RESUMEN
Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.
Asunto(s)
Ácido Ascórbico , Microscopía por Crioelectrón , Transportadores de Sodio Acoplados a la Vitamina C , Humanos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/química , Transporte Biológico , Sodio/metabolismo , Modelos Moleculares , Multimerización de Proteína , Unión Proteica , Células HEK293 , Conformación ProteicaRESUMEN
Alzheimer's disease (AD) is a common neurodegenerative disorder. The number of patients with AD is projected to reach 152 million by 2050. Donepezil, rivastigmine, galantamine, and memantine are the only four drugs currently approved by the United States Food and Drug Administration for AD treatment. However, these drugs can only alleviate AD symptoms. Thus, this research focuses on the discovery of novel lead compounds that possess multitarget regulation of AD etiopathology relating to amyloid cascade. The ascorbic acid structure has been designated as a core functional domain due to several characteristics, including antioxidant activities, amyloid aggregation inhibition, and the ability to be transported to the brain and neurons. Multifunctional ascorbic derivatives were synthesized by copper (I)-catalyzed azide-alkyne cycloaddition reaction (click chemistry). The in vitro and cell-based assays showed that compounds 2c and 5c exhibited prominent multifunctional activities as beta-secretase 1 inhibitors, amyloid aggregation inhibitors, and antioxidant, neuroprotectant, and anti-inflammatory agents. Significant changes in activities promoting neuroprotection and anti-inflammation were observed at a considerably low concentration at a nanomolar level. Moreover, an in silico study showed that compounds 2c and 5c were capable of being permeated across the blood-brain barrier by sodium-dependent vitamin C transporter-2.
Asunto(s)
Proteínas Amiloidogénicas/antagonistas & inhibidores , Antiinflamatorios/farmacología , Ácido Ascórbico/análogos & derivados , Fármacos Neuroprotectores/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Proteínas Amiloidogénicas/metabolismo , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Ácido Ascórbico/química , Ácido Ascórbico/farmacología , Sitios de Unión , Barrera Hematoencefálica , Células Cultivadas , Simulación por Computador , Ciclooxigenasa 2/genética , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Óxido Nítrico Sintasa de Tipo II/genética , Células RAW 264.7 , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Triazoles/farmacologíaRESUMEN
Vitamin C (VitC) is a requisite nutrient for humans and other primates. Extensive research continuously illustrates the applications of VitC in promoting cell reprogramming, fine-tuning embryonic stem cell function, and fighting diseases. Given its chemical reduction property, VitC predominantly acts as an antioxidant to reduce reactive oxygen species (ROS) and as a cofactor for certain dioxygenases involved in epigenetic regulation. Here, we propose that VitC is also a bio-signaling molecule based on the finding that sodium-dependent VitC transporter (SVCT) 2 is a novel receptor-like transporter of VitC that possesses dual activities in mediating VitC uptake and Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 2 signaling pathway. Through interaction, SVCT2 induces JAK2 phosphorylation while transporting VitC into cells. Activated JAK2 phosphorylates the C-terminus of SVCT2, resulting in the recruitment and activation of STAT2. As a highlight, our results suggest that the activation of JAK2 synergistically promotes regulation of VitC in ROS scavenging and epigenetic modifications through phosphorylating pyruvate dehydrogenase kinase 1, ten-eleven translocation enzyme 3, and histone H3 Tyr41. Furthermore, VitC-activated JAK2 exhibits bidirectional effects in regulating cell pluripotency and differentiation. Our results thus reveal that the SVCT2-mediated JAK2 activation facilitates VitC functions in a previously unknown manner.
