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1.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806154

RESUMO

Knowledge on the mechanisms of acid and base secretion in airways has progressed recently. The aim of this review is to summarize the known mechanisms of airway surface liquid (ASL) pH regulation and their implication in lung diseases. Normal ASL is slightly acidic relative to the interstitium, and defects in ASL pH regulation are associated with various respiratory diseases, such as cystic fibrosis. Basolateral bicarbonate (HCO3-) entry occurs via the electrogenic, coupled transport of sodium (Na+) and HCO3-, and, together with carbonic anhydrase enzymatic activity, provides HCO3- for apical secretion. The latter mainly involves CFTR, the apical chloride/bicarbonate exchanger pendrin and paracellular transport. Proton (H+) secretion into ASL is crucial to maintain its relative acidity compared to the blood. This is enabled by H+ apical secretion, mainly involving H+/K+ ATPase and vacuolar H+-ATPase that carry H+ against the electrochemical potential gradient. Paracellular HCO3- transport, the direction of which depends on the ASL pH value, acts as an ASL protective buffering mechanism. How the transepithelial transport of H+ and HCO3- is coordinated to tightly regulate ASL pH remains poorly understood, and should be the focus of new studies.


Assuntos
Bicarbonatos/química , Anidrases Carbônicas/metabolismo , Epitélio/metabolismo , Mucosa Respiratória/metabolismo , Animais , Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Coelhos , Transportadores de Sulfato/metabolismo , Traqueia/metabolismo
2.
Int J Mol Sci ; 22(2)2021 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-33467091

RESUMO

High-capacity tonoplast cation/H+ antiport in plants is partially mediated by a family of CAX transporters. Previous studies have reported that CAX activity is affected by an N-terminal autoinhibitory region. CAXs may be present as heterodimers in plant cells, and this phenomenon necessitates further study. In this study, we demonstrate that there is an interaction between CAX4 and CAX1 as determined by the use of a yeast two-hybrid system and a bimolecular fluorescence complementation assay. More specifically, the N-terminal of CAX4 interacts with CAX1. We further observed the over-expression and either a single or double mutant of CAX1 and CAX4 in response to abiotic stress in Arabidopsis. These results suggest that CAX1 and CAX4 can interact to form a heterodimer, and the N-terminal regions of CAX4 play important roles in vivo; this may provide a foundation for a deep study of CAX4 function in the future.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Estresse Fisiológico , Antiporters/química , Antiporters/genética , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Mutação , Ligação Proteica
3.
Proc Natl Acad Sci U S A ; 117(37): 22974-22983, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873649

RESUMO

Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme's substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.


Assuntos
Álcoois Graxos/metabolismo , Oxirredutases/metabolismo , Antiporters/metabolismo , Biocombustíveis , Ácidos Graxos/metabolismo , Engenharia Metabólica/métodos , Proteínas de Transporte de Cátions Orgânicos/genética , Oxirredutases/fisiologia , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
PLoS One ; 15(5): e0233863, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470053

RESUMO

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Ritmo Circadiano , Proteínas na Dieta/farmacologia , Alimentos , Intestinos/fisiologia , Aminoácidos/metabolismo , Animais , Antiporters/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Grelina/administração & dosagem , Grelina/farmacologia , Insulina/metabolismo , Intestino Delgado/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Simportadores/metabolismo , Serina-Treonina Quinases TOR/metabolismo
5.
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
6.
Food Chem ; 324: 126887, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32339788

RESUMO

Epigenetic regulation and salt ions play essential roles in senescence control, but the underlying regulatory mechanism of senescence has not been thoroughly revealed in broccoli postharvest buds. Here, we found 200 mmol·L-1 NaCl, 400 mmol·L-1 KCl, 40 mmol·L-1 CaCl2 and 0.5 µmol·L-1 Trichostatin-A (TSA, a histone deacetylase inhibitor) delayed the bud senescence. They resulted in significantly inhibiting the malondialdehyde (MDA) content, and dramatically promoting the contents of superoxide dismutase (SOD), peroxidase (POD) and Chlorophyll. Furthermore, the expression of PHEOPHYTINASE (PPH) and NONYELLOWING (NYE1), but not SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), were remarkably repressed by salt ions and TSA. Interestingly, HISTONE DEACETYLASE 9 (HDA9) and CATION/Ca2+ EXCHANGER 1 (CCX1) were down-regulated by NaCl, CaCl2 and TSA. Further assays demonstrated that HDA9 could not interact with CCX1 promoter. It suggested that CCX1 along with HDA9 were involved in inhibiting the senescence of broccoli buds, and regulated aging by indirect interaction.


