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1.
Proc Natl Acad Sci U S A ; 117(41): 25517-25522, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32973095

RESUMO

Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.


Assuntos
Proteínas de Escherichia coli , Prótons , Trocadores de Sódio-Hidrogênio , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Lisina/química , Lisina/genética , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Eletricidade Estática
2.
PLoS One ; 15(8): e0237825, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822435

RESUMO

Cattle temperament is a complex and economically relevant trait. The objective of this study was to identify genomic regions and genes associated with cattle temperament. From a Brahman cattle population of 1,370 animals evaluated for temperament traits (Exit velocity-EV, Pen Score-PS, Temperament Score-TS), two groups of temperament-contrasting animals were identified based on their EV-average values ±1/2 standard deviation (SD). To be considered in the calm group, the EV of females ranged between 0.16-1.82 m/s (n = 50) and the EV of males ranged between 0.4-1.56 m/s (n = 48). Females were classified as temperamental if their EV ranged between 3.13-7.66 m/s (n = 46) and males were classified as temperamental if their EV ranged between 3.05-10.83 m/s (n = 45). Selected animals were genotyped using a total of 139,376 SNPs (GGP-HD-150K), evaluated for their association with EV. The Genome-Wide Association analysis (GWAS) identified fourteen SNPs: rs135340276, rs134895560, rs110190635, rs42949831, rs135982573, rs109393235, rs109531929, rs135087545, rs41839733, rs42486577, rs136661522, rs110882543, rs110864071, rs109722627, (P<8.1E-05), nine of them were located on intergenic regions, harboring seventeen genes, of which only ACER3, VRK2, FANCL and SLCO3A1 were considered candidate associated with bovine temperament due to their reported biological functions. Five SNPs were located at introns of the NRXN3, EXOC4, CACNG4 and SLC9A4 genes. The indicated candidate genes are implicated in a wide range of behavioural phenotypes and complex cognitive functions. The association of the fourteen SNPs on bovine temperament traits (EV, PS and TS) was evaluated; all these SNPs were significant for EV; only some were associated with PS and TS. Fourteen SNPs were associated with EV which allowed the identification of twenty-one candidate genes for Brahman temperament. From a functional point of view, the five intronic SNPs identified in this study, are candidates to address control of bovine temperament, further investigation will probe their role in expression of this trait.


Assuntos
Comportamento Animal , Bovinos/genética , Bovinos/psicologia , Emoções , Temperamento , Ceramidase Alcalina/genética , Animais , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Feminino , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Genótipo , Masculino , Transportadores de Ânions Orgânicos/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Trocadores de Sódio-Hidrogênio/genética , Proteínas de Transporte Vesicular/genética
3.
Am J Physiol Regul Integr Comp Physiol ; 319(4): R412-R427, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32755465

RESUMO

In zebrafish (Danio rerio), a specific ionocyte subtype, the H+-ATPase-rich (HR) cell, is presumed to be a significant site of transepithelial Na+ uptake/acid secretion. During acclimation to environments differing in ionic composition or pH, ionic and acid-base regulations are achieved by adjustments to the activity level of HR cell ion transport proteins. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulations relied on measurements using homogenates derived from the whole body (larvae) or the gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers or transcriptional regulation specifically within individual ionocytes. The goal of the present study was to use fluorescence-activated cell sorting to separate the HR cells from other cellular subpopulations to facilitate the measurement of gene expression of HR cell-specific transporters and enzymes from larvae exposed to low pH (pH 4.0) or low Na+ (5 µM) conditions. The data demonstrate that treatment of larvae with acidic water for 4 days postfertilization caused cell-specific increases in H+-ATPase (atp6v1aa), ca17a, ca15a, nhe3b, and rhcgb mRNA in addition to increases in mRNA linked to cell proliferation. In fish exposed to low Na+, expression of nhe3b and rhcgb was increased owing to HR cell-specific regulation and elevated numbers of HR cells. Thus, the results of this study demonstrate that acclimation to low pH or low Na+ environmental conditions is facilitated by HR cell-specific transcriptional control and by HR cell proliferation.


Assuntos
Transporte de Íons/genética , ATPases Translocadoras de Prótons/genética , Trocadores de Sódio-Hidrogênio/genética , Água , Proteínas de Peixe-Zebra/genética , Animais , Brânquias/metabolismo , Concentração de Íons de Hidrogênio , ATPases Translocadoras de Prótons/metabolismo , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
4.
Nat Cell Biol ; 22(7): 815-827, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601373

RESUMO

Lysosomes serve as cellular degradation and signalling centres that coordinate metabolism in response to intracellular cues and extracellular signals. Lysosomal capacity is adapted to cellular needs by transcription factors, such as TFEB and TFE3, which activate the expression of lysosomal and autophagy genes. Nuclear translocation and activation of TFEB are induced by a variety of conditions such as starvation, lysosome stress and lysosomal storage disorders. How these various cues are integrated remains incompletely understood. Here, we describe a pathway initiated at the plasma membrane that controls lysosome biogenesis via the endocytic regulation of intracellular ion homeostasis. This pathway is based on the exo-endocytosis of NHE7, a Na+/H+ exchanger mutated in X-linked intellectual disability, and serves to control intracellular ion homeostasis and thereby Ca2+/calcineurin-mediated activation of TFEB and downstream lysosome biogenesis in response to osmotic stress to promote the turnover of toxic proteins and cell survival.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Membrana Celular/metabolismo , Endocitose , Lisossomos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Clatrina/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Trocadores de Sódio-Hidrogênio/genética
5.
PLoS One ; 15(7): e0236511, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32722717

