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1.
BMC Med Genet ; 21(1): 97, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32380970

RESUMO

BACKGROUND: Amelogenesis imperfecta (AI) is a highly heterogeneous group of hereditary developmental abnormalities which mainly affects the dental enamel during tooth development in terms of its thickness, structure, and composition. It appears both in syndromic as well as non-syndromic forms. In the affected individuals, the enamel is usually thin, soft, rough, brittle, pitted, chipped, and abraded, having reduced functional ability and aesthetics. It leads to severe complications in the patient, like early tooth loss, severe discomfort, pain, dental caries, chewing difficulties, and discoloration of teeth from yellow to yellowish-brown or creamy type. The study aimed to identify the disease-causing variant in a consanguineous family. METHODS: We recruited a consanguineous Pashtun family of Pakistani origin. Exome sequencing analysis was followed by Sanger sequencing to identify the pathogenic variant in this family. RESULTS: Clinical analysis revealed hypomaturation AI having generalized yellow-brown or creamy type of discoloration in affected members. We identified a novel nonsense sequence variant c.1192C > T (p.Gln398*) in exon-12 of SLC24A4 by using exome sequencing. Later, its co-segregation within the family was confirmed by Sanger sequencing. The human gene mutation database (HGMD, 2019) has a record of five pathogenic variants in SLC24A4, causing AI phenotype. CONCLUSION: This nonsense sequence variant c.1192C > T (p.Gln398*) is the sixth disease-causing variant in SLC24A4, which extends its mutation spectrum and confirms the role of this gene in the morphogenesis of human tooth enamel. The identified variant highlights the critical role of SLC24A4 in causing a rare AI type in humans.


Assuntos
Amelogênese Imperfeita/genética , Antiporters/genética , Cárie Dentária/genética , Predisposição Genética para Doença , Adulto , Amelogênese Imperfeita/epidemiologia , Amelogênese Imperfeita/patologia , Códon sem Sentido/genética , Cárie Dentária/epidemiologia , Cárie Dentária/patologia , Esmalte Dentário/metabolismo , Éxons/genética , Feminino , Humanos , Masculino , Morfogênese/genética , Paquistão/epidemiologia , Linhagem , Perda de Dente/genética , Perda de Dente/fisiopatologia , Sequenciamento Completo do Exoma , Adulto Jovem
2.
Proc Natl Acad Sci U S A ; 117(19): 10313-10321, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341169

RESUMO

The H+/Ca2+ (calcium ion) antiporter (CAX) plays an important role in maintaining cellular Ca2+ homeostasis in bacteria, yeast, and plants by promoting Ca2+ efflux across the cell membranes. However, how CAX facilitates Ca2+ balance in response to dynamic cytosolic Ca2+ perturbations is unknown. Here, we identified a type of Ca2+ "mini-sensor" in YfkE, a bacterial CAX homolog from Bacillus subtilis. The mini-sensor is formed by six tandem carboxylate residues within the transmembrane (TM)5-6 loop on the intracellular membrane surface. Ca2+ binding to the mini-sensor triggers the transition of the transport mode of YfkE from a high-affinity to a low-affinity state. Molecular dynamics simulation and fluorescence resonance energy transfer analysis suggest that Ca2+ binding to the mini-sensor causes an adjacent segment, namely, the exchanger inhibitory peptide (XIP), to move toward the Ca2+ translocation pathway to interact with TM2a in an inward-open cavity. The specific interaction was demonstrated with a synthetic peptide of the XIP, which inhibits YfkE transport and interrupts conformational changes mediated by the mini-sensor. By comparing the apo and Ca2+-bound CAX structures, we propose the following Ca2+ transport regulatory mechanism of YfkE: Ca2+ binding to the mini-sensor induces allosteric conformational changes in the Ca2+ translocation pathway via the XIP, resulting in a rearrangement of the Ca2+-binding transport site in the midmembrane. Since the Ca2+ mini-sensor and XIP sequences are also identified in other CAX homologs and/or Ca2+ transporters, including the mammalian Na+/Ca2+ exchanger (NCX), our study provides a regulatory mechanism for the Ca2+/cation transporter superfamily.