Assuntos
Antioxidantes/metabolismo , Brassica/metabolismo , Regulação para Baixo/efeitos dos fármacos , Histona Desacetilases/metabolismo , Proteínas de Plantas/metabolismo , Sais/farmacologia , Sequência de Aminoácidos , Antiporters/química , Antiporters/genética , Antiporters/metabolismo , Brassica/química , Brassica/classificação , Cloreto de Cálcio/química , Cloreto de Cálcio/farmacologia , Clorofila/metabolismo , Flores/química , Flores/metabolismo , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Íons/química , Filogenia , Sais/química , Alinhamento de Sequência
7.
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
8.
Biochim Biophys Acta Biomembr ; 1862(6): 183225, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126231

RESUMO

NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.


Assuntos
Proteínas de Bactérias/química , Antiportadores de Potássio-Hidrogênio/química , Domínios Proteicos , Vibrio cholerae/química , Trifosfato de Adenosina/metabolismo , Antiporters/química , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Citoplasma/química , Ressonância Magnética Nuclear Biomolecular , Antiportadores de Potássio-Hidrogênio/metabolismo , Conformação Proteica
9.
J Neurosci ; 40(16): 3152-3164, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32156830

RESUMO

Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.


Assuntos
Antiporters/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Animais Geneticamente Modificados , Antiporters/genética , Sinalização do Cálcio/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Trocador de Sódio e Cálcio/genética
10.
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
11.
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
12.
Planta ; 251(3): 71, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32108903

RESUMO

MAIN CONCLUSION: In this study, we show that ectopic expression of either HtNHX1 or HtNHX2, from Helianthus tuberosus plant (located at vacuolar and endosome membranes, respectively), in rice plants could enhance its tolerance to aluminum (Al3+) stress and soil acidity. Plant sodium (potassium)/proton (Na+(K+)/H+ antiporters of the NHX family have been extensively characterized as they are related to the enhancement of salt tolerance. However, no previous study has reported NHX transporter functions in plant tolerance to Al3+ toxicity. In this study, we demonstrate their role as a component of the Al3+ stress tolerance mechanism. We show that the ectopic expression of either HtNHX1 or HtNHX2 , from Helianthus tuberosus plant, in rice (located at vacuole and endosome, respectively) could also enhance rice tolerance to Al3+ stress and soil acidity. Expression of either HtNHX1 or HtNHX2 reduced the inhibitory effect of Al3+ on the rice root elongation rate; both genes were reported to be equally effective in improvement of stress conditions. Expression of HtNHX1 enhanced Al3+-trigged-secretion of citrate acids, rhizosphere acidification, and also reduced K+ efflux from root tissues. In contrast, expression of HtNHX2 prevented Al3+-trigged-decrease of H+ influx into root tissues. Al3+-induced damage of the cell wall extensibility at the root tips was impaired by either HtNHX1 or HtNHX2. Co-expression of HtNHX1 and HtNHX2 further improved rice growth, particularly under the Al3+ stress conditions. The results demonstrate that HtNHX1 and HtNHX2 improved rice tolerance to Al3+ via different mechanisms by altering the K+ and H+ fluxes and the cell wall structure.


Assuntos
Alumínio/toxicidade , Tolerância a Medicamentos/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Oryza/efeitos dos fármacos , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Tolerância ao Sal/fisiologia , Antiporters/genética , Antiporters/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Plasticidade Celular , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Helianthus/metabolismo , Concentração de Íons de Hidrogênio , Meristema/citologia , Meristema/efeitos dos fármacos , Meristema/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Solo , Vacúolos/metabolismo
13.
J Biol Chem ; 295(5): 1350-1365, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31914417

