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1.
Plant Mol Biol ; 102(6): 603-614, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32052233

RESUMO

The WRKY transcription factor family is involved in responding to biotic and abiotic stresses. Its members contain a typical WRKY domain and can regulate plant physiological responses by binding to W-boxes in the promoter regions of downstream target genes. We identified the sweet sorghum SbWRKY50 (Sb09g005700) gene, which encodes a typical class II of the WRKY family protein that localizes to the nucleus and has transcriptional activation activity. The expression of SbWRKY50 in sweet sorghum was reduced by salt stress, and its ectopic expression reduced the salt tolerance of Arabidopsis thaliana plants. Compared with the wild type, the germination rate, root length, biomass and potassium ion content of SbWRKY50 over-expression plants decreased significantly under salt-stress conditions, while the hydrogen peroxide, superoxide anion and sodium ion contents increased. Real-time PCR results showed that the expression levels of AtSOS1, AtHKT1 and genes related to osmotic and oxidative stresses in over-expression strains decreased under salt-stress conditions. Luciferase complementation imaging and yeast one-hybrid assays confirmed that SbWRKY50 could directly bind to the upstream promoter of the SOS1 gene in A. thaliana. However, in sweet sorghum, SbWRKY50 could directly bind to the upstream promoters of SOS1 and HKT1. These results suggest that the new WRKY transcription factor SbWRKY50 participates in plant salt response by controlling ion homeostasis. However, the regulatory mechanisms are different in sweet sorghum and Arabidopsis, which may explain their different salt tolerance levels. The data provide information that can be applied to genetically modifying salt tolerance in different crop varieties.


Assuntos
Homeostase , Tolerância ao Sal/fisiologia , Sorghum/genética , Sorghum/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Proteínas de Transporte , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Potássio/metabolismo , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Sementes , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico , Superóxidos/metabolismo , Simportadores/genética , Simportadores/metabolismo
2.
Plant Mol Biol ; 102(4-5): 553-567, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31989373

RESUMO

KEY MESSAGE: Overexpression of K2-NhaD in transgenic cotton resulted in phenotypes with strong salinity and drought tolerance in greenhouse and field experiments, increased expression of stress-related genes, and improved regulation of metabolic pathways, such as the SOS pathway. Drought and salinity are major abiotic stressors which negatively impact cotton yield under field conditions. Here, a plasma membrane Na+/H+ antiporter gene, K2-NhaD, was introduced into upland cotton R15 using an Agrobacterium tumefaciens-mediated transformation system. Homozygous transgenic lines K9, K17, and K22 were identified by PCR and glyphosate-resistance. TAIL-PCR confirmed that T-DNA carrying the K2-NhaD gene in transgenic lines K9, K17 and K22 was inserted into chromosome 3, 19 and 12 of the cotton genome, respectively. Overexpression of K2-NhaD in transgenic cotton plants grown in greenhouse conditions and subjected to drought and salinity stress resulted in significantly higher relative water content, chlorophyll, soluble sugar, proline levels, and SOD, CAT, and POD activity, relative to non-transgenic plants. The expression of stress-related genes was significantly upregulated, and this resulted in improved regulation of metabolic pathways, such as the salt overly sensitive pathway. K2-NhaD transgenic plants growing under field conditions displayed strong salinity and drought tolerance, especially at high levels of soil salinity and drought. Seed cotton yields in transgenic line were significantly higher than in wild-type plants. In conclusion, the data indicate that K2-NhaD transgenic lines have great potential for the production of stress-tolerant cotton under field conditions.


Assuntos
Gossypium/metabolismo , Proteínas de Plantas/fisiologia , Tolerância ao Sal/genética , Trocadores de Sódio-Hidrogênio/fisiologia , Secas , Gossypium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico , Água/metabolismo
3.
Ecotoxicol Environ Saf ; 188: 109894, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31706239

