International Journal of Molecular Sciences

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Open AccessEditorial

Amino Acids Transport and Metabolism 2.0

by Mariafrancesca Scalise and Cesare Indiveri

Int. J. Mol. Sci. 2020, 21(4), 1212; https://doi.org/10.3390/ijms21041212 - 12 Feb 2020

Cited by 5 | Viewed by 2539

Abstract

This editorial aims to summarize the 19 scientific papers that contributed to this Special Issue. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

Research

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12 pages, 1409 KiB

Open AccessArticle

SLC38A10 (SNAT10) is Located in ER and Golgi Compartments and Has a Role in Regulating Nascent Protein Synthesis

by Rekha Tripathi, Kimia Hosseini, Vasiliki Arapi, Robert Fredriksson and Sonchita Bagchi

Int. J. Mol. Sci. 2019, 20(24), 6265; https://doi.org/10.3390/ijms20246265 - 12 Dec 2019

Cited by 10 | Viewed by 5273

Abstract

The solute carrier (SLC) family-38 of transporters has eleven members known to transport amino acids, with glutamine being a common substrate for ten of them, with SLC38A9 being the exception. In this study, we examine the subcellular localization of SNAT10 in several independent [...] Read more.

The solute carrier (SLC) family-38 of transporters has eleven members known to transport amino acids, with glutamine being a common substrate for ten of them, with SLC38A9 being the exception. In this study, we examine the subcellular localization of SNAT10 in several independent immortalized cell lines and stem cell-derived neurons. Co-localization studies confirmed the SNAT10 was specifically localized to secretory organelles. SNAT10 is expressed in both excitatory and inhibitory neurons in the mouse brain, predominantly in the endoplasmic reticulum, and in the Golgi apparatus. Knock-down experiments of SNAT10, using Slc38a10-specific siRNA in PC12 cells reduced nascent protein synthesis by more than 40%, suggesting that SNAT10 might play a role in signaling pathways that regulate protein synthesis, and may act as a transceptor in a similar fashion to what has been shown previously for SLC38A2 (SNAT2) and SNAT9(SLC38A9). Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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13 pages, 3787 KiB

Open AccessArticle

Broad-Spectrum Amino Acid Transporters ClAAP3 and ClAAP6 Expressed in Watermelon Fruits

by Tianran Shi, Vijay Joshi, Madhumita Joshi, Stanislav Vitha, Holly Gibbs, Kehua Wang and Sakiko Okumoto

Int. J. Mol. Sci. 2019, 20(23), 5855; https://doi.org/10.3390/ijms20235855 - 22 Nov 2019

Cited by 3 | Viewed by 3335

Abstract

Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) [...] Read more.

Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) through the phloem. The amino acid transporters involved in the import of Cit, Arg, and their precursors into developing fruits of watermelon have not been reported. In this study, we have compiled the list of putative amino acid transporters in watermelon and characterized transporters that are expressed in the early stage of fruit development. Using the yeast complementation study, we characterized ClAAP3 (Cla023187) and ClAAP6 (Cla023090) as functional amino acid transporters belonging to the family of amino acid permease (AAP) genes. The yeast growth and uptake assays of radiolabeled amino acid suggested that ClAAP3 and ClAAP6 can transport a broad spectrum of amino acids. Expression of translational fusion proteins with a GFP reporter in Nicotiana benthamiana leaves confirmed the ER- and plasma membrane-specific localization, suggesting the role of ClAAP proteins in the cellular import of amino acids. Based on the gene expression profiles and functional characterization, ClAAP3 and ClAAP6 are expected to play a major role in regulation of amino acid import into developing watermelon fruits. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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20 pages, 2573 KiB

Open AccessArticle

Ceftriaxone Treatment Affects EAAT2 Expression and Glutamatergic Neurotransmission and Exerts a Weak Anticonvulsant Effect in Young Rats

by Aleksey V. Zaitsev, Sergey L. Malkin, Tatyana Y. Postnikova, Ilya V. Smolensky, Olga E. Zubareva, Irina V. Romanova, Maria V. Zakharova, Vladimir B. Karyakin and Vladimir Zavyalov

Int. J. Mol. Sci. 2019, 20(23), 5852; https://doi.org/10.3390/ijms20235852 - 21 Nov 2019

Cited by 13 | Viewed by 4005

Abstract

Epilepsy is a common neurological disorder. Despite the availability of a wide range of antiepileptic drugs, these are unsuccessful in preventing seizures in 20–30% of patients. Therefore, new pharmacological strategies are urgently required to control seizures. Modulation of glutamate uptake may have potential [...] Read more.

