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1.
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
2.
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
3.
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
4.
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
5.
Nature ; 572(7768): 249-253, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367038

RESUMO

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.


Assuntos
Antioxidantes/metabolismo , Lisina/metabolismo , Poliaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters/metabolismo , Cadaverina/metabolismo , Glutamina/metabolismo , Glutationa/metabolismo , NADP/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ornitina Descarboxilase/metabolismo , Oxidantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Elife ; 82019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31339488

RESUMO

The epithelial anion transporter SLC26A9 contributes to airway surface hydration and gastric acid production. Colocalizing with CFTR, SLC26A9 has been proposed as a target for the treatment of cystic fibrosis. To provide molecular details of its transport mechanism, we present cryo-EM structures and a functional characterization of murine Slc26a9. These structures define the general architecture of eukaryotic SLC26 family members and reveal an unusual mode of oligomerization which relies predominantly on the cytosolic STAS domain. Our data illustrates conformational transitions of Slc26a9, supporting a rapid alternate-access mechanism which mediates uncoupled chloride transport with negligible bicarbonate or sulfate permeability. The characterization of structure-guided mutants illuminates the properties of the ion transport path, including a selective anion binding site located in the center of a mobile module within the transmembrane domain. This study thus provides a structural foundation for the understanding of the entire SLC26 family and potentially facilitates their therapeutic exploitation.


Assuntos
Antiporters/metabolismo , Antiporters/ultraestrutura , Cloretos/metabolismo , Microscopia Crioeletrônica , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/ultraestrutura , Animais , Antiporters/química , Sítios de Ligação , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Modelos Moleculares , Domínios Proteicos , Proteolipídeos/metabolismo , Eletricidade Estática , Especificidade por Substrato , Transportadores de Sulfato/química
7.
Biochim Biophys Acta Bioenerg ; 1860(9): 708-716, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31340138

RESUMO

The mitochondrial carnitine/acylcarnitine carrier (CACT) catalyzes an antiport of carnitine and acylcarnitines and also a uniport reaction with a rate of about one tenth with respect to the antiport rate. The antiport process results from the coupling of the two uniport reactions in opposite directions. In this mechanism, the transition of the carrier from the outward open conformation to the inward open one (or vice versa) is much faster for the carrier-substrate complex than for the unbound carrier. To investigate the molecular determinants that couple the binding of the substrate with the conformational transitions, site directed mutagenesis has been employed. The antiport or the uniport reaction was followed as [3H]carnitine uptake in or efflux from proteoliposomes reconstituted with the WT or Trp mutants of the rat CACT. Substitution of each the three Trp residues led to different results. Nearly no variations were observed upon substitution of W192 and/or W296 with Ala. While, substantial alteration of the transport function was observed in the mutants W224A, W224Y and W224F. Mutation of W224 led to the loss of the antiport function while the uniport function was unaltered. In these mutants impairment of the substrate affinity on the external side was also observed. The data highlights that W224 is involved in the coupling of the substrate binding with the matrix gate opening. The experimental data are in line with predictions by homology modeling of the CACT in its cytosolic (c-state) or matrix (m-state) opened conformations.


Assuntos
Antiporters/metabolismo , Carnitina Aciltransferases/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Triptofano/metabolismo , Sequência de Aminoácidos , Animais , Aspergillus nidulans , Transporte Biológico , Carnitina Aciltransferases/química , Carnitina Aciltransferases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Triptofano/química , Triptofano/genética
8.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340538

RESUMO

WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Córtex Cerebral/metabolismo , Nanismo/genética , Epilepsia/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Mitocondriais/genética , Neurogênese/genética , Transtornos Psicomotores/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/genética , Antiporters/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Contagem de Células , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Modelos Animais de Doenças , Nanismo/metabolismo , Nanismo/patologia , Epilepsia/metabolismo , Epilepsia/patologia , Regulação da Expressão Gênica no Desenvolvimento , Mutação em Linhagem Germinativa , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Masculino , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/patologia , Ratos , Ratos Transgênicos , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Oxidorredutase com Domínios WW/deficiência
9.
J Nippon Med Sch ; 86(2): 126-130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130564