RESUMO

Modulation of plant salt tolerance has been drawing great attention. Thymol is a kind of natural chemical that has been developed as anti-microbial reagent and medicine. To date, we still have limited knowledge about thymol-modulated plant physiology. In this work, physiological, histochemical, and biochemical methods were adopted to study thymol-conferred salt resistance in the root of rice (Oryza sativa). Thymol significantly rescued root growth under salt stress. Thymol ameliorated cell membrane damage, oxidative stress, ROS accumulation, and cell death in roots under salt stress. Thymol-attenuated oxidative stress may be resulted from the activation of anti-oxidative capacity, including both enzymatic and non-enzymatic system. Thymol treatment significantly decreased Na+ content in root cells upon salt stress, which might be ascribed to the upregulation of OsSOS1 (salt overly sensitive 1) facilitating Na+ exclusion. In addition, thymol stimulated the expression of genes encoding tonoplast OsNHX (Na+/H+antiporter), which may help root cells to compartmentalize Na+ in vacuole. The results of these works evidenced that thymol was capable of inducing salt tolerance by reestablishing ROS homeostasis and modulating cellular Na+ flux in rice roots. These findings may be applicable to improve crop growth in salinity area.


Assuntos
Antioxidantes/metabolismo , Homeostase/efeitos dos fármacos , Oryza/efeitos dos fármacos , Tolerância ao Sal/efeitos dos fármacos , Sódio/metabolismo , Timol/farmacologia , Íons/metabolismo , Oryza/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Tolerantes a Sal , Trocadores de Sódio-Hidrogênio/metabolismo
4.
BMC Plant Biol ; 19(1): 469, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690290

RESUMO

BACKGROUND: Soybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed. RESULTS: We found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na+ transportation to leaves along with a reduction of Na+ absorption in roots, and the consequent maintenance of a high K+/Na+ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na+ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na+ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition. CONCLUSION: Vacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K+/Na+ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.


Assuntos
Proteínas de Plantas/metabolismo , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Soja/metabolismo , Arabidopsis/genética , Inativação Gênica , Proteínas de Plantas/genética , Potássio/metabolismo , Plantas Tolerantes a Sal/genética , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Soja/genética , Estresse Fisiológico/genética , Regulação para Cima , Vacúolos/metabolismo
5.
Chem Commun (Camb) ; 55(87): 13152-13155, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31617527

RESUMO

We designed a supported lipid bilayer (SLB) biomimetic membrane system that comprised polyaniline (PANI) to support a lipid bilayer membrane that incorporated Na+/H+ transporter proteins (NhaA) to give the system the capability of controllable electrogenic ion transport. The high turnover rate of NhaA (∼105 per min) provides the basis for this PANI-SLB-NhaA system to be a high-speed rechargeable biocapacitor that functions as a low-energy-consuming fast switch for biological engineering applications.


Assuntos
Compostos de Anilina/metabolismo , Materiais Biomiméticos/metabolismo , Técnicas Biossensoriais , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Compostos de Anilina/química , Materiais Biomiméticos/química , Espectroscopia Dielétrica , Eletrodos , Proteínas de Escherichia coli/química , Ouro/química , Ouro/metabolismo , Bicamadas Lipídicas/química , Trocadores de Sódio-Hidrogênio/química
6.
Physiol Rev ; 99(4): 2015-2113, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31507243

RESUMO

Na+/H+ exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na+ and H+ across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na+/H+ exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.


Assuntos
Equilíbrio Ácido-Base , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Conformação Proteica , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Relação Estrutura-Atividade , Distribuição Tecidual
7.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470498

RESUMO

Cancer cells overexpress proton exchangers at the plasma membrane in order acidify the extracellular matrix and maintain the optimal pH for sustaining cancer growth. Among the families of proton exchangers implicated in carcinogenesis, carbonic anhydrases (CAs), monocarboxylate transporters (MCTs), Na+/H+ exchangers (NHEs), sodium bicarbonate cotransporters (NBCs), and vacuolar ATPases (V-ATPases) are highlighted. Considerable research has been carried out into the utility of the understanding of these machineries in the diagnosis and prognosis of several solid tumors. In addition, as therapeutic targets, the interference of their functions has contributed to the discovery or optimization of cancer therapies. According to recent reports, the study of these mechanisms seems promising in the particular case of oral squamous cell carcinoma (OSCC). In the present review, the latest advances in these fields are summarized, in particular, the usefulness of proton exchangers as potential prognostic biomarkers and therapeutic targets in OSCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias Bucais/metabolismo , Prótons , Carcinoma de Células Escamosas/patologia , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Bucais/patologia , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Nanomedicina Teranóstica/métodos , ATPases Vacuolares Próton-Translocadoras/metabolismo
8.
Hypertension ; 74(3): 526-535, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31352824