Epilepsy is a common neurological disorder. Despite the availability of a wide range of antiepileptic drugs, these are unsuccessful in preventing seizures in 20–30% of patients. Therefore, new pharmacological strategies are urgently required to control seizures. Modulation of glutamate uptake may have potential in the treatment of pharmacoresistant forms of epilepsy. Previous research showed that the antibiotic ceftriaxone (CTX) increased the expression and functional activity of excitatory amino acid transporter 2 (EAAT2) and exerted considerable anticonvulsant effects. However, other studies did not confirm a significant anticonvulsant effect of CTX administration. We investigated the impacts of CTX treatment on EAAT expression and glutamatergic neurotransmission, as well its anticonvulsant action, in young male Wistar rats. As shown by a quantitative real-time polymerase chain reaction (qPCR) assay and a Western blot analysis, the mRNA but not the protein level of EAAT2 increased in the hippocampus following CTX treatment. Repetitive CTX administration had only a mild anticonvulsant effect on pentylenetetrazol (PTZ)-induced convulsions in a maximal electroshock threshold test (MEST). CTX treatment did not affect the glutamatergic neurotransmission, including synaptic efficacy, short-term facilitation, or the summation of excitatory postsynaptic potentials (EPSPs) in the hippocampus and temporal cortex. However, it decreased the field EPSP (fEPSP) amplitudes evoked by intense electrical stimulation. In conclusion, in young rats, CTX treatment did not induce overexpression of EAAT2, therefore exerting only a weak antiseizure effect. Our data provide new insight into the effects of modulation of EAAT2 expression on brain functioning. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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15 pages, 82364 KiB

Open AccessArticle

Inducible Slc7a7 Knockout Mouse Model Recapitulates Lysinuric Protein Intolerance Disease

by Susanna Bodoy, Fernando Sotillo, Meritxell Espino-Guarch, Maria Pia Sperandeo, Aida Ormazabal, Antonio Zorzano, Gianfranco Sebastio, Rafael Artuch and Manuel Palacín

Int. J. Mol. Sci. 2019, 20(21), 5294; https://doi.org/10.3390/ijms20215294 - 24 Oct 2019

Cited by 19 | Viewed by 4418

Abstract

Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y⁺LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from [...] Read more.

Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y⁺LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7^−/−). The Slc7a7^−/− model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7^−/− mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7^−/− model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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9 pages, 547 KiB

Open AccessCommunication

L-Alanine Exporter, AlaE, of Escherichia coli Functions as a Safety Valve to Enhance Survival under Feast Conditions

by Satoshi Katsube, Tasuke Ando and Hiroshi Yoneyama

Int. J. Mol. Sci. 2019, 20(19), 4942; https://doi.org/10.3390/ijms20194942 - 7 Oct 2019

Cited by 7 | Viewed by 4049

Abstract

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine [...] Read more.