RESUMO

Pseudomyogenic hemangioendothelioma (PMHE) is a new entity. It is an intermediate soft tissue tumor clinically and/or histopathologically mimicking some other high-grade malignant tumors and some inflammatory diseases. We report a case of PMHE on the left plantar surface of a 28-year-old woman. Histopathological examination of the resected specimen revealed spindle and epithelioid cells with plump and atypical nuclei proliferated in the dermis and subcutaneous fat tissue with marked fibroplasia. Both spindle and epithelioid cells had abundant eosinophilic cytoplasm. Neoplastic cells were diffusely positive for AE1/AE3, CK7, vimentin, CD31, FLI-1, ERG, and INI-1. From those findings, we made the diagnosis of PMHE. We describe the main points of differentiation between PMHE and diseases that have similar clinical and/or histopathological findings, including cellular dermatofibroma, spindle cell squamous cell carcinoma, epithelioid sarcoma, epithelioid hemangioendothelioma, epithelioid angiosarcoma, nodular or proliferative fasciitis, and granulomatous fibrosing granulation tissue due to a ruptured epidermal cyst.


Assuntos
Hemangioendotelioma Epitelioide/patologia , Neoplasias de Tecidos Moles/patologia , Adulto , Antiporters/metabolismo , Diagnóstico Diferencial , Feminino , Hemangioendotelioma Epitelioide/diagnóstico , Hemangioendotelioma Epitelioide/genética , Humanos , Queratinas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína SMARCB1/metabolismo , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/genética , Regulador Transcricional ERG/metabolismo , Vimentina/metabolismo
10.
PLoS One ; 14(4): e0214862, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30951542

RESUMO

The effects of hyperuricemia on the expression of kidney drug transporters and on the pharmacokinetics of several substrate drugs were examined. We first established a rat model of hyperuricemia without marked symptoms of chronic kidney failure by 10-day co-administration of oxonic acid (uricase inhibitor) and adenine (biosynthetic precursor of uric acid). These hyperuricemic rats showed plasma uric acid concentrations of up to 6 mg/dL, which is similar to the serum uric acid level in hyperuricemic humans, with little change of inulin clearance. The mRNA levels of multidrug and toxin extrusion 1 (Mate1, Slc47a1), organic anion transporter 1 (Oat1, Slc22a6), organic cation transporter 2 (Oct2, Slc22a2), urate transporter 1 (Urat1, Slc22a12) and peptide transporter 1 (Pept1, Slc15a1) were significantly decreased in kidney of hyperuricemic rats. Since Oct2, Mate1 and Oat1 are important for renal drug elimination, we next investigated whether the pharmacokinetics of their substrates, metformin, cephalexin and creatinine, were altered. The plasma concentration of metformin was not affected, while its kidney tissue accumulation was significantly increased. The plasma concentration and kidney tissue accumulation of cephalexin and the plasma concentration of creatinine were also increased. Furthermore, the protein expression of kidney Mate1 was decreased in hyperuricemic rats. Accordingly, although multiple factors may influence renal handling of these drugs, these observations can be accounted for, at least in part, by downregulation of Mate1-mediated apical efflux from tubular cells and Oct2-mediated basolateral uptake. Our results suggest that hyperuricemia could alter the disposition of drugs that are substrates of Mate1 and/or Oct2.


Assuntos
Antiporters/genética , Antiporters/metabolismo , Hiperuricemia/genética , Hiperuricemia/metabolismo , Rim/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/genética , Transportador 2 de Cátion Orgânico/metabolismo , Adenina/administração & dosagem , Animais , Cefalexina/sangue , Cefalexina/farmacocinética , Creatinina/sangue , Creatinina/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Hiperuricemia/sangue , Rim/efeitos dos fármacos , Masculino , Metformina/sangue , Metformina/farmacocinética , Ácido Oxônico/administração & dosagem , Farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ácido Úrico/administração & dosagem
11.
Acta Biochim Pol ; 66(1): 111-114, 2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30793712

