvasive alternative administration method that avoids adverse effects arised from peak plasma drug concentrations. Although human and animal skin represents the most reliable models to perform penetration studies, they involve a series of ethical issues and restrictions. For these reasons new in vitro approaches based on artificially reconstructed human skin or “human skin equivalents” are being developed as possible alternatives for transdermal testing. There is little information, however, on the efficiency of such new in vitro methods on cutaneous penetration of active ingredients. The objective of the current study was to investigate the sildenafil citrate loaded in three commercial transdermal vehicles using 3D full-thickness skin equivalent and compare the results with the permeability experiments using porcine skin. Our results demonstrated that, while the formulation plays an imperative role in an appropriate dermal uptake of sildenafil citrate, the D coefficient results obtained by using the 3D skin equivalent are comparable to those obtained by using the porcine skin when a simple drug suspension is applied (1.17 × 10-10 ± 0.92 × 10-10 cm2/s vs 3.5 × 102 ± 3.3 × 102 cm2/s), suggesting that in such case, this 3D skin model can be a valid alternative for ex-vivo skin absorption experiments.

The effects of excess salt intake on the kidney metabolism in normal Sprague Dawley rats.

Introduction: This study aimed to determine the effects of a high salt diet upon the metabolism of O 2 and substances in the kidney in normal Sprague Dawley (SD) rats. Methods were developed enabling the intermittent collection of renal arteriovenous (A-Vr) blood and urine samples and to measure renal blood flow (RBF) and arterial pressure (BP) in conscious freely moving rats with GFR determined in a separate group of conscious rats. Other groups were studied to obtain cortical (Cx) and outer medullary (OM) tissue samples for associated transcriptomic and metabolomic profiles. Methods: SD rats were instrumented with a renal arterial ultrasonic flow probe, a femoral arterial and renal venous catheters, and RBF and BP measured continuously (24/7). Arterial (A) and renal venous (Vr) blood and urine were collected repeatedly before and 7, 14, and 21 days after increasing the salt diet from 0.4% (LS) to 4% (HS) NaCl. GFR was measured in age-matched rats and used as control shams for normalization. Blood O 2 content was determined by radiometry. A global metabolomic analysis was performed on plasma and Cx and OM tissue samples at JAX laboratories (Thermo Q-Exactive Orbitrap coupled to Vanquich UPLC system) and together with mRNAseq analysis for Cx and OM by Novogene Inc. Results: BP increased less than 5 mmHg by day 3 of the HS diet while RBF rose nearly 20% (P<0.05) and both were sustained at this level throughout the study. O 2 delivery, O 2 consumption (VO 2 ) and O 2 extraction rose over the 21 days of the HS diet (P<0.05). Total GFR increased from 0.64 to 0.83 mL/min/100 g bw (P<0.05) at HS 7 and remained elevated at HS 21. Whole kidney Na + reabsorption and kidney VO 2 were highly correlated positively (r=0.99). A sparse partial least squares discriminant analysis (sPLS-DA) of metabolomics for each of the 4 modes (C18 +/- and HILIC +/-) revealed clear distinctions between LS, HS14, and HS21 within Cx and OM. The mRNAseq tissue analysis found that the HS diet resulted in reduced expression of genes related to the TCA cycle and metabolism of amino acids and fatty acids. This was consistent with the metabolomic data showing a reduction of tissue citrate and succinate levels. In contrast, glycolysis-related genes including hexokinase, pyruvate kinase and lactate dehydrogenase were upregulated in the Cx tissue by HS. Minimal changes in metabolic pathways were observed in the OM tissue. A-Vr x RBF enabled metabolomic flux determination which showed the release of glucose on LS fed rats which was not evident with the HS diet. Uptake of citrate which was seen on LS was less evident on HS but there was a clear tendency observed for increased release of lactate, pyruvate, and α-ketoglutarate. Conclusions: Normal SD rats fed a HS underwent changes of RBF, O 2 extraction and utilization of metabolic substrates, most notably transitioning ATP production predominantly driven from TCA cycle to non-TCA cycle pathways which we are currently exploring more deeply. HL-116264, HL-151587 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mapping the Metabolic Niche of Citrate Metabolism and SLC13A5.