RESUMO

The present study directly tested the hypothesis that the NHE3 (Na+/H+ exchanger 3) in the proximal tubules of the kidney is required for the development of Ang II (angiotensin II)-induced hypertension using PT-Nhe3-/- (proximal tubule-specific NHE3 knockout) mice. Specifically, PT-Nhe3-/- mice were generated using the SGLT2-Cre/Nhe3loxlox approach, whereas Ang II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT-Nhe3-/- mice. Under basal conditions, systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were significantly lower in male and female PT-Nhe3-/- than WT mice (P<0.01). A high pressor, 1.5 mg/kg per day, intraperitoneal or a slow pressor dose of Ang II, 0.5 mg/kg per day, intraperitoneal for 2 weeks significantly increased systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure in male and female WT mice (P<0.01), but the hypertensive response to Ang II was markedly attenuated in male and female PT-Nhe3-/- mice (P<0.01). Ang II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in Ang II-infused PT-Nhe3-/- mice (P<0.01). AT1 receptor blocker losartan completely blocked Ang II-induced hypertension in both WT and PT-Nhe3-/- mice (P<0.01). However, inhibition of nitric oxide synthase with L-NG-Nitroarginine methyl ester had no effect on Ang II-induced hypertension in WT or PT-Nhe3-/- mice (not significant). Furthermore, Ang II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney may be a therapeutical target in hypertension induced by Ang II or with increased NHE3 expression in the proximal tubules.


Assuntos
Angiotensina II/farmacologia , Túbulos Renais Proximais/metabolismo , Losartan/administração & dosagem , Receptor Tipo 1 de Angiotensina/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Animais , Modelos Animais de Doenças , Feminino , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Injeções Intraperitoneais , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Knockout , Distribuição Aleatória , Valores de Referência , Trocadores de Sódio-Hidrogênio/metabolismo , Resultado do Tratamento
9.
Plant Sci ; 285: 14-25, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203878

RESUMO

Intracellular Na+/H+ antiporters (NHXs) play important roles in plant tolerance to salt stress. However, plant NHXs functioning in salt tolerance and the underlying physiological mechanisms remain poorly understood. In this report, we report the identification and functional characterization of PbrNHX2 isolated from Pyrus betulaefolia. PbrNHX2 expression levels were induced by salt, and dehydration, but was unaffected by cold. PbrNHX2 was localized in the tonoplast. Overexpression of PbrNHX2 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to salt tolerance, whereas down-regulation of PbrNHX2 in Pyrus betulaefolia by virus-induced gene silencing (VIGS) resulted in elevated salt sensitivity. The transgenic lines contained lower levels of Na+, higher levels of K+, and higher K/Na ratio, whereas they were changed in an opposite way when PbrNHX2 was silenced. In addition, the transgenic plants accumulated lower levels of reactive oxygen species compared with wild type, accompanied by higher activities of three antioxidant enzymes. Taken together, the data demonstrate that PbrNHX2 plays a positive role in salt tolerance and that it holds a great potential for engineering salt tolerance in crops.


Assuntos
Genes de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Pyrus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Plantas Tolerantes a Sal/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Inativação Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Pyrus/genética , Pyrus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Estresse Salino , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/fisiologia , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/fisiologia , Tabaco
10.
Int J Mol Sci ; 20(12)2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208046