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine movement across the inner membrane. We identified the novel gene alaE, encoding an L-alanine exporter. To elucidate the physiological function of L-Alanine exporter, AlaE, we determined the susceptibility of alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655, to L-alanyl-L-alanine (Ala-Ala), which shows toxicity to the L-alanine-nonmetabolizing variant lacking alaE. The dadA-deficient mutant has a similar minimum inhibitory concentration (MIC) (>1.25 mg/mL) to that observed in MG1655. However, alaE- and alaE/dadA-deficient mutants had MICs of 0.04 and 0.0025 mg/mL, respectively. The results suggested that the efficacy of AlaE to relieve stress caused by toxic intracellular accumulation of L-alanine was higher than that of DadA. Consistent with this, the intracellular level of alanine in the alaE-mutant was much higher than that in MG1655 and the dadA-mutant. We, therefore, conclude that AlaE functions as a ‘safety-valve’ to prevent the toxic level accumulation of intracellular L-alanine under a peptide-rich environment, such as within the animal intestine. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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29 pages, 6176 KiB

Open AccessArticle

Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo

by Sabrina Petralla, Luis Emiliano Peña-Altamira, Eleonora Poeta, Francesca Massenzio, Marco Virgili, Simona Nicole Barile, Luigi Sbano, Emanuela Profilo, Mariangela Corricelli, Alberto Danese, Carlotta Giorgi, Rita Ostan, Miriam Capri, Paolo Pinton, Ferdinando Palmieri, Francesco Massimo Lasorsa and Barbara Monti

Int. J. Mol. Sci. 2019, 20(18), 4486; https://doi.org/10.3390/ijms20184486 - 11 Sep 2019

Cited by 15 | Viewed by 4477

Abstract

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate–aspartate NADH shuttle (MAS), expressed [...] Read more.

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate–aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor βs (TGFβs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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15 pages, 2051 KiB

Open AccessArticle

Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium

by Carmela Vigorito, Evgeniya Anishchenko, Luigi Mele, Giovanna Capolongo, Francesco Trepiccione, Miriam Zacchia, Patrizia Lombari, Rosanna Capasso, Diego Ingrosso and Alessandra F. Perna

Int. J. Mol. Sci. 2019, 20(9), 2269; https://doi.org/10.3390/ijms20092269 - 8 May 2019

Cited by 16 | Viewed by 3560

Abstract

(1) The beneficial effects of hydrogen sulfide (H₂S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H₂S biosynthesis, previously used as a marker for H₂S [...] Read more.

(1) The beneficial effects of hydrogen sulfide (H₂S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H₂S biosynthesis, previously used as a marker for H₂S production, is dramatically increased in circulation in uremia, while H₂S release is impaired. Thus, lanthionine could be classified as a novel uremic toxin. Our research was aimed at defining the mechanism(s) for lanthionine toxicity. (2) The effect of lanthionine on H₂S release was tested by a novel lead acetate strip test (LAST) in EA.hy926 cell cultures. Effects of glutathione, as a redox agent, were assayed. Levels of sulfane sulfur were evaluated using the SSP4 probe and flow cytometry. Protein content and glutathionylation were analyzed by Western Blotting and immunoprecipitation, respectively. Gene expression and miRNA levels were assessed by qPCR. (3) We demonstrated that, in endothelial cells, lanthionine hampers H₂S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine-β-synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca²⁺ levels. (4) Lanthionine-induced alterations in cell cultures, which involve both sulfur amino acid metabolism and calcium homeostasis, are consistent with uremic dysfunctional characteristics and further support the uremic toxin role of this amino acid. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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16 pages, 1752 KiB

Open AccessArticle

Transcriptional Regulation Factors of the Human Mitochondrial Aspartate/Glutamate Carrier Gene, Isoform 2 (SLC25A13): USF1 as Basal Factor and FOXA2 as Activator in Liver Cells

by Paolo Convertini, Simona Todisco, Francesco De Santis, Ilaria Pappalardo, Dominga Iacobazzi, Maria Antonietta Castiglione Morelli, Yvonne N. Fondufe-Mittendorf, Giuseppe Martelli, Ferdinando Palmieri and Vittoria Infantino

Int. J. Mol. Sci. 2019, 20(8), 1888; https://doi.org/10.3390/ijms20081888 - 16 Apr 2019

Cited by 19 | Viewed by 5904

Abstract

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 [...] Read more.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (−442 bp/−19 bp), as well as an enhancer region (−968 bp/−768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC). Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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14 pages, 3613 KiB

Open AccessArticle

Unique Regulation of Enterocyte Brush Border Membrane Na-Glutamine and Na-Alanine Co-Transport by Peroxynitrite during Chronic Intestinal Inflammation

by Subha Arthur, Palanikumar Manoharan, Shanmuga Sundaram, M Motiur Rahman, Balasubramanian Palaniappan and Uma Sundaram

Int. J. Mol. Sci. 2019, 20(6), 1504; https://doi.org/10.3390/ijms20061504 - 26 Mar 2019

Cited by 9 | Viewed by 4128

Abstract

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is [...] Read more.