RESUMO

INTRODUCTION: Small cell lung carcinoma (SCLC) is an aggressive pulmonary neoplasm of neuroendocrine origin. Keratins form a large group of intermediate filaments, which are major structural proteins in epithelial cells and carcinomas. SCLC shows a wide spectrum of keratin expression, from very strong to completely negative. A prognostic role of keratin expression in SCLC is unknown. MATERIAL AND METHODS: Tumor tissue microarray samples from a unique series of 82 SCLC patients who underwent pulmonary resection were stained with keratin specific antibodies AE1/AE3 and CAM5.2. The percentage o1f positively stained cells and their staining pattern (diffusely membranous, partially membranous and dot-like) were evaluated. The median expression value was used for the distinction between keratin-negative and -positive patients. Overall survival in respective groups was compared using the log-rank test. Multivariate Cox proportional hazards regression analysis was performed adjusting for age, gender, tumor site, tumor stage, and tumor histology. RESULTS: edian expression of AE1/AE3 and CAM5.2 was 80% and 90%, respectively. Five cases were completely negative for AE1/AE3 and three for Cam5.2. Median overall survival for patients with stronger and weaker AE1/AE3 staining was 24.7 and 13.8 months, respectively (p=0.019). There was no difference in survival in relation to the CAM5.2 expression (p=0.44). In multivariate analysis adjusted for CAM5.2, T and N stage, gender and age at diagnosis, stronger AE1/AE3 expression was an independent predictor of increased survival (HR 0.50; 95% CI, 0.27-0.94; p=0.031). CONCLUSION: High expression of AE1/AE3 is a favorable prognostic factor in surgically treated SCLC. The applicability of this finding to a typical patient population treated with non-surgical methods warrants further studies.


Assuntos
Queratinas/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Antiporters/metabolismo , Biomarcadores/metabolismo , Feminino , Humanos , Pulmão/metabolismo , Pulmão/cirurgia , Masculino , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/cirurgia , Análise Serial de Tecidos
12.
Arch Biochem Biophys ; 664: 117-126, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30738038

RESUMO

Extracellular cystine (CYC) uptake by xC- antiporter is important for the cell viability. Especially in cancer cells, the upregulation of xC- activity is observed, which protects these cells from intracellular oxidative stress. Hence, inhibition of the CYC uptake may eventually lead to cancer cell death. Up to now, the molecular level mechanism of the CYC uptake by xC- antiporter has not been studied in detail. In this study, we applied several different simulation techniques to investigate the transport of CYC through xCT, the light subunit of the xC- antiporter, which is responsible for the CYC and glutamate translocation. Specifically, we studied the permeation of CYC across three model systems, i.e., outward facing (OF), occluded (OCC) and inward facing (IF) configurations of xCT. We also investigated the effect of mutation of Cys327 to Ala within xCT, which was also studied experimentally in literature. This allowed us to qualitatively compare our computation results with experimental observations, and thus, to validate our simulations. In summary, our simulations provide a molecular level mechanism of the transport of CYC across the xC- antiporter, more specifically, which amino acid residues in the xC- antiporter play a key role in the uptake, transport and release of CYC.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Antiporters/metabolismo , Cistina/metabolismo , Alanina/metabolismo , Substituição de Aminoácidos , Sistema y+ de Transporte de Aminoácidos/química , Arginina/metabolismo , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Transporte Proteico
13.
Am J Respir Cell Mol Biol ; 60(6): 705-716, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30742493

RESUMO

Bicarbonate facilitates mucin unpacking and bacterial killing; however, its transport mechanisms in the airways are not well understood. cAMP stimulates anion efflux through the cystic fibrosis (CF) transmembrane conductance regulator (CFTR; ABCC7) anion channel, and this is defective in CF. The anion exchanger pendrin (SLC26A4) also mediates HCO3- efflux and is upregulated by proinflammatory cytokines. Here, we examined pendrin and CFTR expression and their contributions to HCO3- secretion by human nasal and bronchial epithelia. In native tissue, both proteins were most abundant at the apical pole of ciliated surface cells with little expression in submucosal glands. In well-differentiated primary nasal and bronchial cell cultures, IL-4 dramatically increased pendrin mRNA levels and apical immunostaining. Exposure to low-Cl- apical solution caused intracellular alkalinization (ΔpHi) that was enhanced fourfold by IL-4 pretreatment. ΔpHi was unaffected by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) or CFTR inhibitor CFTRinh-172, but was reduced by adenoviral shRNA targeting pendrin. Forskolin increased ΔpHi, and this stimulation was prevented by CFTRinh-172, implicating CFTR, yet forskolin only increased ΔpHi after pendrin expression had been induced by IL-4. The dependence of ΔpHi on pendrin suggests there is minimal electrical coupling between Cl- and HCO3- fluxes and that CFTR activation increases anion exchange-mediated HCO3- influx. Conversely, inducing pendrin expression increased forskolin-stimulated, CFTRinh-172-sensitive current by approximately twofold in epithelial and nonepithelial cells. We conclude that pendrin mediates most HCO3- secretion across airway surface epithelium during inflammation and enhances electrogenic Cl- secretion via CFTR, as described for other SLC26A transporters.