RESUMO

Metabolic reprogramming in cancer cells can increase their dependence on metabolic substrates such as glucose. As such, the vulnerability of cancer cells to glucose deprivation creates an attractive opportunity for therapeutic intervention. Because it is not possible to starve tumors of glucose in vivo, here we sought to identify the mechanisms in glucose deprivation-induced cancer cell death and then designed inhibitor combinations to mimic glucose deprivation-induced cell death. Using metabolomic profiling, we found that cells undergoing glucose deprivation-induced cell death exhibited dramatic accumulation of intracellular l-cysteine and its oxidized dimer, l-cystine, and depletion of the antioxidant GSH. Building on this observation, we show that glucose deprivation-induced cell death is driven not by the lack of glucose, but rather by l-cystine import. Following glucose deprivation, the import of l-cystine and its subsequent reduction to l-cysteine depleted both NADPH and GSH pools, thereby allowing toxic accumulation of reactive oxygen species. Consistent with this model, we found that the glutamate/cystine antiporter (xCT) is required for increased sensitivity to glucose deprivation. We searched for glycolytic enzymes whose expression is essential for the survival of cancer cells with high xCT expression and identified glucose transporter type 1 (GLUT1). Testing a drug combination that co-targeted GLUT1 and GSH synthesis, we found that this combination induces synthetic lethal cell death in high xCT-expressing cell lines susceptible to glucose deprivation. These results indicate that co-targeting GLUT1 and GSH synthesis may offer a potential therapeutic approach for targeting tumors dependent on glucose for survival.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Glucose/metabolismo , Neoplasias/metabolismo , Antiporters/metabolismo , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisteína/metabolismo , Cistina/metabolismo , Dimerização , Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/biossíntese , Glutationa/metabolismo , Humanos , Metaboloma/genética , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Medicamentos Sintéticos/farmacologia
14.
J Ethnopharmacol ; 252: 112581, 2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31968215

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. AIM OF THE STUDY: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). MATERIALS AND METHODS: The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. RESULTS: In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. CONCLUSIONS: A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.


Assuntos
Aconitina/análogos & derivados , Aconitum , Antiporters/metabolismo , Barreira Hematoencefálica/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Aconitina/farmacologia , Animais , Linhagem Celular , Humanos , Masculino , Camundongos Endogâmicos ICR , Proteínas de Transporte de Cátions Orgânicos/genética , Prótons , RNA Interferente Pequeno/genética
15.
Biochem Biophys Res Commun ; 524(1): 89-95, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980167

RESUMO

Glycogen storage disease type Ib (GSD-Ib), caused by a deficiency in glucose-6-phosphate transporter (G6PT), is characterized by disrupted glucose homeostasis, inflammatory bowel disease, neutropenia, and neutrophil dysfunction. The purpose of this study was to investigate the role of G6PT on macrophage functions and metabolism. Peritoneal macrophages of G6pt-/- mice were lower in number and their effector functions including migration, superoxide production, and phagocytosis were impaired. To investigate the underlying mechanisms of macrophage dysfunction, the G6PT gene was mutated in porcine alveolar macrophage 3D4/31 cells using the CRISPR/Cas9 technology. The G6PT-deficient macrophages exhibited significant decline in cell growth, bactericidal activity, and antiviral response. These phenotypes are associated with the impaired glycolysis and mitochondrial oxidative phosphorylation. We therefore propose that the G6PT-mediated metabolism is essential for effector functions of macrophage, the immune deficiencies observed in GSD-Ib extend beyond neutropenia and neutrophil dysfunction, and future therapeutic targets aimed both the neutrophils and macrophages may be necessary.


Assuntos
Antiporters/genética , Antiporters/metabolismo , Doença de Depósito de Glicogênio Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proliferação de Células , Glucose/metabolismo , Glicólise , Humanos , Macrófagos/citologia , Camundongos , Mitocôndrias/metabolismo , Modelos Animais , Mutação , Neutrófilos/metabolismo , Oxirredução , Fenótipo , Fosforilação , Suínos
16.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936580