RESUMO

The salt overly sensitive 1 (SOS1) gene encodes the plasma membrane Na+/H+ antiporter, SOS1, that is mainly responsible for extruding Na+ from the cytoplasm and reducing the Na+ content in plants under salt stress and is considered a vital determinant in conferring salt tolerance to the plant. However, studies on the salt tolerance function of the TrSOS1 gene of recretohalophytes, such as Tamarix, are limited. In this work, the effects of salt stress on cotton seedlings transformed with tobacco-rattle-virus-based virus-induced gene silencing (VIGS) of the endogenous GhSOS1 gene, or Agrobacterium rhizogenes strain K599-mediated TrSOS1-transgenic hairy root composite cotton plants exhibiting VIGS of GhSOS1 were first investigated. Then, with Arabidopsis thaliana AtSOS1 as a reference, differences in the complementation effect of TrSOS1 or GhSOS1 in a yeast mutant were compared under salt treatment. Results showed that compared to empty-vector-transformed plants, GhSOS1-VIGS-transformed cotton plants were more sensitive to salt stress and had reduced growth, insufficient root vigor, and increased Na+ content and Na+/K+ ratio in roots, stems, and leaves. Overexpression of TrSOS1 enhanced the salt tolerance of hairy root composite cotton seedlings exhibiting GhSOS1-VIGS by maintaining higher root vigor and leaf relative water content (RWC), and lower Na+ content and Na+/K+ ratio in roots, stems, and leaves. Transformations of TrSOS1, GhSOS1, or AtSOS1 into yeast NHA1 (Na+/H+ antiporter 1) mutant reduced cellular Na+ content and Na+/K+ ratio, increased K+ level under salt stress, and had good growth complementation in saline conditions. In particular, the ability of TrSOS1 or GhSOS1 to complement the yeast mutant was better than that of AtSOS1. This may indicate that TrSOS1 is an effective substitute and confers enhanced salt tolerance to transgenic hairy root composite cotton seedlings, and even the SOS1 gene from salt-tolerant Tamarix or cotton may have higher efficiency than salt-sensitive Arabidopsis in regulating Na+ efflux, maintaining Na+ and K+ homeostasis, and therefore contributing to stronger salt tolerance.


Assuntos
Inativação Gênica , Gossypium/genética , Brotos de Planta/genética , Tolerância ao Sal/genética , Plântula/genética , Trocadores de Sódio-Hidrogênio/genética , Tamaricaceae/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Plântula/virologia , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico/genética
11.
Cell Mol Life Sci ; 76(19): 3783-3800, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31165202

RESUMO

In the male reproductive tract, ionic equilibrium is essential to maintain normal spermatozoa production and, hence, the reproductive potential. Among the several ions, HCO3- and H+ have a central role, mainly due to their role on pH homeostasis. In the male reproductive tract, the major players in pH regulation and homeodynamics are carbonic anhydrases (CAs), HCO3- membrane transporters (solute carrier 4-SLC4 and solute carrier 26-SLC26 family transporters), Na+-H+ exchangers (NHEs), monocarboxylate transporters (MCTs) and voltage-gated proton channels (Hv1). CAs and these membrane transporters are widely distributed throughout the male reproductive tract, where they play essential roles in the ionic balance of tubular fluids. CAs are the enzymes responsible for the production of HCO3- which is then transported by membrane transporters to ensure the maturation, storage, and capacitation of the spermatozoa. The transport of H+ is carried out by NHEs, Hv1, and MCTs and is essential for the electrochemical balance and for the maintenance of the pH within the physiological limits along the male reproductive tract. Alterations in HCO3- production and transport of ions have been associated with some male reproductive dysfunctions. Herein, we present an up-to-date review on the distribution and role of the main intervenient on pH homeodynamics in the fluids throughout the male reproductive tract. In addition, we discuss their relevance for the establishment of the male reproductive potential.


Assuntos
Genitália Masculina/metabolismo , Concentração de Íons de Hidrogênio , Animais , Bicarbonatos/metabolismo , Anidrases Carbônicas/metabolismo , Fertilidade , Genitália Masculina/química , Homeostase , Humanos , Canais Iônicos/metabolismo , Bombas de Íon/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
12.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067690

RESUMO

Out-of-hospital sudden cardiac arrest is a major public health problem with an overall survival of less than 5%. Upon cardiac arrest, cessation of coronary blood flow rapidly leads to intense myocardial ischemia and activation of the sarcolemmal Na+-H+ exchanger isoform-1 (NHE-1). NHE-1 activation drives Na+ into cardiomyocytes in exchange for H+ with its exchange rate intensified upon reperfusion during the resuscitation effort. Na+ accumulates in the cytosol driving Ca2+ entry through the Na+-Ca2+ exchanger, eventually causing cytosolic and mitochondrial Ca2+ overload and worsening myocardial injury by compromising mitochondrial bioenergetic function. We have reported clinically relevant myocardial effects elicited by NHE-1 inhibitors given during resuscitation in animal models of ventricular fibrillation (VF). These effects include: (a) preservation of left ventricular distensibility enabling hemodynamically more effective chest compressions, (b) return of cardiac activity with greater electrical stability reducing post-resuscitation episodes of VF, (c) less post-resuscitation myocardial dysfunction, and (d) attenuation of adverse myocardial effects of epinephrine; all contributing to improved survival in animal models. Mechanistically, NHE-1 inhibition reduces adverse effects stemming from Na+-driven cytosolic and mitochondrial Ca2+ overload. We believe the preclinical work herein discussed provides a persuasive rationale for examining the potential role of NHE-1 inhibitors for cardiac resuscitation in humans.