Assuntos
Antiporters/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Antiporters/genética , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Mutação , Conformação Proteica , Homologia de Sequência , Trocador de Sódio e Cálcio/genética
3.
Am J Physiol Cell Physiol ; 318(6): C1136-C1143, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32293934

RESUMO

The transport of electrolytes and fluid by the intestinal epithelium is critical in health to maintain appropriate levels of fluidity of the intestinal contents. The transport mechanisms that underlie this physiological process are also subject to derangement in various digestive disease states, such as diarrheal illnesses. This article summarizes the 2019 Hans Ussing Lecture of the Epithelial Transport Group of the American Physiological Society and discusses some pathways by which intestinal transport is dysregulated, particularly in the setting of infection with the diarrheal pathogen, Salmonella, and in patients treated with small-molecule inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFr-TKI). The burdensome diarrhea in patients infected with Salmonella may be attributable to decreased expression of the chloride-bicarbonate exchanger downregulated in adenoma (DRA) that participates in electroneutral NaCl absorption. This outcome is possibly secondary to increased epithelial proliferation and/or decreased epithelial differentiation that occurs following infection. Conversely, the diarrheal side effects of cancer treatment with EGFr-TKI may be related to the known ability of EGFr-associated signaling to reduce calcium-dependent chloride secretion. Overall, the findings described may suggest targets for therapeutic intervention in a variety of diarrheal disease states.


Assuntos
Antiporters/metabolismo , Diarreia/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antineoplásicos/toxicidade , Diferenciação Celular , Proliferação de Células , Diarreia/induzido quimicamente , Diarreia/microbiologia , Diarreia/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Permeabilidade , Inibidores de Proteínas Quinases/toxicidade , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia
4.
Biochim Biophys Acta Bioenerg ; 1861(7): 148185, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32171794

RESUMO

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.


Assuntos
Antiporters/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Subunidades Proteicas/metabolismo , Força Próton-Motriz/fisiologia , Sequência de Aminoácidos , Antiporters/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Prótons , Sais/química , Thermus thermophilus/metabolismo , Água/química
5.
Proc Natl Acad Sci U S A ; 117(9): 4732-4740, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32075917

RESUMO

Multidrug and toxic compound extrusion (MATE) transporters are ubiquitous ion-coupled antiporters that extrude structurally and chemically dissimilar cytotoxic compounds and have been implicated in conferring multidrug resistance. Here, we integrate double electron-electron resonance (DEER) with functional assays and site-directed mutagenesis of conserved residues to illuminate principles of ligand-dependent alternating access of PfMATE, a proton-coupled MATE from the hyperthermophilic archaeon Pyrococcus furiosus Pairs of spin labels monitoring the two sides of the transporter reconstituted into nanodiscs reveal large-amplitude movement of helices that alter the orientation of a putative substrate binding cavity. We found that acidic pH favors formation of an inward-facing (IF) conformation, whereas elevated pH (>7) and the substrate rhodamine 6G stabilizes an outward-facing (OF) conformation. The lipid-dependent PfMATE isomerization between OF and IF conformation is driven by protonation of a previously unidentified intracellular glutamate residue that is critical for drug resistance. Our results can be framed in a mechanistic model of transport that addresses central aspects of ligand coupling and alternating access.


Assuntos
Antiporters/química , Antiporters/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Antiporters/genética , Resistência a Múltiplos Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas de Transporte de Cátions Orgânicos/genética , Conformação Proteica , Prótons , Pyrococcus furiosus/metabolismo
6.
Am J Physiol Cell Physiol ; 318(2): C392-C405, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774702

RESUMO

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H+(OH-) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H+(OH-) transport in the absence of ammonia. We quantitated H+ flux by monitoring changes in intracellular pH (pHi) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH4Cl. Our results demonstrate that SLC4A11 mediated conductive H+(OH-) transport that was stimulated by raising the extracellular pH (pHe). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pHe. In studies using increasing NH4Cl concentrations with equal NH4+ extracellular and intracellular concentrations, the shift in the reversal potential (Erev) due to the addition of ammonia was compatible with NH3-H+ transport competing with H+(OH-) rather than NH3-nH+ (n ≥ 2) transport. The increase in equivalent H+(OH-) flux observed in the presence of a transcellular H+ gradient was also compatible with SLC4A11-mediated NH3-H+ flux. The NH3 versus Erev data fit a theoretical model suggesting that NH3-H+ and H+(OH-) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H+(OH-) transport and ammonia-induced whole cell currents were blocked suggesting that the H+(OH-) and NH3-H+ transport processes share common features involving the SLC4A11 transport mechanism.