Asunto(s)
Ácido Ascórbico/metabolismo , Janus Quinasa 2/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/genética , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Animales , Ácido Ascórbico/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Dioxigenasas/genética , Epigénesis Genética/efectos de los fármacos , Células HEK293 , Histonas/metabolismo , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Dominios Proteicos , Factor de Transcripción STAT2/genética , Transducción de Señal/efectos de los fármacos , Transportadores de Sodio Acoplados a la Vitamina C/químicaRESUMEN
In this study, a novel brain targeting ascorbic acid (AA) derivative with "lock-in" function was designed and synthesized as a liposome ligand to prepare novel liposomes to achieve the effective delivery of drug formulations to brain via glucose transporter 1 (GLUT1) and the Na+-dependent vitamin C transporter (SVCT2). The liposome was prepared and characterized in terms of the particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cell cytotoxicity. The preliminary evaluation in vivo demonstrated that the AA-thiamine disulfide system (TDS)-coated liposome had an improved targeting ability and significantly increased the brain concentration of docetaxel (DTX) as compared to the naked docetaxel, the non-coated and the AA-coated liposomes. The relative uptake efficiency and concentration efficiency were enhanced by 3.24- and 5.62-fold compared to that of the naked docetaxel, respectively. Both distribution data and pharmacokinetic parameters suggested that the ascorbic acid thiamine disulfide delivery system was a promising carrier to enhance central nervous system (CNS) drug's delivery ability into brain.
Asunto(s)
Antineoplásicos/química , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Docetaxel/química , Liposomas/química , Animales , Antineoplásicos/farmacología , Encéfalo , Docetaxel/farmacología , Composición de Medicamentos/métodos , Transportador de Glucosa de Tipo 1/química , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Ratones , Estructura Molecular , Albúmina Sérica Bovina/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Relación Estructura-Actividad , Propiedades de Superficie , Tiamina/análogos & derivados , Tiamina/química , Distribución TisularRESUMEN
Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.
Asunto(s)
Ácido Ascórbico/química , Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Transportadores de Sodio Acoplados a la Vitamina C/química , Secuencia de Aminoácidos , Ácido Ascórbico/biosíntesis , Ácido Ascórbico/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Cristalografía por Rayos X , Humanos , Ligandos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/ultraestructura , Microscopía Electrónica de Transmisión , Unión Proteica , Transportadores de Sodio Acoplados a la Vitamina C/metabolismoRESUMEN
Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.
Asunto(s)
Expresión Génica , Transportadores de Sodio Acoplados a la Vitamina C/genética , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Animales , Humanos , Oocitos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/aislamiento & purificación , Xenopus laevisRESUMEN
We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.
Asunto(s)
Membrana Celular/metabolismo , Modelos Biológicos , Señales de Clasificación de Proteína , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Secuencia de Aminoácidos , Animales , Polaridad Celular , Secuencia Conservada , Perros , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células de Riñón Canino Madin Darby , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/genéticaRESUMEN
Vitamin C (ascorbic acid) is required for the synthesis of collagen, carnitine, catecholamine and the neurotransmitter norepinephrine. Vitamin C also plays an important role in protection against oxidative stress. Transporters for vitamin C and its oxidized form dehydroascorbate (DHA) are crucial to keep vitamin concentrations optimal in the body. The human SLC23 family consists of the Na(+)-dependent vitamin C transporters SVCT1 (SLC23A1) and SVCT2 (SLC23A2) and the orphan transporter SVCT3 (SLC23A3). Phylogenetically, the SLC23 family belongs to the nucleobase-ascorbate transporter family although no specificity for nucleobases has yet been demonstrated for the human members of this family. In fact, the SVCT1 and SVCT2 transporters are rather specific for ascorbic acid. SVCT1 is expressed in epithelial tissues such as intestine, where it contributes to the maintenance of whole-body ascorbic acid levels, whereas the expression of SVCT2 is relatively widespread either to protect metabolically active cells and specialized tissues from oxidative stress or to deliver ascorbic acid to tissues that are in high demand of the vitamin for enzymatic reactions. DHA, the oxidized form of ascorbic acid is taken up and distributed in the body by facilitated transport via members of the SLC2/GLUT family (GLUT1, GLUT3, and GLUT4). Although, the main focus of this review is on the SLC23 family of ascorbic acid transporters, transporters of DHA and nucleobases are also briefly discussed for completeness.