Assuntos
Parada Cardíaca/tratamento farmacológico , Isquemia Miocárdica/genética , Trocadores de Sódio-Hidrogênio/genética , Fibrilação Ventricular/tratamento farmacológico , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Parada Cardíaca/genética , Parada Cardíaca/patologia , Humanos , Modelos Animais , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Sarcolema/metabolismo , Sarcolema/patologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Fibrilação Ventricular/genética , Fibrilação Ventricular/patologia
13.
Int J Mol Sci ; 20(10)2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100786

RESUMO

In Arabidopsis, the salt overly sensitive (SOS) pathway, consisting of calcineurin B-like protein 4 (CBL4/SOS3), CBL-interacting protein kinase 24 (CIPK24/SOS2) and SOS1, has been well defined as a crucial mechanism to control cellular ion homoeostasis by extruding Na+ to the extracellular space, thus conferring salt tolerance in plants. CBL10 also plays a critical role in salt tolerance possibly by the activation of Na+ compartmentation into the vacuole. However, the functional relationship of the SOS and CBL10-regulated processes remains unclear. Here, we analyzed the genetic interaction between CBL4 and CBL10 and found that the cbl4 cbl10 double mutant was dramatically more sensitive to salt as compared to the cbl4 and cbl10 single mutants, suggesting that CBL4 and CBL10 each directs a different salt-tolerance pathway. Furthermore, the cbl4 cbl10 and cipk24 cbl10 double mutants were more sensitive than the cipk24 single mutant, suggesting that CBL10 directs a process involving CIPK24 and other partners different from the SOS pathway. Although the cbl4 cbl10, cipk24 cbl10, and sos1 cbl10 double mutants showed comparable salt-sensitive phenotype to sos1 at the whole plant level, they all accumulated much lower Na+ as compared to sos1 under high salt conditions, suggesting that CBL10 regulates additional unknown transport processes that play distinct roles from the SOS1 in Na+ homeostasis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Tolerância ao Sal/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica de Plantas , Homeostase , Mutação , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Salino/fisiologia , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Vacúolos/metabolismo
14.
PLoS Pathog ; 15(4): e1007575, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31002735

RESUMO

High-risk human papillomavirus (HPV) E6 proteins associate with the cellular ubiquitin ligase E6-Associated Protein (E6AP), and then recruit both p53 and certain cellular PDZ proteins for ubiquitination and degradation by the proteasome. Low-risk HPV E6 proteins also associate with E6AP, yet fail to recruit p53 or PDZ proteins; their E6AP-dependent targets have so far been uncharacterized. We found a cellular PDZ protein called Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) is targeted for degradation by both high and low-risk HPV E6 proteins as well as E6 proteins from diverse non-primate mammalian species. NHERF1 was degraded by E6 in a manner dependent upon E6AP ubiquitin ligase activity but independent of PDZ interactions. A novel structural domain of E6, independent of the p53 recognition domain, was necessary to associate with and degrade NHERF1, and the NHERF1 EB domain was required for E6-mediated degradation. Degradation of NHERF1 by E6 activated canonical Wnt/ß-catenin signaling, a key pathway that regulates cell growth and proliferation. Expression levels of NHERF1 increased with increasing cell confluency. This is the first study in which a cellular protein has been identified that is targeted for degradation by both high and low-risk HPV E6 as well as E6 proteins from diverse animal papillomaviruses. This suggests that NHERF1 plays a role in regulating squamous epithelial growth and further suggests that the interaction of E6 proteins with NHERF1 could be a common therapeutic target for multiple papillomavirus types.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Fosfoproteínas/genética , Filogenia , Complexo de Endopeptidases do Proteassoma , Proteólise , Proteínas Repressoras/genética , Trocadores de Sódio-Hidrogênio/genética , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Proteína Wnt1/genética , beta Catenina/genética
15.
Nat Commun ; 10(1): 1742, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988359

RESUMO

Na+/H+ antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.