The small molecule citrate is a key molecule that is synthesized de novo and involved in diverse biochemical pathways influencing cell metabolism and function. Citrate is highly abundant in the circulation, and cells take up extracellular citrate via the sodium-dependent plasma membrane transporter NaCT encoded by the SLC13A5 gene. Citrate is critical to maintaining metabolic homeostasis and impaired NaCT activity is implicated in metabolic disorders. Though citrate is one of the best known and most studied metabolites in humans, little is known about the consequences of altered citrate uptake and metabolism. Here, we review recent findings on SLC13A5, NaCT, and citrate metabolism and discuss the effects on metabolic homeostasis and SLC13A5-dependent phenotypes. We discuss the "multiple-hit theory" and how stress factors induce metabolic reprogramming that may synergize with impaired NaCT activity to alter cell fate and function. Furthermore, we underline how citrate metabolism and compartmentalization can be quantified by combining mass spectrometry and tracing approaches. We also discuss species-specific differences and potential therapeutic implications of SLC13A5 and NaCT. Understanding the synergistic impact of multiple stress factors on citrate metabolism may help to decipher the disease mechanisms associated with SLC13A5 citrate transport disorders.

Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

Structural characterization of the Aspergillus niger citrate transporter CexA uncovers the role of key residues S75, R192 and Q196

The Aspergillus niger CexA transporter belongs to the DHA1 (Drug-H+ antiporter) family. CexA homologs are exclusively found in eukaryotic genomes, and CexA is the sole citrate exporter to have been functionally characterized in this family so far. In the present work, we expressed CexA in Saccharomyces cerevisiae, demonstrating its ability to bind isocitric acid, and import citrate at pH 5.5 with low affinity. Citrate uptake was independent of the proton motive force and compatible with a facilitated diffusion mechanism. To unravel the structural features of this transporter, we then targeted 21 CexA residues for site-directed mutagenesis. Residues were identified by a combination of amino acid residue conservation among the DHA1 family, 3D structure prediction, and substrate molecular docking analysis. S. cerevisiae cells expressing this library of CexA mutant alleles were evaluated for their capacity to grow on carboxylic acid-containing media and transport of radiolabeled citrate. We also determined protein subcellular localization by GFP tagging, with seven amino acid substitutions affecting CexA protein expression at the plasma membrane. The substitutions P200A, Y307A, S315A, and R461A displayed loss-of-function phenotypes. The majority of the substitutions affected citrate binding and translocation. The S75 residue had no impact on citrate export but affected its import, as the substitution for alanine increased the affinity of the transporter for citrate. Conversely, expression of CexA mutant alleles in the Yarrowia lipolytica cex1Δ strain revealed the involvement of R192 and Q196 residues in citrate export. Globally, we uncovered a set of relevant amino acid residues involved in CexA expression, export capacity and import affinity.

Elucidating the effect of iron acquisition systems in Klebsiella pneumoniae on susceptibility to the novel siderophore-cephalosporin cefiderocol.

Cefiderocol (CFDC) is a novel siderophore-cephalosporin, effective against multidrug-resistant Gram-negative bacteria. As it has a siderophore side chain, it can utilize iron acquisition systems for penetration of the bacterial outer membrane. We aimed to elucidate the role of siderophores and iron uptake receptors in defining Klebsiella pneumoniae susceptibility to CFDC. Initially, 103 K. pneumoniae strains were characterized for susceptibility to different antibiotics including CFDC. CFDC minimum inhibitory concentrations (MIC) were determined in iron-depleted and iron-enriched conditions. Iron uptake genes including siderophores, their receptors, ferric citrate (fecA) and iron uptake (kfu) receptors were detected by PCR in all the strains. For 10 selected strains, gene expression was tested in iron-depleted media with or without CFDC treatment and compared to expression in iron-enriched conditions. CFDC exhibited 96.1% susceptibility, being superior to all the other antibiotics (MIC50: 0.5 and MIC90: 4 μg/ml). Only three strains (2.9%) were intermediately susceptible and a pandrug resistant strain (0.97%) was resistant to CFDC (MIC: 8 and 256 μg/ml, respectively). The presence of kfu and fecA had a significant impact on CFDC MIC, especially when co-produced, and if coupled with yersiniabactin receptor (fyuA). CFDC MICs were negatively correlated with enterobactin receptor (fepA) expression and positively correlated with expression of kfu and fecA. Thus, fepA was associated with increased susceptibility to CFDC, while kfu and fecA were associated with reduced susceptibility to CFDC. CFDC MICs increased significantly in iron-enriched media, with reduced expression of siderophore receptors, hence, causing less drug uptake. Iron acquisition systems have a significant impact on CFDC activity, and their altered expression is a factor leading to reduced susceptibility. Iron concentration is also a major player affecting CFDC susceptibility; therefore, it is essential to explore possible ways to improve the drug activity to facilitate its use to treat infections in iron-rich sites.