Assuntos
Amônia/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Transporte de Íons/fisiologia , Bicarbonatos/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Sódio/metabolismo
8.
Adv Exp Med Biol ; 1131: 857-879, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646537

RESUMO

In Drosophila photoreceptor cells, Ca2+ exerts regulatory functions that control the shape, duration, and amplitude of the light response. Ca2+ also orchestrates light adaptation allowing Drosophila to see in light intensity regimes that span several orders of magnitude ranging from single photons to bright sunlight. The prime source for Ca2+ elevation in the cytosol is Ca2+ influx from the extracellular space through light-activated TRP channels. This Ca2+ influx is counterbalanced by constitutive Ca2+ extrusion via the Na+/Ca2+ exchanger, CalX. The light-triggered rise in intracellular Ca2+ exerts its regulatory functions through interaction with about a dozen well-characterized Ca2+ and Ca2+/CaM binding proteins. In this review we will discuss the dynamic changes in Ca2+ concentration upon illumination of photoreceptor cells. We will present the proteins that are known to interact with Ca2+ (/CaM) and elucidate the physiological functions of these interactions.


Assuntos
Cálcio , Drosophila , Células Fotorreceptoras de Invertebrados , Transdução de Sinais , Animais , Antiporters/metabolismo , Cálcio/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/metabolismo , Luz , Células Fotorreceptoras de Invertebrados/fisiologia
9.
Nat Commun ; 10(1): 5080, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704944

RESUMO

Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker's yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10-100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr90. Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts.


Assuntos
Proteínas de Transporte/genética , Engenharia Metabólica/métodos , Metais Pesados/metabolismo , Saccharomyces cerevisiae/genética , Antiporters/genética , Antiporters/metabolismo , Arsênico/metabolismo , Biodegradação Ambiental , Cádmio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cromo/metabolismo , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas SLC31/genética , Proteínas SLC31/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrôncio/metabolismo , Radioisótopos de Estrôncio/metabolismo
10.
BMC Med Genet ; 20(1): 188, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31775791

RESUMO

BACKGROUND: Gorlin-Chaudhry-Moss syndrome (GCMS) and Fontaine-Farriaux syndrome (FFS) are extremely rare genetic disorders that share similar clinical manifestations. Because a de novo missense mutation of the solute carrier family 25 member 24 (SLC25A24) gene was suggested to be the common genetic basis of both syndromes, it has been proposed recently that they be integrated into a single disorder under the name of Fontaine progeroid syndrome (FPS). CASE PRESENTATION: A 9-year-old Korean girl presented with typical clinical features of FPS. She had generalized loose skin with decreased subcutaneous fat, skin wrinkling on the forehead and limbs, skull deformities and a peculiar facial appearance with microphthalmia and midface hypoplasia, anomalies of the digits and nails, a large umbilical hernia and a nearly normal developmental outcome. She exhibited prenatal and postnatal growth retardation together with short stature, and records showed that her height and weight were invariably under - 2.0 SD from birth to the age of 10 years. SLC25A24 analysis revealed a heterozygous mutation reported previously, NM_013386:c.650G > A, p.[Arg217His]. After screening her family for the identified mutation, she was confirmed as being a de novo case of FPS caused by an SLC25A24 mutation. CONCLUSION: We describe a Korean girl with typical clinical findings of FPS and a de novo mutation in SLC25A24, as well as 10 years of clinical follow-up, including growth and developmental achievements.


