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1.
Adv Exp Med Biol ; 1173: 21-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456203

RESUMO

Iron is an essential trace element in the human body, but excess iron is toxic as it contributes to oxidative damage. To keep iron concentration within the optimal physiologic range, iron metabolism at the cellular level and the whole systemic level are tightly regulated. Balance of iron homeostasis depends on the expression levels and activities of iron carriers, iron transporters, and iron regulatory and storage proteins. Divalent metal transporter 1 (DMT1) at the apical membrane of intestinal enterocyte brings in non-heme iron from the diet, whereas ferroportin 1 (FPN1) at the basal membrane exports iron into the circulation. Plasma transferrin (Tf) then carries iron to various tissues and cells. After binding to transferrin receptor 1 (TfR1), the complex is endocytosed into the cell, where iron enters the cytoplasm via DMT1 on the endosomal membrane. Free iron is either utilized in metabolic processes, such as synthesis of hemoglobin and Fe-S cluster, or sequestered in the cytosolic ferritin, serving as a cellular iron store. Excess iron can be exported from the cell via FPN1. The liver-derived peptide hepcidin plays a major regulatory role in controlling FPN1 level in the enterocyte, and thus controls the whole-body iron absorption. Inside the cells, iron regulatory proteins (IRPs) modulate the expressions of DMT1, TfR1, ferritin, and FPN1 via binding to the iron-responsive element (IRE) in their mRNAs. Both the release of hepcidin and the IRP-IRE interaction are coordinated with the fluctuation of the cellular iron level. Therefore, an adequate and steady iron supplement is warranted for the utilization of cells around the body. Investigations on the molecular mechanisms of cellular iron metabolism and regulation could advance the fields of iron physiology and pathophysiology.


Assuntos
Ferro/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Enterócitos/metabolismo , Ferritinas/metabolismo , Homeostase , Humanos , Sobrecarga de Ferro , Receptores da Transferrina/metabolismo , Fatores de Transcrição/metabolismo , Transferrina/metabolismo
2.
World J Microbiol Biotechnol ; 35(8): 124, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346773

RESUMO

Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.


Assuntos
Candida glabrata/genética , Proteínas de Transporte de Cátions/genética , Proteínas Fúngicas/genética , Candida glabrata/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Homeostase , Concentração de Íons de Hidrogênio , Potássio/metabolismo , Sódio/metabolismo
3.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
4.
Life Sci ; 233: 116697, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351968

RESUMO

AIMS: The present study investigated if berberine might induce Zrt-Irt-like protein 14 (ZIP14) and affect zinc redistribution to protect intestinal barrier in sepsis. MAIN METHODS: Rodent model of sepsis was induced by cecal ligation and puncture (CLP). Plasma endotoxin was assayed by LAL test and plasma zinc was measured by flame atomic spectrophotometer. Gut mucosal permeability was determined by plasma FITC-dextran. Zinc content and ZIP14 mRNA in gut mucosa were assayed by spectrophotometer and qRT-PCR, respectively. Tight junction integrity of Caco-2 was evaluated by transepithelial electrical resistance (TEER). Tight junction (TJ) protein expression was detected by Western blotting. KEY FINDINGS: Berberine and zinc gluconate pretreatment to CLP rats improved survival rate, reduced plasma endotoxin level, alleviated hypozincemia, increased zinc accumulation and ZIP14 mRNA expression in the intestinal mucosa. Berberine and zinc gluconate pretreatment decreased CLP-elicited intestinal hyperpermeability to FITC-dextran. These effects of berberine in vivo were abolished by AG1024. In vitro, lipopolysaccharide (LPS) repressed zinc transfer into Caco-2 cells exposed to zinc gluconate. Berberine and IGF-I treatment increased ZIP14 protein expression and promoted zinc transfer into Caco-2 cells exposed to zinc gluconate plus LPS. Berberine treatment induced TJ protein (claudin-1 and occludin) and raised TEER in LPS-treated Caco-2 cells. These effects of berberine in vitro were partially inhibited by ZIP14 siRNA. SIGNIFICANCE: The present study reveals that berberine induces ZIP14 expression and affects zinc re- distribution to protect intestinal barrier in sepsis, which is partially linked with the activation of IGF-I signaling.