A specialized metabolic pathway partitions citrate in hydroxyapatite to impact mineralization of bones and teeth.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na+-dependent citrate transporter solute carrier family 13 member 5 (Slc13a5) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5-/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5-deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

A Novel and Cross-Species Active Mammalian INDY (NaCT) Inhibitor Ameliorates Hepatic Steatosis in Mice with Diet-Induced Obesity.

Mammalian INDY (mINDY, NaCT, gene symbol SLC13A5) is a potential target for the treatment of metabolically associated fatty liver disease (MAFLD). This study evaluated the effects of a selective, cross-species active, non-competitive, non-substrate-like inhibitor of NaCT. First, the small molecule inhibitor ETG-5773 was evaluated for citrate and succinate uptake and fatty acid synthesis in cell lines expressing both human NaCT and mouse Nact. Once its suitability was established, the inhibitor was evaluated in a diet-induced obesity (DIO) mouse model. DIO mice treated with 15 mg/kg compound ETG-5773 twice daily for 28 days had reduced body weight, fasting blood glucose, and insulin, and improved glucose tolerance. Liver triglycerides were significantly reduced, and body composition was improved by reducing fat mass, supported by a significant reduction in the expression of genes for lipogenesis such as SREBF1 and SCD1. Most of these effects were also evident after a seven-day treatment with the same dose. Further mechanistic investigation in the seven-day study showed increased plasma β-hydroxybutyrate and activated hepatic adenosine monophosphate-activated protein kinase (AMPK), reflecting findings from Indy (−/−) knockout mice. These results suggest that the inhibitor ETG-5773 blocked citrate uptake mediated by mouse and human NaCT to reduce liver steatosis and body fat and improve glucose regulation, proving the concept of NaCT inhibition as a future liver treatment for MAFLD.

Development of a novel lactic acid bacteria starter culture approach: From insect microbiome to plant-based fermentations

Lactic acid bacteria (LAB) are a broad group of gram-positive bacteria that have actively been used worldwide in food productions due to their nutritional, technological, bioprotection and flavour-related characteristics, which define them as powerful biological tools to be used in fermented food applications. LAB are ubiquitous in nature and some species have been found in fructose-rich niches, such as insect microbiomes. Twenty-eight LAB species were isolated from Apis mellifera, Bombus spp. and Andrena nigroaenea insect sources. Lactococcus lactis was the most widespread species in all insect species. A phenotypical characterization analysis based on carbohydrate metabolism, subproduct and acid production, pectinolytic activity, citrate uptake, phytate degradation and diacetyl production was carried out. Based on this analysis, two strains belonging to Leuconostoc pseudomesenteroides and L.lactis species were found to be the most suitable strains for use in common food plant matrices. The former showed an ability to significantly catabolize raffinose, maltose and citrate, which are present in soy beverages, while the latter was able to produce high concentrations of diacetyl and lactic acid, which are relevant for the generation of plant dairy alternatives, and noticeably degraded phytic acid, pectin and sucrose mostly found in bean, cereal and fruit-based plant matrices.

The Role of Citrate Homeostasis in Merkel Cell Carcinoma Pathogenesis.

Simple SummaryMerkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer. Despite important progress, overall understanding of the events that drive MCC carcinogenesis remains incomplete. We discovered that the plasma membrane citrate transporter (pmCiC) is upregulated in Merkel cell carcinoma cell lines. Cancer cells import extracellular citrate via pmCiC to support their metabolism, which is critical to support proliferation and metastatic spread. In this study, we show that inhibition of pmCiC can decrease the growth rate of Merkel cell carcinoma cell lines. Targeting pmCiC and thereby the tumor metabolism should be considered further as a potential anti-cancer therapy.Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.