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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10 pages, 6985 KiB

Open AccessArticle

Volta Phase Plate Cryo-EM Structure of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2

by Jean-Marc Jeckelmann and Dimitrios Fotiadis

Int. J. Mol. Sci. 2019, 20(4), 931; https://doi.org/10.3390/ijms20040931 - 21 Feb 2019

Cited by 13 | Viewed by 5411

Abstract

Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two [...] Read more.

Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two subunits are linked by a conserved disulfide bridge. Several human diseases are associated with loss of function or overexpression of specific HATs making them drug targets. The human HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is specific for the transport of large neutral L-amino acids and specific amino acid-related compounds. Human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris and pure recombinant protein purified. Here we present the first cryo-electron microscopy (cryo-EM) 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex. The structure could be determined at ~13 Å resolution using direct electron detector and Volta phase plate technologies. The 3D-map displays two prominent densities of different sizes. The available X-ray structure of the 4F2hc ectodomain fitted nicely into the smaller density revealing the relative position of 4F2hc with respect to LAT2 and the membrane plane. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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18 pages, 3651 KiB

Open AccessArticle

Regulatory Aspects of the Vacuolar CAT2 Arginine Transporter of S. lycopersicum: Role of Osmotic Pressure and Cations

by Jessica Cosco, Teresa M. R. Regina, Mariafrancesca Scalise, Michele Galluccio and Cesare Indiveri

Int. J. Mol. Sci. 2019, 20(4), 906; https://doi.org/10.3390/ijms20040906 - 19 Feb 2019

Cited by 7 | Viewed by 3928

Abstract

Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from S. lycopersicum (SlCAT2) [...] Read more.

Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from S. lycopersicum (SlCAT2) that was previously overexpressed in E. coli and reconstituted in proteoliposomes for transport assay as [³H]Arg uptake. The orientation of the reconstituted transporter has been attempted and current data support the hypothesis that the protein is inserted in the liposome in the same orientation as in the vacuole. SlCAT2 activity was dependent on the pH, with an optimum at pH 7.5. SlCAT2 transport activity was stimulated by the increase of internal osmolality from 0 to 175 mOsmol while the activity was inhibited by the increase of external osmolality. K⁺, Na⁺, and Mg²⁺ present on the external side of proteoliposomes at physiological concentrations, inhibited the transport activity; differently, the cations had no effect when included in the internal proteoliposome compartment. This data highlighted an asymmetric regulation of SlCAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the SlCAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter GkApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of SlCAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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16 pages, 1736 KiB

Open AccessArticle

Silencing of Transcription Factor Sp1 Promotes SN1 Transporter Regulation by Ammonia in Mouse Cortical Astrocytes

by Katarzyna Dąbrowska and Magdalena Zielińska

Int. J. Mol. Sci. 2019, 20(2), 234; https://doi.org/10.3390/ijms20020234 - 9 Jan 2019

Cited by 6 | Viewed by 4447

Abstract

The involvement of the astrocytic SN1 (SNAT3) transporter in ammonia-induced l-glutamine retention was recently documented in mouse-cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its [...] Read more.