Asunto(s)
Ácido Ascórbico/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Ácido Deshidroascórbico/metabolismo , Humanos , Cinética , Datos de Secuencia MolecularRESUMEN
Vitamin C (ascorbic acid, AA) is an antioxidant that acts as a free radical scavenger and cofactor for several important enzymatic reactions, thus being important for normal cellular functions, growth and development. Accumulation of AA in cells depends on two types of sodium-dependent vitamin C transporters (SVCTs), designed as SVCT1 and SVCT2. In human, they are the products of SLC23A1 and SLC23A2 genes, respectively. In the present work, the molecular cloning of the cDNAs corresponding to slc23a1 and slc23a2 in a teleost fish, the Senegalese sole (Solea senegalensis Kaup, 1858) is first described. Sequence analysis of the predicted polypeptides revealed a conserved topology with those of mammals with important motifs involved in structure and function, being also present in svct1 and svct2. Phylogenetic analyses including a range of vertebrate SVCTs suggest that both transporters are the result of an ancient gene duplication event that occurred prior to the divergence of tetrapods and teleosts, which took place 450 million years ago. Expression profiles in juvenile tissues and during larval development were analyzed using a real-time PCR approach. In juvenile fish, slc23a1 was strongly expressed in intestine, whereas slc23a2 exhibited a widespread distribution in tissues. Transcripts of both genes were detected at early developmental stages, probably representing mRNAs of maternal origin. A possible regulation by their own substrate was detected after first uptakes of AA from diet in both genes. During metamorphosis, both slc23a1 and slc23a2 were down-regulated, the former in a thyroid hormone (TH) dependent way. This pattern coincided with a significant reduction in the AA content of larvae during metamorphosis. These results are interpreted in a physiological context of general reduction in the metabolism of metamorphic larvae. Data presented here provide the first step toward a better understanding of the physiological role of SVCTs in teleost fish.
Asunto(s)
Peces Planos/genética , Regulación de la Expresión Génica , Transportadores de Sodio Acoplados a la Vitamina C/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Ácido Ascórbico/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Larva/efectos de los fármacos , Larva/genética , Metamorfosis Biológica/efectos de los fármacos , Metamorfosis Biológica/genética , Datos de Secuencia Molecular , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Senegal , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Hormonas Tiroideas/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Vitamin C, or ascorbic acid, is important as an antioxidant and participates in numerous cellular functions. Although it circulates in plasma in micromolar concentrations, it reaches millimolar concentrations in most tissues. These high ascorbate cellular concentrations are thought to be generated and maintained by the SVCT2 (Slc23a2), a specific transporter for ascorbate. The vitamin is also readily recycled from its oxidized forms inside cells. Neurons in the central nervous system (CNS) contain some of the highest ascorbic acid concentrations of mammalian tissues. Intracellular ascorbate serves several functions in the CNS, including antioxidant protection, peptide amidation, myelin formation, synaptic potentiation, and protection against glutamate toxicity. The importance of the SVCT2 for CNS function is supported by the finding that its targeted deletion in mice causes widespread cerebral hemorrhage and death on post-natal day 1. Neuronal ascorbate content as maintained by this protein also has relevance for human disease, since ascorbate supplements decrease infarct size in ischemia-reperfusion injury models of stroke, and since ascorbate may protect neurons from the oxidant damage associated with neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. The aim of this review is to assess the role of the SVCT2 in regulating neuronal ascorbate homeostasis and the extent to which ascorbate affects brain function and antioxidant defenses in the CNS.