Binding of Citrate-Fe3+ to Plastic Culture Dishes, an Artefact Useful as a Simple Technique to Screen for New Iron Chelators.

NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe3+), citrate uptake/binding as monitored by the association of [14C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe3+-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe3+ chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe3+ chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe3+ and prevent the formation and adsorption of citrate-Fe3+ to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.

The fciTABC and feoABI systems contribute to ferric citrate acquisition in Stenotrophomonas maltophilia

BackgroundStenotrophomonas maltophilia, a member of γ-proteobacteria, is a ubiquitous environmental bacterium that is recognized as an opportunistic nosocomial pathogen. FecABCD system contributes to ferric citrate acquisition in Escherichia coli. FeoABC system, consisting of an inner membrane transporter (FeoB) and two cytoplasmic proteins (FeoA and FeoC), is a well-known ferrous iron transporter system in γ-proteobacteria. As revealed by the sequenced genome, S. maltophilia appears to be equipped with several iron acquisition systems; however, the understanding of these systems is limited. In this study, we aimed to elucidate the ferric citrate acquisition system of S. maltophilia.MethodsCandidate genes searching and function validation are the strategy for elucidating the genes involved in ferric citrate acquisition. The candidate genes responsible for ferric citrate acquisition were firstly selected using FecABCD of E. coli as a reference, and then revealed by transcriptome analysis of S. maltophilia KJ with and without 2,2′-dipyridyl (DIP) treatment. Function validation was carried out by deletion mutant construction and ferric citrate utilization assay. The bacterial adenylate cyclase two-hybrid system was used to verify intra-membrane protein–protein interaction.ResultsSmlt2858 and Smlt2356, the homologues of FecA and FecC/D of E. coli, were first considered; however, deletion mutant construction and functional validation ruled out their involvement in ferric citrate acquisition. FciA (Smlt1148), revealed by its upregulation in DIP-treated KJ cells, was the outer membrane receptor for ferric citrate uptake. The fciA gene is a member of the fciTABC operon, in which fciT, fciA, and fciC participated in ferric citrate acquisition. Uniquely, the Feo system of S. maltophilia is composed of a cytoplasmic protein FeoA, an inner membrane transporter FeoB, and a predicted inner membrane protein FeoI. The intra-membrane protein–protein interaction between FeoB and FeoI may extend the substrate profile of FeoB to ferric citrate. FeoABI system functioned as an inner membrane transporter of ferric citrate.ConclusionsThe FciTABC and FeoABI systems contribute to ferric citrate acquisition in S. maltophilia.

L-Arginine and Cardioactive Arginine Derivatives as Substrates and Inhibitors of Human and Mouse NaCT/Nact

The uptake transporter NaCT (gene symbol SLC13A5) is expressed in liver and brain and important for energy metabolism and brain development. Substrates include tricarboxylic acid cycle intermediates, e.g., citrate and succinate. To gain insights into the substrate spectrum of NaCT, we tested whether arginine and the cardioactive L-arginine metabolites asymmetric dimethylarginine (ADMA) and L-homoarginine are also transported by human and mouse NaCT/Nact. Using HEK293 cells overexpressing human or mouse NaCT/Nact we characterized these substances as substrates. Furthermore, inhibition studies were performed using the arginine derivative symmetric dimethylarginine (SDMA), the NaCT transport inhibitor BI01383298, and the prototypic substrate citrate. Arginine and the derivatives ADMA and L-homoarginine were identified as substrates of human and mouse NaCT. Transport of arginine and derivatives mediated by human and mouse NaCT were dose-dependently inhibited by SDMA. Whereas BI01383298 inhibited only human NaCT-mediated citrate uptake, it inhibits the uptake of arginine and derivatives mediated by both human NaCT and mouse Nact. In contrast, the prototypic substrate citrate inhibited the transport of arginine and derivatives mediated only by human NaCT. These results demonstrate a so far unknown link between NaCT/Nact and L-arginine and its cardiovascular important derivatives.

NaCT/SLC13A5 facilitates citrate import and metabolism under nutrient-limited conditions

SUMMARYCitrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.