Assuntos
Berberina/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Coinfecção/prevenção & controle , Gluconatos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Sepse/prevenção & controle , Tirfostinas/farmacologia , Zinco/metabolismo , Animais , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Coinfecção/metabolismo , Coinfecção/microbiologia , Humanos , Masculino , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar , Sepse/metabolismo , Sepse/microbiologia , Transdução de Sinais/efeitos dos fármacos
5.
BMC Plant Biol ; 19(1): 250, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185911

RESUMO

BACKGROUND: Cadmium (Cd) is a widespread toxic heavy metal pollutant in agricultural soil, and Cd accumulation in rice grains is a major intake source of Cd for Asian populations that adversely affect human health. However, the molecular mechanism underlying Cd uptake, translocation and accumulation has not been fully understood in rice plants. RESULTS: In this study, a mutant displaying extremely low Cd accumulation (lcd1) in rice plant and grain was generated by EMS mutagenesis from indica rice cultivar 9311 seeds. The candidate SNPs associated with low Cd accumulation phenotype in the lcd1 mutant were identified by MutMap and the transcriptome changes between lcd1 and WT under Cd exposure were analyzed by RNA-seq. The lcd1 mutant had lower Cd uptake and accumulation in rice root and shoot, as well as less growth inhibition compared with WT in the presence of 5 µM Cd. Genetic analysis showed that lcd1 was a single locus recessive mutation. The SNP responsible for low Cd accumulation in the lcd1 mutant located at position 8,887,787 on chromosome 7, corresponding to the seventh exon of OsNRAMP5. This SNP led to a Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 in the lcd1 mutant. A total of 1208 genes were differentially expressed between lcd1 and WT roots under Cd exposure, and DEGs were enriched in transmembrane transport process GO term. Increased OsHMA3 expression probably adds to the effect of OsNRAMP5 mutation to account for the significant decreases in Cd accumulation in rice plant and grain of the lcd1 mutant. CONCLUSIONS: An extremely low Cd mutant lcd1 was isolated and identified using MutMap and RNA-seq. A Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 is likely responsible for low Cd accumulation in the lcd1 mutant. This work provides more insight into the mechanism of Cd uptake and accumulation in rice, and will be helpful for developing low Cd accumulation rice by marker-assisted breeding.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Oryza/genética , Proteínas de Plantas/genética , Poluentes do Solo/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Oryza/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência
6.
Gene ; 710: 399-405, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200088

RESUMO

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.


Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não Traduzidas
7.
BMC Plant Biol ; 19(1): 239, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170918

RESUMO

BACKGROUND: Ammonium transporters (AMTs), a family of proteins transporting ammonium salt and its analogues, have been studied in many aspects. Although numerous studies have found that ammonium affects the interaction between plants and pathogens, the role of AMTs remains largely unknown, especially that of the AMT2-type AMTs. RESULTS: In the present study, we found that the concentration of ammonium in wheat leaves decreased after infection with Puccinia striiformis f. sp. tritici (Pst), the causal agent of stripe rust. Then, an AMT2-type ammonium transporter gene induced by Pst was identified and designated as TaAMT2;3a. Transient expression assays indicated that TaAMT2;3a was located to the cell and nuclear membranes. TaAMT2;3a successfully complemented the function of a yeast mutant defective in NH4+ transport, indicating its ammonium transport capacity. Function of TaAMT2;3a in wheat-Pst interaction was further analyzed by barley stripe mosaic virus (BSMV)-induced gene silencing. Pst growth was significantly retarded in TaAMT2;3a-knockdown plants, in which ammonium in leaves were shown to be induced at the early stage of infection. Histological observation showed that the hyphal length, the number of hyphal branches and haustorial mother cells decreased in the TaAMT2;3a knockdown plants, leading to the impeded growth of rust pathogens. CONCLUSIONS: The results clearly indicate that the induction of AMT2-type ammonium transporter gene TaAMT2;3a may facilitates the nitrogen uptake from wheat leaves by Pst, thereby contribute to the infection of rust fungi.