Assuntos
Antiporters/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas Mitocondriais/genética , Fenótipo , Progéria/patologia , Criança , Feminino , Seguimentos , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Progéria/genética , República da Coreia , Síndrome
11.
J Neuromuscul Dis ; 6(4): 485-501, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31594244

RESUMO

BACKGROUND: Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed. OBJECTIVE: A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause. METHODS: Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed. RESULTS: Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress. CONCLUSIONS: This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/genética , Ácido Aspártico/genética , Ácido Glutâmico/genética , Mitocôndrias/genética , Mutação/genética , Animais , Ácido Aspártico/metabolismo , Dermatomiosite/metabolismo , Cães , Ácido Glutâmico/metabolismo , Humanos , Mitocôndrias/metabolismo , Miosite/genética , Oxirredução , Polimiosite/metabolismo
12.
Elife ; 82019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31637997

RESUMO

Transporters from bacteria to humans contain inverted repeat domains thought to arise evolutionarily from the fusion of smaller membrane protein genes. Association between these domains forms the functional unit that enables transporters to adopt distinct conformations necessary for function. The small multidrug resistance (SMR) family provides an ideal system to explore the role of mutations in altering conformational preference since transporters from this family consist of antiparallel dimers that resemble the inverted repeats present in larger transporters. Here, we show using NMR spectroscopy how a single conservative mutation introduced into an SMR dimer is sufficient to change the resting conformation and function in bacteria. These results underscore the dynamic energy landscape for transporters and demonstrate how conservative mutations can influence structure and function.


Assuntos
Antiporters/química , Antiporters/genética , Antiporters/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Modelos Teóricos , Mutagênese , Conformação Proteica
13.
Mediators Inflamm ; 2019: 7583760, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582903

RESUMO

Diabetic heart dysfunctions during cardiac surgeries have revealed several clinical problems associated with ion imbalance. However, the mechanism of ion imbalance mediated by cardioplegia and a diabetic heart is largely unclear. We hypothesized that ion transporters might be regulated differently in the diabetic heart and that the differentially regulated ion transporters may involve in ion imbalance of the diabetic heart after cardioplegic arrest. In this study, we modified the Langendorff-free cardioplegia method and identified the involved ion transporters after cardioplegia-induced arrest between wild type and db/db heart. Enhanced expression of Na+-K+-2Cl- cotransporter 1 (NKCC1) was observed in the db/db heart compared to the wild type heart. Enhanced NKCC1 activity was observed in the left ventricle of db/db mice compared to that of wild type after cardioplegia-induced arrest. The expression and activity of Slc26a6, a dominant Cl-/HCO3 - exchanger in cardiac tissues, were enhanced in left ventricle strips of db/db mice compared to that of wild type. The Cl- transporting activity in left ventricle strips of db/db mice was dramatically increased as compared to that of wild type. Interestingly, expression of Slc26a6, as well as carbonic anhydrase IV as a supportive enzyme of Slc26a6, was increased in db/db cardiac strips compared to wild type cardiac strips. Thus, the enhanced Cl- transporting activity and expression by NKCC1 and Slc26a6 in db/db cardiac tissues after cardioplegia-induced arrest provide greater insight into enhanced acidosis and Cl- movement-mediated db/db heart dysfunction. Thus, we suggested that enhanced Cl- influx and HCO3 - efflux through NKCC1 and Slc26a6 offer more acidic circumstances in the diabetic heart after cardioplegic arrest. These transporters should be considered as potential therapeutic targets to develop the next generation of cardioplegia solution for protection against ischemia-reperfusion injury in diabetic hearts.


Assuntos
Antiporters/metabolismo , Parada Cardíaca Induzida/efeitos adversos , Parada Cardíaca/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Coração , Parada Cardíaca/etiologia , Concentração de Íons de Hidrogênio , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 2 da Família 12 de Carreador de Soluto/genética , Transportadores de Sulfato/genética
14.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514314

RESUMO

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 ßs (TGFßs). 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.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Mitocôndrias/metabolismo , Células Precursoras de Oligodendrócitos/citologia , Células Precursoras de Oligodendrócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Inativação Gênica , Lactatos/metabolismo , Ventrículos Laterais/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Neurônios/metabolismo , Fator de Crescimento Derivado de Plaquetas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
15.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544789