Asunto(s)
Ácido Ascórbico/metabolismo , Sistema Nervioso Central/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/fisiología , Animales , Ácido Ascórbico/fisiología , Transporte Biológico/fisiología , Regulación de la Expresión Génica , Humanos , Ratones , Conformación Proteica , Transportadores de Sodio Acoplados a la Vitamina C/química , Relación Estructura-ActividadRESUMEN
In order to improve oral absorption, a novel prodrug of saquinavir (Saq), ascorbyl-succinic-saquinavir (AA-Su-Saq) targeting sodium dependent vitamin C transporter (SVCT) was synthesized and evaluated. Aqueous solubility, stability and cytotoxicity were determined. Affinity of AA-Su-Saq towards efflux pump P-glycoprotein (P-gp) and recognition of AA-Su-Saq by SVCT were studied. Transepithelial permeability across polarized MDCK-MDR1 and Caco-2 cells were determined. Metabolic stability of AA-Su-Saq in rat liver microsomes was investigated. AA-Su-Saq appears to be fairly stable in both DPBS and Caco-2 cells with half lives of 9.65 and 5.73 h, respectively. Uptake of [(3)H]Saquinavir accelerated by 2.7 and 1.9 fold in the presence of 50 µM Saq and AA-Su-Saq in MDCK-MDR1 cells. Cellular accumulation of [(14)C]AA diminished by about 50-70% relative to control in the presence of 200 µM AA-Su-Saq in MDCK-MDR1 and Caco-2 cells. Uptake of AA-Su-Saq was lowered by 27% and 34% in the presence of 5mM AA in MDCK-MDR1 and Caco-2 cells, respectively. Absorptive permeability of AA-Su-Saq was elevated about 4-5 fold and efflux index reduced by about 13-15 fold across the polarized MDCK-MDR1 and Caco-2 cells. Absorptive permeability of AA-Su-Saq decreased 44% in the presence of 5mM AA across MDCK-MDR1 cells. AA-Su-Saq was devoid of cytotoxicity over the concentration range studied. AA-Su-Saq significantly enhanced the metabolic stability but lowered the affinity towards CYP3A4. In conclusion, prodrug modification of Saq through conjugation to AA via a linker significantly raised the absorptive permeability and metabolic stability. Such modification also caused significant evading of P-gp mediated efflux and CYP3A4 mediated metabolism. SVCT targeted prodrug approach can be an attractive strategy to enhance the oral absorption and systemic bioavailability of anti-HIV protease inhibitors.
Asunto(s)
Ácido Ascórbico/análogos & derivados , Inhibidores de la Proteasa del VIH/síntesis química , Terapia Molecular Dirigida/métodos , Profármacos/metabolismo , Saquinavir/análogos & derivados , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Ácido Ascórbico/análisis , Ácido Ascórbico/síntesis química , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Transporte Biológico , Células CACO-2 , Línea Celular , Citocromo P-450 CYP3A , Composición de Medicamentos , Estabilidad de Medicamentos , Epitelio/metabolismo , Inhibidores de la Proteasa del VIH/análisis , Inhibidores de la Proteasa del VIH/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Microsomas Hepáticos/metabolismo , Permeabilidad , Profármacos/síntesis química , Ratas , Saquinavir/análisis , Saquinavir/síntesis química , Saquinavir/química , Saquinavir/metabolismo , Sodio/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Solubilidad , Vitaminas/metabolismoRESUMEN
The ascorbate transporters SVCT1 and SVCT2 are crucial for maintaining intracellular ascorbate concentrations in most cell types. Although the two transporter isoforms are highly homologous, they have different physiologic functions. The SVCT1 is located primarily in epithelial cells and has its greatest effect in reabsorbing ascorbate in the renal tubules. The SVCT2 is located in most non-epithelial tissues, with the highest expression in brain and neuroendocrine tissues. These transporters are hydrophobic membrane proteins that have a high affinity and are highly selective for ascorbate. Their ability to concentrate ascorbate inside cells is driven by the sodium gradient across the plasma membrane as generated by Na+/K+ ATPase. They can concentrate ascorbate 20 to 60-fold over plasma ascorbate concentrations. Ascorbate transport on these proteins is regulated at the transcriptional, translational and post-translational levels. Available studies show that transporter function is acutely regulated by protein kinases A and C, whereas transporter expression is increased by low intracellular ascorbate and associated oxidative stress. The knockout of the SVCT2 in mice is lethal on day 1 of life, and almost half of SVCT1 knockout mice do not survive to weaning. These findings confirm the importance both of cellular ascorbate and of the two transport proteins as key to maintaining intracellular ascorbate. LINKED ARTICLES This article is part of a themed section on Transporters. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2011.164.issue-7.