Assuntos
Basidiomycota/fisiologia , Proteínas de Transporte de Cátions/genética , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Triticum/genética , Triticum/microbiologia , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo
8.
BMC Plant Biol ; 19(1): 283, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248369

RESUMO

BACKGROUND: Metal homeostasis is critical for plant growth, development and adaptation to environmental stresses and largely governed by a variety of metal transporters. The plant ZIP (Zn-regulated transporter, Iron-regulated transporter-like Protein) family proteins belong to the integral membrane transporters responsible for uptake and allocation of essential and non-essential metals. However, whether the ZIP family members mediate metal efflux and its regulatory mechanism remains unknown. RESULTS: In this report, we provided evidence that OsZIP1 is a metal-detoxified transporter through preventing excess Zn, Cu and Cd accumulation in rice. OsZIP1 is abundantly expressed in roots throughout the life span and sufficiently induced by excess Zn, Cu and Cd but not by Mn and Fe at transcriptional and translational levels. Expression of OsZIP-GFP fusion in rice protoplasts and tobacco leaves shows that OsZIP1 resides in the endoplasmic reticulum (ER) and plasma membrane (PM). The yeast (Saccharomyces cerevisiae) complementation test shows that expression of OsZIP1 reduced Zn accumulation. Transgenic rice overexpressing OsZIP1 grew better under excess metal stress but accumulated less of the metals in plants. In contrast, both oszip1 mutant and RNA interference (RNAi) lines accumulated more metal in roots and contributed to metal sensitive phenotypes. These results suggest OsZIP1 is able to function as a metal exporter in rice when Zn, Cu and Cd are excess in environment. We further identified the DNA methylation of histone H3K9me2 of OsZIP1 and found that OsZIP1 locus, whose transcribed regions imbed a 242 bp sequence, is demethylated, suggesting that epigenetic modification is likely associated with OsZIP1 function under Cd stress. CONCLUSION: OsZIP1 is a transporter that is required for detoxification of excess Zn, Cu and Cd in rice.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/genética , Proteínas de Plantas/genética , Zinco/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estresse Fisiológico
9.
Toxicol Lett ; 313: 50-59, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31238089

RESUMO

Iron overload causes osteoporosis by enhancing osteoclastic bone resorption. During differentiation, osteoclasts demand high energy and contain abundant mitochondria. In mitochondria, iron is used for the synthesis of Fe-S clusters to support mitochondria biogenesis and electron transport chain. Moreover, mitochondrial reactive oxygen species (ROS) play an important role in osteoclastogenesis. Activation of MAPKs (ERK1/2, JNK, and p38) by ROS is essential and contribute to osteoclast differentiation. How iron chelation impairs electron transport chain and ROS dependent MAPKs activation during osteoclast differentiation is unknown. This study aimed to determine the direct effects of iron chelation on osteoclast differentiation, electron transport chain and MAPKs activation. In the present study, we found that when iron chelator, deferoxamine (DFO), was added, a dose-dependent inhibition of osteoclast differentiation and bone resorption was observed. Supplementation of transferrin-bound iron recovered osteoclastogenesis. Iron chelation resulted in a marked decrease in ferritin level, and increased expression of transferrin receptor 1 and ferroportin. As an iron chelator, DFO negatively affected mitochondrial function through decreasing activities of all the complexes. Expressions of mitochondrial subunits encoded both by mitochondrial and nuclear DNA were decreased. DFO augmented production of mitochondrial ROS, but inhibited the phosphorylation of ERK1/2, JNK, and p38, even in the presence of hydrogen peroxide. These results suggest that iron chelation directly inhibits iron-uptake stimulated osteoclast differentiation and suppresses electron transport chain. Iron chelation negatively regulates MAPKs activation, and this negative regulation is independent on ROS stimulation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Desferroxamina/farmacologia , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoclastos/efeitos dos fármacos , Animais , Reabsorção Óssea , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Ferritinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Osteoclastos/enzimologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Comput Biol Chem ; 80: 498-511, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176140

RESUMO

Magnesium (Mg) is an important micronutrient for various physiological processes in plants. In this study, putative Magnesium Transporter (MGT) genes have been identified in Solanum lycopersicum, Solanum tuberosum, Brachypodium distachyon, Fagaria vesca, Brassica juncea and were classified into 5 distinct groups based on their sequence homology. MGT genes are very diverse and possess very low sequence identity within its family. However, the Gly-Met-Asn (GMN) signature motif is present in most of the genes which are believed to be essential for Mg2+ recognition. In S. lycopersicum, different physiological root growth pattern was observed in both Mg excess and deficient conditions. Quantitative RT-PCR gene expression study shows that most of the SlMGT genes were upregulated in response to Mg deficient condition.