Assuntos
Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Pulmão/metabolismo , Mucosa Respiratória/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/metabolismo , Linhagem Celular , Antiportadores de Cloreto-Bicarbonato/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Interleucina-4/genética , Interleucina-4/metabolismo , Transporte de Íons/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Transportadores de Sulfato/genética
14.
PLoS Genet ; 15(2): e1008007, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30807572

RESUMO

Cystic Fibrosis (CF) exhibits morbidity in several organs, including progressive lung disease in all patients and intestinal obstruction at birth (meconium ileus) in ~15%. Individuals with the same causal CFTR mutations show variable disease presentation which is partly attributed to modifier genes. With >6,500 participants from the International CF Gene Modifier Consortium, genome-wide association investigation identified a new modifier locus for meconium ileus encompassing ATP12A on chromosome 13 (min p = 3.83x10(-10)); replicated loci encompassing SLC6A14 on chromosome X and SLC26A9 on chromosome 1, (min p<2.2x10(-16), 2.81x10(-11), respectively); and replicated a suggestive locus on chromosome 7 near PRSS1 (min p = 2.55x10(-7)). PRSS1 is exclusively expressed in the exocrine pancreas and was previously associated with non-CF pancreatitis with functional characterization demonstrating impact on PRSS1 gene expression. We thus asked whether the other meconium ileus modifier loci impact gene expression and in which organ. We developed and applied a colocalization framework called the Simple Sum (SS) that integrates regulatory and genetic association information, and also contrasts colocalization evidence across tissues or genes. The associated modifier loci colocalized with expression quantitative trait loci (eQTLs) for ATP12A (p = 3.35x10(-8)), SLC6A14 (p = 1.12x10(-10)) and SLC26A9 (p = 4.48x10(-5)) in the pancreas, even though meconium ileus manifests in the intestine. The meconium ileus susceptibility locus on chromosome X appeared shifted in location from a previously identified locus for CF lung disease severity. Using the SS we integrated the lung disease association locus with eQTLs from nasal epithelia of 63 CF participants and demonstrated evidence of colocalization with airway-specific regulation of SLC6A14 (p = 2.3x10(-4)). Cystic Fibrosis is realizing the promise of personalized medicine, and identification of the contributing organ and understanding of tissue specificity for a gene modifier is essential for the next phase of personalizing therapeutic strategies.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Antiporters/genética , Fibrose Cística/genética , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla/métodos , ATPase Trocadora de Hidrogênio-Potássio/genética , Transportadores de Sulfato/genética , Tripsina/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antiporters/metabolismo , Fibrose Cística/metabolismo , Feminino , Regulação da Expressão Gênica , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Pulmão/metabolismo , Masculino , Especificidade de Órgãos , Pâncreas Exócrino/metabolismo , Transportadores de Sulfato/metabolismo , Tripsina/metabolismo
15.
Mol Cell ; 73(5): 1056-1065.e7, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30738704

RESUMO

The mitochondrial inner membrane harbors a large number of metabolite carriers. The precursors of carrier proteins are synthesized in the cytosol and imported into mitochondria by the translocase of the outer membrane (TOM) and the carrier translocase of the inner membrane (TIM22). Molecular chaperones in the cytosol and intermembrane space bind to the hydrophobic precursors to prevent their aggregation. We report that the major metabolite channel of the outer membrane, termed porin or voltage-dependent anion channel (VDAC), promotes efficient import of carrier precursors. Porin interacts with carrier precursors arriving in the intermembrane space and recruits TIM22 complexes, thus ensuring an efficient transfer of the precursors to the inner membrane translocase. Porin channel mutants impaired in metabolite transport are not disturbed in carrier import into mitochondria. We conclude that porin serves distinct functions as outer membrane channel for metabolites and as coupling factor for protein translocation into the inner membrane.


Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters/genética , Antiporters/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Porinas/genética , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1323-1331, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30716472

RESUMO

BACKGROUND: We analyzed the CFTR response to VX-809/VX-770 drugs in conditionally reprogrammed cells (CRC) of human nasal epithelium (HNE) from F508del/F508del patients based on SNP rs7512462 in the Solute Carrier Family 26, Member 9 (SLC26A9; MIM: 608481) gene. METHODS: The Isc-eq measurements of primary nasal epithelial cells from F508del/F508del patients (n = 12) for CFTR function were performed in micro Ussing chambers and compared with non-CF controls (n = 2). Data were analyzed according to the rs7512462 genotype which were determined by real-time PCR. RESULTS: The CRC-HNE cells from F508del/F508del patients evidenced high variability in the basal levels of CFTR function. Also, the rs7512462*C allele showed an increased basal CFTR function and higher responses to VX-809 + VX-770. The rs7512462*CC + CT genotypes together evidenced CFTR function levels of 14.89% relatively to wt/wt (rs7512462*CT alone-15.29%) i.e., almost double of rs7512462*TT (7.13%). Furthermore, sweat [Cl-] and body mass index of patients also evidenced an association with the rs7512462 genotype. CONCLUSION: The CFTR function can be performed in F508del/F508del patient-derived CRC-HNEs and its function and responses to VX-809 + VX-770 combination as well as clinical data, are all associated with the rs7512462 variant, which partially sheds light on the generally inter-individual phenotypic variability and in personalized responses to CFTR modulator drugs.


Assuntos
Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Antiporters/genética , Benzodioxóis/farmacologia , Agonistas dos Canais de Cloreto/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Quinolonas/farmacologia , Transportadores de Sulfato/genética , Alelos , Antiporters/metabolismo , Sequência de Bases , Índice de Massa Corporal , Estudos de Casos e Controles , Reprogramação Celular , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Cultura em Câmaras de Difusão , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Genótipo , Humanos , Modelos Biológicos , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Deleção de Sequência , Transportadores de Sulfato/metabolismo , Suor/química
17.
Plant Physiol ; 179(4): 1569-1580, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30710051

RESUMO

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking µ- or σ-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxΦ sorting motifs, which are in a cytosolic loop of BOR1. A yeast two-hybrid assay supported the interaction of the AP2 complex µ-subunit with the C-terminal tail but not with the YxxΦ motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Boro/metabolismo , Endocitose/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Antiporters/análise , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Polaridade Celular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico , Técnicas do Sistema de Duplo-Híbrido
18.
J Biol Chem ; 294(9): 3181-3191, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30622138

RESUMO

Milk is a hallmark of mammals that is critical for normal growth and development of offspring. During biosynthesis of lactose in the Golgi complex, H+ is produced as a by-product, and there is no known mechanism for maintaining luminal pH within the physiological range. Here, using conditional, tissue-specific knockout mice, immunostaining, and biochemical assays, we test whether the putative H+/Ca2+/Mn2+ exchanger known as TMEM165 (transmembrane protein 165) participates in normal milk production. We find TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and for normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased osmosis-mediated dilution of the milk caused by the decreased biosynthesis of lactose. When normalized to total protein levels, only calcium and manganese levels were significantly lower in the milk from TMEM165-deficient dams than control dams. These findings suggest that TMEM165 supplies Ca2+ and Mn2+ to the Golgi complex in exchange for H+ to sustain the functions of lactose synthase and potentially other glycosyl-transferases. Our findings highlight the importance of cation and pH homeostasis in the Golgi complex of professional secretory cells and the critical role of TMEM165 in this process.


Assuntos
Antiporters/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Complexo de Golgi/metabolismo , Leite/metabolismo , Animais , Antiporters/deficiência , Antiporters/genética , Peso Corporal , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Feminino , Técnicas de Inativação de Genes , Lactação , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/fisiologia , Camundongos , Osmose
19.
Proc Natl Acad Sci U S A ; 116(4): 1241-1250, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30626647

RESUMO

Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-K M hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-K M hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (∼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.


Assuntos
Antiporters/metabolismo , Glucose-6-Fosfatase/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Neutropenia/metabolismo , Fosforilação/fisiologia , Animais , Morte Celular/fisiologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Ratos Wistar
20.
Biopharm Drug Dispos ; 40(1): 3-11, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30488476

RESUMO

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250 mg/kg) in rats. Metformin (100 mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15 mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0 â†’ 600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.


Assuntos
Hiperuricemia/metabolismo , Hipoglicemiantes/farmacocinética , Metformina/farmacocinética , Animais , Antiporters/metabolismo , Linhagem Celular , Humanos , Hiperuricemia/induzido quimicamente , Hiperuricemia/patologia , Hipoglicemiantes/sangue , Hipoglicemiantes/urina , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Masculino , Metformina/sangue , Metformina/urina , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Ácido Oxônico , Ratos Wistar , Proteínas Recombinantes/metabolismo , Distribuição Tecidual , Ácido Úrico/sangue
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