The involvement of the astrocytic SN1 (SNAT3) transporter in ammonia-induced l-glutamine retention was recently documented in mouse-cultured astrocytes. Here we investigated the involvement of specificity protein 1 (Sp1) transcription factor in SN1 regulation in ammonium chloride (“ammonia”)-treated astrocytes. Sp1 expression and its cellular localization were determined using real-time qPCR, Western blot, and confocal microscopy. Sp1 binding to Snat3 promoter was analyzed by chromatin immunoprecipitation. The role of Sp1 in SN1 expression and SN1-mediated [³H]glutamine uptake in ammonia-treated astrocytes was verified using siRNA and mithramycin A. The involvement of protein kinase C (PKC) isoforms in Sp1 level/phosphorylation status was verified using siRNA technology. Sp1 translocation to the nuclei and its enhanced binding to the Snat3 promoter, along with Sp1 dependence of system N-mediated [³H]glutamine uptake, were observed in astrocytes upon ammonia exposure. Ammonia decreased the level of phosphorylated Sp1, and the effect was reinforced by long-term incubation with PKC modulator, phorbol 12-myristate 13-acetate, which is a treatment likely to dephosphorylate Sp1. Furthermore, silencing of the PKCδ isoform appears to enhance the ammonia effect on the Sp1 level. Collectively, the results demonstrate the regulatory role of Sp1 in regulation of SN1 expression and activity in ammonia-treated astrocytes and implicate altered Sp1 phosphorylation status in this capacity. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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26 pages, 8101 KiB

Open AccessArticle

Discovery of Potent Inhibitors for the Large Neutral Amino Acid Transporter 1 (LAT1) by Structure-Based Methods

by Natesh Singh, Mariafrancesca Scalise, Michele Galluccio, Marcus Wieder, Thomas Seidel, Thierry Langer, Cesare Indiveri and Gerhard F. Ecker

Int. J. Mol. Sci. 2019, 20(1), 27; https://doi.org/10.3390/ijms20010027 - 21 Dec 2018

Cited by 35 | Viewed by 8991

Abstract

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically [...] Read more.

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure−activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC₅₀ value in the sub-μM range. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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Review

Jump to: Editorial, Research

27 pages, 342 KiB

Open AccessReview

Amino Acid Transport Defects in Human Inherited Metabolic Disorders

by Raquel Yahyaoui and Javier Pérez-Frías

Int. J. Mol. Sci. 2020, 21(1), 119; https://doi.org/10.3390/ijms21010119 - 23 Dec 2019

Cited by 40 | Viewed by 7461

Abstract

Amino acid transporters play very important roles in nutrient uptake, neurotransmitter recycling, protein synthesis, gene expression, cell redox balance, cell signaling, and regulation of cell volume. With regard to transporters that are closely connected to metabolism, amino acid transporter-associated diseases are linked to [...] Read more.

Amino acid transporters play very important roles in nutrient uptake, neurotransmitter recycling, protein synthesis, gene expression, cell redox balance, cell signaling, and regulation of cell volume. With regard to transporters that are closely connected to metabolism, amino acid transporter-associated diseases are linked to metabolic disorders, particularly when they involve different organs, cell types, or cell compartments. To date, 65 different human solute carrier (SLC) families and more than 400 transporter genes have been identified, including 11 that are known to include amino acid transporters. This review intends to summarize and update all the conditions in which a strong association has been found between an amino acid transporter and an inherited metabolic disorder. Many of these inherited disorders have been identified in recent years. In this work, the physiological functions of amino acid transporters will be described by the inherited diseases that arise from transporter impairment. The pathogenesis, clinical phenotype, laboratory findings, diagnosis, genetics, and treatment of these disorders are also briefly described. Appropriate clinical and diagnostic characterization of the underlying molecular defect may give patients the opportunity to avail themselves of appropriate therapeutic options in the future. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

11 pages, 817 KiB

Open AccessReview

Excitatory Amino Acid Transporters (EAATs): Glutamate Transport and Beyond

by Simona Magi, Silvia Piccirillo, Salvatore Amoroso and Vincenzo Lariccia

Int. J. Mol. Sci. 2019, 20(22), 5674; https://doi.org/10.3390/ijms20225674 - 13 Nov 2019

Cited by 76 | Viewed by 6508

Abstract

Na⁺-dependent excitatory amino acid transporters (EAATs) are the major transport mechanisms for extracellular glutamate removal in the central nervous system (CNS). The primary function assigned to EAATs is the maintenance of low extracellular glutamate levels, thus allowing glutamate to be used [...] Read more.