Assuntos
Proteínas de Transporte de Cátions/genética , Genoma , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Magnésio/metabolismo , Magnoliopsida/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Reação em Cadeia da Polimerase em Tempo Real , Sementes/genética , Homologia de Sequência , Estresse Fisiológico/genética , Regulação para Cima
11.
BMC Plant Biol ; 19(1): 206, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31109290

RESUMO

BACKGROUND: In order to grow, plants rely on soil nutrients which can vary both spatially and temporally depending on the environment, the soil type or the microbial activity. An essential nutrient is nitrogen, which is mainly accessible as nitrate and ammonium. Many studies have investigated transport genes for these ions in Arabidopsis thaliana and recently in crop species, including Maize, Rice and Barley. However, in most crop species, an understanding of the participants in nitrate and ammonium transport across the soil plant continuum remains undefined. RESULTS: We have mapped a non-exhaustive set of putative nitrate and ammonium transporters in maize. The selected transporters were defined based on previous studies comparing nitrate transport pathways conserved between Arabidopsis and Zea mays (Plett D et. al, PLOS ONE 5:e15289, 2010). We also selected genes from published studies (Gu R et. al, Plant and Cell Physiology, 54:1515-1524, 2013, Garnett T et. al, New Phytol 198:82-94, 2013, Garnett T et. al, Frontiers in Plant Sci 6, 2015, Dechorgnat J et. al, Front Plant Sci 9:531, 2018). To analyse these genes, the plants were grown in a semi-hydroponic system to carefully control nitrogen delivery and then harvested at both vegetative and reproductive stages. The expression patterns of 26 putative nitrogen transporters were then tested. Six putative genes were found not expressed in our conditions. Transcripts of 20 other genes were detected at both the vegetative and reproductive stages of maize development. We observed the expression of nitrogen transporters in all organs tested: roots, young leaves, old leaves, silks, cobs, tassels and husk leaves. We also followed the gene expression response to nitrogen starvation and resupply and uncovered mainly three expression patterns: (i) genes unresponsiveness to nitrogen supply; (ii) genes showing an increase of expression after nitrogen starvation; (iii) genes showing a decrease of expression after nitrogen starvation. CONCLUSIONS: These data allowed the mapping of putative nitrogen transporters in maize at both the vegetative and reproductive stages of development. No growth-dependent expression was seen in our conditions. We found that nitrogen transporter genes were expressed in all the organs tested and in many cases were regulated by the availability of nitrogen supplied to the plant. The gene expression patterns in relation to organ specificity and nitrogen availability denote a speciality of nitrate and ammonium transporter genes and their probable function depending on the plant organ and the environment.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Proteínas de Plantas/genética , Transcriptoma , Zea mays/genética , Proteínas de Transporte de Ânions/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Nitrogênio/deficiência , Proteínas de Plantas/metabolismo , Zea mays/metabolismo
12.
Oxid Med Cell Longev ; 2019: 7623023, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049138

RESUMO

Iron homeostasis in the cardiac tissue as well as the involvement of the hepcidin-ferroportin (HAMP-FPN) axis in this process and in cardiac functionality are not fully understood. Imbalance of iron homeostasis occurs in several cardiac diseases, including iron-overload cardiomyopathies such as Friedreich's ataxia (FRDA, OMIM no. 229300), a hereditary neurodegenerative disorder. Exploiting the induced pluripotent stem cells (iPSCs) technology and the iPSC capacity to differentiate into specific cell types, we derived cardiomyocytes of a FRDA patient and of a healthy control subject in order to study the cardiac iron homeostasis and the HAMP-FPN axis. Both CTR and FRDA iPSCs-derived cardiomyocytes express cardiac differentiation markers; in addition, FRDA cardiomyocytes maintain the FRDA-like phenotype. We found that FRDA cardiomyocytes show an increase in the protein expression of HAMP and FPN. Moreover, immunofluorescence analysis revealed for the first time an unexpected nuclear localization of FPN in both CTR and FRDA cardiomyocytes. However, the amount of the nuclear FPN was less in FRDA cardiomyocytes than in controls. These and other data suggest that iron handling and the HAMP-FPN axis regulation in FRDA cardiac cells are hampered and that FPN may have new, still not fully understood, functions. These findings underline the complexity of the cardiac iron homeostasis.