Assuntos
Pyrococcus abyssi/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons , Modelos Moleculares , Prótons , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
16.
Nat Commun ; 10(1): 1199, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867421

RESUMO

Calcium is a universal secondary messenger that triggers many cellular responses. However, it is unclear how a calcium signal is coordinately decoded by different calcium sensors, which in turn regulate downstream targets to fulfill a specific physiological function. Here we show that SOS2-LIKE PROTEIN KINASE5 (PKS5) can negatively regulate the Salt-Overly-Sensitive signaling pathway in Arabidopsis. PKS5 can interact with and phosphorylate SOS2 at Ser294, promote the interaction between SOS2 and 14-3-3 proteins, and repress SOS2 activity. However, salt stress promotes an interaction between 14-3-3 proteins and PKS5, repressing its kinase activity and releasing inhibition of SOS2. We provide evidence that 14-3-3 proteins bind to Ca2+, and that Ca2+ modulates 14-3-3-dependent regulation of SOS2 and PKS5 kinase activity. Our results suggest that a salt-induced calcium signal is decoded by 14-3-3 and SOS3/SCaBP8 proteins, which selectively activate/inactivate the downstream protein kinases SOS2 and PKS5 to regulate Na+ homeostasis by coordinately mediating plasma membrane Na+/H+ antiporter and H+-ATPase activity.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Tolerância ao Sal/fisiologia , Proteínas 14-3-3/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Fosforilação , Plantas Geneticamente Modificadas , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/genética , ATPases Translocadoras de Prótons/metabolismo , Estresse Salino/fisiologia , Transdução de Sinais/fisiologia , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
17.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832374

RESUMO

HKT1 and SOS1 are two key Na⁺ transporters that modulate salt tolerance in plants. Although much is known about the respective functions of HKT1 and SOS1 under salt conditions, few studies have examined the effects of HKT1 and SOS1 mutations on the expression of other important Na⁺ and K⁺ transporter genes. This study investigated the physiological parameters and expression profiles of AtHKT1;1, AtSOS1, AtHAK5, AtAKT1, AtSKOR, AtNHX1, and AtAVP1 in wild-type (WT) and athkt1;1 and atsos1 mutants of Arabidopsis thaliana under 25 mM NaCl. We found that AtSOS1 mutation induced a significant decrease in transcripts of AtHKT1;1 (by 56⁻62% at 6⁻24 h), AtSKOR (by 36⁻78% at 6⁻24 h), and AtAKT1 (by 31⁻53% at 6⁻24 h) in the roots compared with WT. This led to an increase in Na⁺ accumulation in the roots, a decrease in K⁺ uptake and transportation, and finally resulted in suppression of plant growth. AtHKT1;1 loss induced a 39⁻76% (6⁻24 h) decrease and a 27⁻32% (6⁻24 h) increase in transcripts of AtSKOR and AtHAK5, respectively, in the roots compared with WT. At the same time, 25 mM NaCl decreased the net selective transport capacity for K⁺ over Na⁺ by 92% in the athkt1;1 roots compared with the WT roots. Consequently, Na⁺ was loaded into the xylem and delivered to the shoots, whereas K⁺ transport was restricted. The results indicate that AtHKT1;1 and AtSOS1 not only mediate Na⁺ transport but also control ion uptake and the spatial distribution of Na⁺ and K⁺ by cooperatively regulating the expression levels of relevant Na⁺ and K⁺ transporter genes, ultimately regulating plant growth under salt stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Homeostase , Estresse Salino , Trocadores de Sódio-Hidrogênio/genética , Simportadores/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Canais de Potássio/genética , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores/metabolismo
18.
Biomed Res Int ; 2019: 3702783, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834261