Na⁺-dependent excitatory amino acid transporters (EAATs) are the major transport mechanisms for extracellular glutamate removal in the central nervous system (CNS). The primary function assigned to EAATs is the maintenance of low extracellular glutamate levels, thus allowing glutamate to be used as a signaling molecule in the brain and to avoid excitotoxicity. However, glutamate has other recognized functions. For instance, it is a key anaplerotic substrate for the tricarboxylic acid (TCA) cycle, as it can be converted to α-ketoglutarate by transaminases or glutamate dehydrogenase. Furthermore, glutamate is a precursor of the main antioxidant glutathione, which plays a pivotal role in preventing oxidative cell death. Therefore, glutamate signaling/use is at the crossroad of multiple metabolic pathways and accordingly, it can influence a plethora of cell functions, both in health and disease. Here, we provide an overview of the main functions of glutamate and its transport systems, analyzing its role as a neurotransmitter and at the same time, the possible metabolic fates it can undergo in the intracellular milieu. Specifically, the metabolic role of glutamate and the molecular machinery proposed to metabolically support its transport will be further analyzed. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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37 pages, 1614 KiB

Open AccessReview

Excitatory Amino Acid Transporters in Physiology and Disorders of the Central Nervous System

by Anna R. Malik and Thomas E. Willnow

Int. J. Mol. Sci. 2019, 20(22), 5671; https://doi.org/10.3390/ijms20225671 - 12 Nov 2019

Cited by 106 | Viewed by 9156

Abstract

Excitatory amino acid transporters (EAATs) encompass a class of five transporters with distinct expression in neurons and glia of the central nervous system (CNS). EAATs are mainly recognized for their role in uptake of the amino acid glutamate, the major excitatory neurotransmitter. EAATs-mediated [...] Read more.

Excitatory amino acid transporters (EAATs) encompass a class of five transporters with distinct expression in neurons and glia of the central nervous system (CNS). EAATs are mainly recognized for their role in uptake of the amino acid glutamate, the major excitatory neurotransmitter. EAATs-mediated clearance of glutamate released by neurons is vital to maintain proper glutamatergic signalling and to prevent toxic accumulation of this amino acid in the extracellular space. In addition, some EAATs also act as chloride channels or mediate the uptake of cysteine, required to produce the reactive oxygen speciesscavenger glutathione. Given their central role in glutamate homeostasis in the brain, as well as their additional activities, it comes as no surprise that EAAT dysfunctions have been implicated in numerous acute or chronic diseases of the CNS, including ischemic stroke and epilepsy, cerebellar ataxias, amyotrophic lateral sclerosis, Alzheimer’s disease and Huntington’s disease. Here we review the studies in cellular and animal models, as well as in humans that highlight the roles of EAATs in the pathogenesis of these devastating disorders. We also discuss the mechanisms regulating EAATs expression and intracellular trafficking and new exciting possibilities to modulate EAATs and to provide neuroprotection in course of pathologies affecting the CNS. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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13 pages, 646 KiB

Open AccessReview

Computer-Aided Strategies for Determining the Amino Acid Composition of Medium for Chinese Hamster Ovary Cell-Based Biomanufacturing Platforms

by Bergthor Traustason, Matthew Cheeks and Duygu Dikicioglu

Int. J. Mol. Sci. 2019, 20(21), 5464; https://doi.org/10.3390/ijms20215464 - 2 Nov 2019

Cited by 9 | Viewed by 6034

Abstract

Chinese hamster ovary (CHO) cells are used for the production of the majority of biopharmaceutical drugs, and thus have remained the standard industry host for the past three decades. The amino acid composition of the medium plays a key role in commercial scale [...] Read more.