Assuntos
Cardiomiopatias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ataxia de Friedreich/metabolismo , Hepcidinas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Ferro/metabolismo , Miócitos Cardíacos/metabolismo , Cardiomiopatias/patologia , Ataxia de Friedreich/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Miócitos Cardíacos/patologia
13.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052156

RESUMO

Cystic fibrosis (CF) is a genetic disorder affecting several organs including airways. Bacterial infection, inflammation and iron dysbalance play a major role in the chronicity and severity of the lung pathology. The aim of this study was to investigate the effect of lactoferrin (Lf), a multifunctional iron-chelating glycoprotein of innate immunity, in a CF murine model of Pseudomonas aeruginosa chronic lung infection. To induce chronic lung infection, C57BL/6 mice, either cystic fibrosis transmembrane conductance regulator (CFTR)-deficient (Cftrtm1UNCTgN(FABPCFTR)#Jaw) or wild-type (WT), were intra-tracheally inoculated with multidrug-resistant MDR-RP73 P. aeruginosa embedded in agar beads. Treatments with aerosolized bovine Lf (bLf) or saline were started five minutes after infection and repeated daily for six days. Our results demonstrated that aerosolized bLf was effective in significantly reducing both pulmonary bacterial load and infiltrated leukocytes in infected CF mice. Furthermore, for the first time, we showed that bLf reduced pulmonary iron overload, in both WT and CF mice. In particular, at molecular level, a significant decrease of both the iron exporter ferroportin and iron storage ferritin, as well as luminal iron content was observed. Overall, bLf acts as a potent multi-targeting agent able to break the vicious cycle induced by P. aeruginosa, inflammation and iron dysbalance, thus mitigating the severity of CF-related pathology and sequelae.


Assuntos
Anti-Infecciosos/uso terapêutico , Fibrose Cística/terapia , Lactoferrina/uso terapêutico , Pneumonia/terapia , Administração por Inalação , Animais , Anti-Infecciosos/administração & dosagem , Proteínas de Transporte de Cátions/metabolismo , Bovinos , Fibrose Cística/complicações , Fibrose Cística/genética , Ferritinas/metabolismo , Lactoferrina/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/etiologia , Pneumonia/microbiologia , Pseudomonas aeruginosa/patogenicidade
14.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052176

RESUMO

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Canais de Potássio/metabolismo , Saccharomyces cerevisiae/genética , Canais de Sódio/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Canais de Potássio/genética , Saccharomyces cerevisiae/metabolismo , Canais de Sódio/genética
15.
Planta ; 250(2): 667-674, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31104129

RESUMO

MAIN CONCLUSION: Mercury accumulation in Arabidopsis shoots is accelerated by endodermis specific expression of fusion proteins of a bacterial mercury transporter MerC and a plant SNARE SYP121 under control of SCARECROW promoter. We previously demonstrated that the CaMV 35S RNA promoter (p35S)-driven ubiquitous expression of a bacterial mercury transporter MerC, fused with SYP121, an Arabidopsis SNARE protein increases mercury accumulation of Arabidopsis. To establish an improved fine-tuned mercury transport system in plants for phytoremediation, the present study generated and characterized transgenic Arabidopsis plants expressing MerC-SYP121 specifically in the root endodermis, which is a crucial cell type for root element uptake. We generated four independent transgenic Arabidopsis lines expressing a transgene encoding mCherry-MerC-SYP121 under the control of the endodermis-specific SCARECROW promoter (hereafter pSCR lines). Quantitative real-time PCR analysis showed that expression levels of the transgene in roots of the pSCR lines were 3-23% of the p35S driven-overexpressing line. Confocal microscopy analysis showed that mCherry-MerC-SYP121 was dominantly expressed in the endodermis of the meristematic zone as well as in the mature zone of the pSCR roots. Mercury accumulation in shoots of the pSCR lines exposed to inorganic mercury was overall higher than the wild-type and comparable to the p35S over-expressing line. These results suggest that endodermis-specific expression of the MerC-SYP121 fusion proteins in plant roots sufficiently enhances mercury uptake and accumulation into shoots, which would be an ideal phenotype for phytoremediation of mercury-contaminated environments.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mercúrio/metabolismo , Proteínas Qa-SNARE/metabolismo , Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Meristema/genética , Meristema/metabolismo , Especificidade de Órgãos , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusão
16.
Planta ; 250(2): 549-561, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31119363