RESUMO

The endosomal-type Na+, K+/H+ antiporters (NHXs) play important roles in K+, vesicle pH homeostasis, and protein trafficking in plant. However, the structure governing ion transport mechanism and the key residues related to the structure-function of the endosomal-type NHXs remain unclear. Here, the structure-function relationship of the only endosomal-type NHX from mulberry, MnNHX6, was investigated by homology modeling, mutagenesis, and localization analyses in yeast. The ectopic expression of MnNHX6 in arabidopsis and Nhx1 mutant yeast can enhance their salt tolerance. MnNHX6's three-dimensional structure, established by homology modeling, was supported by empirical, phylogenetic, and experimental data. Structure analysis showed that MnNHX6 contains unusual 13 transmembrane helices, but the structural core formed by TM5-TM12 assembly is conserved. Localization analysis showed that MnNHX6 has the same endosomal localization as yeast Nhx1/VPS44, and Arg402 is important for protein stability of MnNHX6. Mutagenesis analysis demonstrated MnNHX6 contains a conserved cation binding mechanism and a similar charge-compensated pattern as NHE1, but shares a different role in ion selectivity than the vacuolar-type NHXs. These results improve our understanding of the role played by the structure-function related key residues of the plant endosomal-type NHXs, and provide a basis for the ion transport mechanism study of endosomal-type NHXs.


Assuntos
Antiporters/química , Antiporters/metabolismo , Endossomos/metabolismo , Morus/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Sequência Conservada , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação/genética , Fenótipo , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Tolerância ao Sal , Relação Estrutura-Atividade
17.
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
18.
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
19.
Urolithiasis ; 48(1): 1-8, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31201468

RESUMO

Oxalobacter sp. promotion of enteric oxalate excretion, correlating with reductions in urinary oxalate excretion, was previously reported in rats and mice, but the mechanistic basis for this affect has not been described. The main objective of the present study was to determine whether the apical oxalate transport proteins, PAT1 (slc26a6) and DRA (slc26a3), are involved in mediating the Oxalobacter-induced net secretory flux across colonized mouse cecum and distal colon. We measured unidirectional and net fluxes of oxalate across tissues removed from colonized PAT1 and DRA knockout (KO) mice and also across two double knockout (dKO) mouse models with primary hyperoxaluria, type 1 (i.e., deficient in alanine-glyoxylate aminotransferase; AGT KO), including PAT1/AGT dKO and DRA/AGT dKO mice compared to non-colonized mice. In addition, urinary oxalate excretion was measured before and after the colonization procedure. The results demonstrate that Oxalobacter can induce enteric oxalate excretion in the absence of either apical oxalate transporter and urinary oxalate excretion was reduced in all colonized genotypes fed a 1.5% oxalate-supplemented diet. We conclude that there are other, as yet unidentified, oxalate transporters involved in mediating the directional changes in oxalate transport across the Oxalobacter-colonized mouse large intestine.


Assuntos
Antiporters/metabolismo , Mucosa Intestinal/metabolismo , Oxalatos/metabolismo , Oxalobacter formigenes/metabolismo , Transportadores de Sulfato/metabolismo , Animais , Antiporters/genética , Ceco/metabolismo , Ceco/microbiologia , Colo/metabolismo , Colo/microbiologia , Fezes/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Knockout , Oxalobacter formigenes/isolamento & purificação , Eliminação Renal , Transportadores de Sulfato/genética , Simbiose
20.
FEBS Lett ; 594(1): 3-18, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31705665

RESUMO

Cellular metabolism generally refers to biochemical processes that produce or consume energy within the cell. Recent studies have established that aberrant metabolic states caused by internal or external stresses and genetic mutations are intertwined with several human pathologies. Gaining insight into these metabolic alterations is, therefore, essential for understanding the pathophysiology of various diseases. Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by hypoglycemia, excessive glycogen accumulation in the liver and kidney, neutropenia, neutrophil dysfunction, and inflammatory bowel disease. GSD-Ib is caused by a deficiency of glucose-6-phosphate transporter (G6PT). Recently, it was reported that deficiency of G6PT also leads to the aberrant proliferation and differentiation of mesenchymal stem cells and impaired regulatory T-cell function. This review describes the broad impact of altered cellular metabolism resulting from a lack of G6PT activity on cellular function and considers the prospects of developing novel approaches for GSD-Ib treatment.


Assuntos
Antiporters/metabolismo , Doença de Depósito de Glicogênio Tipo I/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Antiporters/genética , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo I/imunologia , Doença de Depósito de Glicogênio Tipo I/patologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Linfócitos T/imunologia
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