RESUMO

Increased metabolism accelerates local acid production in cancer tissue. The mechanisms eliminating acidic waste products from human colon cancer tissue represent promising therapeutic targets for pharmacological manipulation in order to improve prognosis for the increasing number of patients with colon cancer. We sampled biopsies of human colonic adenocarcinomas and matched normal colon tissue from patients undergoing colon cancer surgery. We measured steady-state intracellular pH and rates of net acid extrusion in freshly isolated human colonic crypts based on fluorescence microscopy. Net acid extrusion was almost entirely (>95%) Na+-dependent. The capacity for net acid extrusion was increased and steady-state intracellular pH elevated around 0.5 in crypts from colon cancer tissue compared with normal colon tissue irrespective of whether they were investigated in the presence or absence of CO2/HCO3 -. The accelerated net acid extrusion from the human colon cancer tissue was sensitive to the Na+/H+-exchange inhibitor cariporide. We conclude that enhanced net acid extrusion via Na+/H+-exchange elevates intracellular pH in human colon cancer tissue.


Assuntos
Ácidos/metabolismo , Neoplasias do Colo/genética , Trocadores de Sódio-Hidrogênio/genética , Ácidos/química , Bicarbonatos/metabolismo , Dióxido de Carbono/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Guanidinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Íons/química , Íons/metabolismo , Masculino , Microscopia de Fluorescência , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia , Ativação Transcricional/genética
19.
Plant Physiol Biochem ; 139: 161-170, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30897507

RESUMO

Soil salinization is a major abiotic stress condition that affects about half of global agricultural lands. Salinity leads to osmotic shock, ionic imbalance and/or toxicity and build-up of reactive oxygen species. Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electro-neutral exchange of K⁺/Na⁺ for H⁺ and are implicated in cell expansion, development, pH/ion homeostasis and salt tolerance. Porteresia coarctata is a salt secreting halophytic wild rice that thrives in the coastal-riverine interface. P. coarctata NHX1 (PcNHXI) expression is induced by salinity in P. coarctata roots and shows high sequence identity to Oryza sativa NHX1. PcNHX1 confers hygromycin and Li+ sensitivity and Na+ tolerance transport in a yeast strain lacking sodium transport systems. Additionally, transgenic PcNHX1 expressing tobacco seedlings (PcNHX1 promoter) show significant growth advantage under increasing concentrations of NaCl and MS salts. Etiolated PcNHX1 seedlings also exhibit significantly elongated hypocotyl lengths in 100 mM NaCl. PcNHX1 expression in transgenic tobacco roots increases under salinity, similar to expression in P. coarctata roots. Under incremental salinity, transgenic lines show reduction in leaf Na+, stem specific accumulation of Na+ and K+ (unaltered Na+/K+ ratios). PcNHX1 transgenic plants also show enhanced chlorophyll content and reduced malondialdehyde (MDA) production in leaves under salinity. The above data suggests that PcNHX1 overexpression (controlled by PcNHX1p) enhances stem specific accumulation of Na+, thereby protecting leaf tissues from salt induced injury.


Assuntos
Hipocótilo/crescimento & desenvolvimento , Proteínas de Plantas/genética , Caules de Planta/metabolismo , Poaceae/genética , Trocadores de Sódio-Hidrogênio/genética , Sódio/metabolismo , Clorofila/metabolismo , Genes de Plantas/genética , Genes de Plantas/fisiologia , Malondialdeído/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Poaceae/fisiologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Tabaco
20.
Biol Chem ; 400(10): 1359-1370, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30738013

RESUMO

Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette family of proteins because it has evolved into a channel. Mutations in CFTR cause cystic fibrosis, the most common genetic disease in people of European origin. The F508del mutation is found in about 90% of patients and here we present data that suggest its main effect is on CFTR stability rather than on the three-dimensional (3D) folded state. A survey of recent cryo-electron microscopy studies was carried out and this highlighted differences in terms of CFTR conformation despite similarities in experimental conditions. We further studied CFTR structure under various phosphorylation states and with the CFTR-interacting protein NHERF1. The coexistence of outward-facing and inward-facing conformations under a range of experimental conditions was suggested from these data. These results are discussed in terms of structural models for channel gating, and favour the model where the mostly disordered regulatory-region of the protein acts as a channel plug.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Microscopia Crioeletrônica , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Conformação Proteica , Estabilidade Proteica , Proteólise , Trocadores de Sódio-Hidrogênio/metabolismo
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