RESUMO

MAIN CONCLUSION: OsHAK16 mediates K uptake and root-to-shoot translocation in a broad range of external K concentrations, thereby contributing to the maintenance of K homeostasis and salt tolerance in the rice shoot. The HAK/KUP/KT transporters have been widely associated with potassium (K) transport across membranes in both microbes and plants. Here, we report the physiological function of OsHAK16, a member belonging to the HAK/KUP/KT family in rice (Oryza sativa L.). Transcriptional expression of OsHAK16 was up-regulated by K deficiency or salt stress. OsHAK16 is localized at the plasma membrane. OsHAK16 knockout (KO) dramatically reduced root K net uptake rate and growth at both 0.1 mM and 1 mM K supplies, while OsHAK16 overexpression (OX) increased total K uptake and growth only at 0.1 mM K level. OsHAK16-KO decreased the rate of rubidium (Rb) uptake and translocation compared to WT at both 0.2 mM and 1 mM Rb levels. OsHAK16 disruption decreased while its overexpression increased K concentration in root slightly but in shoot remarkably. The relative distribution of total K between shoot and root decreased by 30% in OsHAK16-KO lines and increased by 30% in its OX lines compared to WT. OsHAK16-KO diminished K uptake and K/Na ratio, while OsHAK16-OX improved K uptake and translocation from root to shoot, resulting in increased sensitivity and tolerance to salt stress, respectively. Expression of OsHAK16 enhanced the growth of high salt-sensitive yeast mutant by increasing its K but no Na content. Taking all these together, we conclude that OsHAK16 plays crucial roles in maintaining K homeostasis and salt tolerance in rice shoot.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/genética , Potássio/metabolismo , Tolerância ao Sal , Proteínas de Transporte de Cátions/genética , Homeostase , Transporte de Íons , Oryza/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia
17.
Inorg Chem ; 58(9): 5932-5942, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-30986048

RESUMO

Cluster 2 (288HDDDNAHAHTH298) from Neisseria meningitidis ZnuD is a flexible loop that captures zinc(II) ions, acting as a "fishing net". We describe its Zn(II) and Cu(II) binding capabilities, focusing on the thermodynamics of such interactions and comparing them with the complexes of the 1MAHHHHHHL9-NH2 region. Copper(II) complexes with the studied ZnuD regions are thermodynamically more stable than the zinc(II) ones-Cu(II) complexes dominate in solution even in close to physiological ratios of the studied metal ions (a 10-fold excess of Zn(II) over Cu(II)). While the binding of native Zn(II) has no significant impact on the structure of its transporter, Cu(II) binding induces a conformational change of cluster 2 to a polyproline II-like helix. To the best of our knowledge, this is the first evidence of a copper(II)-induced formation of a polyproline II-like structure in a sequence that does not contain proline residues. Cu(II) coordination also changes the structure of an intracellular, N-terminal, His-rich region, folding it to an α helix.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Neisseria meningitidis/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Cobre/química , Humanos , Infecções Meningocócicas/microbiologia , Modelos Moleculares , Neisseria meningitidis/química , Ligação Proteica , Termodinâmica , Zinco/química
18.
Nat Commun ; 10(1): 1791, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996251

RESUMO

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Zinco/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Encéfalo/patologia , Encéfalo/cirurgia , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/cirurgia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutagênese , Mutação , Fosforilação/fisiologia , Alinhamento de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
Mol Biotechnol ; 61(6): 442-450, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980224

RESUMO

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K+ transporter (HKT)'s play an important role in reducing the phytotoxicity of Na+. Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na+/K+ concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema.


Assuntos
Arabidopsis/genética , Brassicaceae/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Simportadores/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Sequência de Bases , Brassicaceae/efeitos dos fármacos , Brassicaceae/crescimento & desenvolvimento , Brassicaceae/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Teste de Complementação Genética , Transporte de Íons/efeitos dos fármacos , Mutação , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Potássio/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Especificidade da Espécie , Estresse Fisiológico/genética , Simportadores/metabolismo
20.
Mol Biotechnol ; 61(6): 451-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997666

RESUMO

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
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