RESUMO

In view of the documented association of solute carrier family 19 member 1 (SLC19A1) G80A (R27H) polymorphism with the risk for different types of cancers and systemic lupus erythematosus (SLE), we have reanalysed the case-control study on breast cancer to ascertain the conditions in which this polymorphic variant exerts the risk of breast cancer. Association statistics have revealed that this polymorphism exerts the risk for breast cancer under the conditions of low folate intake, and in the absence of well-documented protective polymorphism in cytosolic serine hydroxymethyltransferase. To substantiate this observation, we have developed a homology model of SLC19A1 using glycerol-3-phosphate transporter (d1pw4a) as a template where 73% of the residues were modelled at 90% confidence while 162 residues were modelled ab initio. The wild and mutant proteins shared same topology in S3, S5, S6, S7, S11 and S12 transmembrane domains. The topology varied at S1 (28-43 residue vs 28-44 residue), S2 (66-87 residue vs 69-87 residue), S4 (117-140 residue vs 117-139 residue), S8 (305-325 residue vs 305-324 residue), S9 (336-356 residue vs 336-355residue), and S10 (361-386 residue vs 361-385 residue) transmembrane domains between wild versus mutant proteins. S2 domain is shortened by three amino acid residues in the mutant while in other domains the difference corresponds to one amino acid residue. The 3DLigandSite analysis revealed that the metallic-ligand-binding sites at 273Trp, 277Asn, 379Leu, 439Phe and 442Leu are although unaffected, there is a loss of active sites corresponding to nonmetallic ligand binding. Tetrahydrofolate and methotrexate have lesser affinity towards the mutant protein than the wild protein. To conclude, the R27H polymorphism affects the secondary and tertiary structures of SLC19A1 with the significant loss in ligand-binding sites.


Assuntos
Neoplasias da Mama/genética , Proteína Carregadora de Folato Reduzido/química , Proteína Carregadora de Folato Reduzido/genética , Antiporters/química , Sítios de Ligação/genética , Estudos de Casos e Controles , Simulação por Computador , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Humanos , Metotrexato/química , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/química , Polimorfismo Genético , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Fatores de Risco
16.
Am J Physiol Cell Physiol ; 317(6): C1153-C1160, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31532720

RESUMO

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.


Assuntos
Células Acinares/metabolismo , Antiporters/genética , Bicarbonatos/metabolismo , Líquidos Corporais/metabolismo , Pâncreas Exócrino/metabolismo , Transportadores de Sulfato/genética , Células Acinares/citologia , Animais , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antiporters/deficiência , Aquaporina 5/genética , Aquaporina 5/metabolismo , Peso Corporal , Linhagem Celular , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Transporte de Íons , Masculino , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Tamanho do Órgão , Pâncreas Exócrino/citologia , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Transportadores de Sulfato/deficiência
17.
Arch Biochem Biophys ; 674: 108114, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31557465

RESUMO

We performed computer simulations to investigate the effect of oxidation on the extracellular cystine (CYC) uptake by the xC- antiporter. The latter is important for killing of cancer cells. Specifically, applying molecular dynamics (MD) simulations we studied the transport of CYC across xCT, i.e., the light subunit of the xC- antiporter, in charge of bidirectional transport of CYC and glutamate. We considered the outward facing (OF) configuration of xCT, and to study the effect of oxidation, we modified the Cys327 residue, located in the vicinity of the extracellular milieu, to cysteic acid (CYO327). Our computational results showed that oxidation of Cys327 results in a free energy barrier for CYC translocation, thereby blocking the access of CYC to the substrate binding site of the OF system. The formation of the energy barrier was found to be due to the conformational changes in the channel. Analysis of the MD trajectories revealed that the reorganization of the side chains of the Tyr244 and CYO327 residues play a critical role in the OF channel blocking. Indeed, the calculated distance between Tyr244 and either Cys327 or CYO327 showed a narrowing of the channel after oxidation. The obtained free energy barrier for CYC translocation was found to be 33.9kJmol-1, indicating that oxidation of Cys327, by e.g., cold atmospheric plasma, is more effective in inhibiting the xC- antiporter than in the mutation of this amino acid to Ala (yielding a barrier of 32.4kJmol-1). The inhibition of the xC- antiporter may lead to Cys starvation in some cancer cells, eventually resulting in cancer cell death.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Antiporters/metabolismo , Cistina/metabolismo , Sistema y+ de Transporte de Aminoácidos/química , Antiporters/química , Transporte Biológico , Cisteína/química , Cistina/química , Humanos , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Oxirredução , Fosfatidilcolinas/química , Conformação Proteica , Termodinâmica
18.
Exp Eye Res ; 188: 107782, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31491427