Chinese hamster ovary (CHO) cells are used for the production of the majority of biopharmaceutical drugs, and thus have remained the standard industry host for the past three decades. The amino acid composition of the medium plays a key role in commercial scale biologics manufacturing, as amino acids constitute the building blocks of both endogenous and heterologous proteins, are involved in metabolic and non-metabolic pathways, and can act as main sources of nitrogen and carbon under certain conditions. As biomanufactured proteins become increasingly complex, the adoption of model-based approaches become ever more popular in complementing the challenging task of medium development. The extensively studied amino acid metabolism is exceptionally suitable for such model-driven analyses, and although still limited in practice, the development of these strategies is gaining attention, particularly in this domain. This paper provides a review of recent efforts. We first provide an overview of the widely adopted practice, and move on to describe the model-driven approaches employed for the improvement and optimization of the external amino acid supply in light of cellular amino acid demand. We conclude by proposing the likely prevalent direction the field is heading towards, providing a critical evaluation of the current state and the future challenges and considerations. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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24 pages, 1898 KiB

Open AccessReview

Mitochondrial Carriers for Aspartate, Glutamate and Other Amino Acids: A Review

by Magnus Monné, Angelo Vozza, Francesco Massimo Lasorsa, Vito Porcelli and Ferdinando Palmieri

Int. J. Mol. Sci. 2019, 20(18), 4456; https://doi.org/10.3390/ijms20184456 - 10 Sep 2019

Cited by 47 | Viewed by 6784

Abstract

Members of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates [...] Read more.

Members of the mitochondrial carrier (MC) protein family transport various molecules across the mitochondrial inner membrane to interlink steps of metabolic pathways and biochemical processes that take place in different compartments; i.e., are localized partly inside and outside the mitochondrial matrix. MC substrates consist of metabolites, inorganic anions (such as phosphate and sulfate), nucleotides, cofactors and amino acids. These compounds have been identified by in vitro transport assays based on the uptake of radioactively labeled substrates into liposomes reconstituted with recombinant purified MCs. By using this approach, 18 human, plant and yeast MCs for amino acids have been characterized and shown to transport aspartate, glutamate, ornithine, arginine, lysine, histidine, citrulline and glycine with varying substrate specificities, kinetics, influences of the pH gradient, and capacities for the antiport and uniport mode of transport. Aside from providing amino acids for mitochondrial translation, the transport reactions catalyzed by these MCs are crucial in energy, nitrogen, nucleotide and amino acid metabolism. In this review we dissect the transport properties, phylogeny, regulation and expression levels in different tissues of MCs for amino acids, and summarize the main structural aspects known until now about MCs. The effects of their disease-causing mutations and manipulation of their expression levels in cells are also considered as clues for understanding their physiological functions. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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14 pages, 267 KiB

Open AccessReview

The L-Type Amino Acid Transporter LAT1—An Emerging Target in Cancer

by Pascal Häfliger and Roch-Philippe Charles

Int. J. Mol. Sci. 2019, 20(10), 2428; https://doi.org/10.3390/ijms20102428 - 16 May 2019

Cited by 154 | Viewed by 8882

Abstract

Chronic proliferation is a major hallmark of tumor cells. Rapidly proliferating cancer cells are highly dependent on nutrients in order to duplicate their cell mass during each cell division. In particular, essential amino acids are indispensable for proliferating cancer cells. Their uptake across [...] Read more.

Chronic proliferation is a major hallmark of tumor cells. Rapidly proliferating cancer cells are highly dependent on nutrients in order to duplicate their cell mass during each cell division. In particular, essential amino acids are indispensable for proliferating cancer cells. Their uptake across the cell membrane is tightly controlled by membrane transporters. Among those, the L-type amino acid transporter LAT1 (SLC7A5) has been repeatedly found overexpressed in a vast variety of cancers. In this review, we summarize the most recent advances in our understanding of the role of LAT1 in cancer and highlight preclinical studies and drug developments underlying the potential of LAT1 as therapeutic target. Full article

(This article belongs to the Special Issue Amino Acids Transport and Metabolism 2.0)

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