RESUMO

The cornea is an important tissue that refracts light, and the corneal endothelium prevents edema of the corneal stroma by acting as a barrier and a pump for the transport of essential molecules/ions. Sodium bicarbonate transporter-like protein 11 (SLC4A11) is a transporter present in the corneal endothelium, and its mutation causes corneal endothelial disease. Here, we aimed to investigate the degradation pathway of SLC4A11. Quantitative PCR analysis revealed that two variants of SLC4A11 transcripts, variant 2 (SLC4A11-B) and variant 3 (SLC4A11-C), were expressed in human corneal endothelial tissues. Transient overexpression of these variants in HEK293T cells revealed that SLC4A11-B abundantly localized to the cell membrane. Furthermore, SLC4A11-B-transfected HEK293T cells expressed the mature glycosylated forms and immature non-glycosylated forms of SLC4A11. Cycloheximide chase experiments revealed that mature SLC4A11 showed high degradation stability; however, degradation of immature SLC4A11-B was significantly faster than that of immature SLC4A11-C. Therefore, we performed further degradation analysis of the SLC4A11 mutants, which are classified into ER-retained and cell surface-associated mutants similar to the wild type. Compared to the wild type, ER-retained mutants S213P and W240P showed delayed degradation but the cell surface-associated mutants showed minimal degradation. Further analysis using proteasome inhibitors revealed that degradation of immature SLC4A11 was delayed after treatment with the proteasome inhibitors, MG-132 and bortezomib, and was mediated by poly-ubiquitination. Moreover, the degradation of immature SLC4A11 protein was suppressed by Eeyarestatin I, an ER-associated protein degradation (ERAD) inhibitor. Collectively, these data suggest that SLC4A11 protein is degraded via ERAD.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Degradação Associada com o Retículo Endoplasmático/fisiologia , Epitélio Posterior/metabolismo , Western Blotting , Membrana Celular/metabolismo , Células HEK293 , Homeostase , Humanos , Plasmídeos , Reação em Cadeia da Polimerase , Dobramento de Proteína , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
19.
Nat Chem Biol ; 15(10): 945-948, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501590

RESUMO

Helical membrane proteins are typically assumed to attain stable transmembrane topologies immediately upon co-translational membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transmembrane helix flips in and out of the membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, membrane topology can display rapid dynamics in vivo and can be regulated by post-translational assembly.


Assuntos
Antiporters/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/fisiologia , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Antiporters/genética , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Variação Genética , Proteínas de Membrana/metabolismo , Plasmídeos , Conformação Proteica
20.
J Dermatol ; 46(11): 1027-1030, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31486119

RESUMO

Oculocutaneous albinism (OCA) is a rare and heterogeneous disorder characterized by hypopigmentation of the skin, hair and eyes. Thirty OCA type 6 (OCA6) patients with 24 mutations in SLC24A5 have been reported across various populations; however, only one patient has been identified in a Chinese population. This study identifies two novel SLC24A5 frame-shift variants in two unrelated Chinese patients and both are predicted to be pathogenic by American College of Medical Genetics guidelines. The genotypes and phenotypes of all three Chinese OCA6 patients are unique compared with those identified in other populations. All of the mutations identified to date in Chinese OCA6 patients are predicted to be non-functional, a finding that is useful in guiding genetic diagnosis and counseling for OCA6 in China.


Assuntos
Albinismo Oculocutâneo/genética , Antiporters/genética , Albinismo Oculocutâneo/etnologia , Grupo com Ancestrais do Continente Asiático , Pré-Escolar , Feminino , Mutação da Fase de Leitura , Humanos , Lactente
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