RESUMO

The severe side effects of chemosynthetic anti-diarrhea drugs have created an interest in low-toxic alternative plant-derived compounds. FengLiao consists of Polygonum hydropiper Linn. and Daphniphyllum calycinum Bench., and is widely used in China to treat diarrhea due to low levels of toxicity. In this study, the effects of FengLiao were analyzed in a castor oil-induced diarrhea model, using the anti-diarrhea drug, loperamide, as the positive control. The effects were evaluated using stool characteristics and the expression levels of various diarrhea-related factors in the jejunum and liver, as well as changes in the microbiota of the jejunum. The symptoms of diarrhea and stool consistency were improved through FengLiao and loperamide treatment. Furthermore, FengLiao down-regulated alpha 1-acid glycoprotein (AGP) and C-reactive protein (CRP) levels, and up-regulated transferrin (TRF) mRNA levels in the liver, and down-regulated Aquaporin 3 (AQP3) and Na+/H+ exchanger isoform 8 (NHE8) expression in the epithelial cells of the jejunum. It also increased the relative abundance of Bifidobacterium, Aerococcus, Corynebacterium_1 and Pseudomonas, and lowered the Firmicutes/Bacteroidetes (F/B) ratio, which maintained the balance between immunity and intestinal health. Taken together, FengLiao alleviated castor oil-induced diarrhea by altering gut microbiota, and levels of jejunum epithelial transport proteins and acute phase proteins.


Assuntos
Proteínas da Fase Aguda/genética , Aquaporinas/genética , Diarreia/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/genética , Animais , Óleo de Rícino/toxicidade , Daphniphyllum/química , Diarreia/genética , Diarreia/microbiologia , Medicamentos de Ervas Chinesas/uso terapêutico , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Jejuno/microbiologia , Camundongos , Polygonum/química
6.
J Plant Physiol ; 251: 153217, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32574916

RESUMO

F-box protein, one of the building blocks of the SCF complex, functions in substrate recognition of the SCF subtype of E3 ubiquitin ligase. However, the role of F-box protein in salt stress is largely elusive in plants. Here, we report the characterization of an Arabidopsis salt-tolerant mutant est1 with significantly reduced sodium content and higher Na+/H+ antiporter activity after NaCl treatment compared to the wild-type. Over-expression of EST1 resulted in increased sensitivity to salt stress, suggesting that EST1 may act as a negative regulator for salt tolerance in Arabidopsis. EST1 encodes an F-box protein, which interacts with ASK4, ASK14, and ASK18, and is likely targeted to the endoplasmic reticulum. In addition, EST1 interacts with MKK4 and negatively regulates MKK4 protein levels and the activity of the plasma membrane Na+/H+ antiporter. Our findings demonstrate the existence of an EST1-MKK4 module that mediates salt sensitivity by regulating the activity of the plasma membrane Na+/H+ antiporter. These results provide important information for engineering salt-tolerant crops.


Assuntos
Arabidopsis/fisiologia , Proteínas F-Box/genética , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Trocadores de Sódio-Hidrogênio/genética , Arabidopsis/genética , Membrana Celular , Proteínas F-Box/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-32428567

RESUMO

To understand the role of 14-3-3 beta/alpha in hypoosmotic regulation of turbot (Scophthalmus maximus), we characterized the 14-3-3 beta/alpha gene and analyzed the tissue distribution and its gene transcriptional patterns in the main expressed tissues under low salt stress. The 14-3-3 beta/alpha cDNA is 892 bp in length, incorporating an ORF of 774 bp with a putative primary structure of 257 residues. The deduced amino acid sequences shared highly conserved structures with other eukaryotes. Quantitative real-time PCR results showed that the 14-3-3 beta/alpha transcripts were widely expressed in various tissues of turbot, with most abundant in the gill (P < .05), to a lesser extent in the kidney, intestine, brain and spleen, and at low levels in the pituitary and other tissues examined. And the expression of turbot 14-3-3 beta/alpha exhibited a trend of increasing first and then decreasing with the time of stress under low salt stress, and the highest value appeared in 12 h (P < .05). After injecting different concentrations of dsRNA, the mRNA expression of 14-3-3 gene decreased significantly during the monitoring period, and the best interference effect was achieved 12 h after injecting 4 µg/g dsRNA. For the first time, the gene was silenced in fish by intramuscular injection of dsRNA. It also provides a new and effective way to study gene function at the individual level. Moreover, the mRNA interference of 14-3-3 beta/alpha would cause changes in the expression of several ion channel proteins, for example, the decrease of Na+-K+-ATPase and Na+-H+-exchanger and the increase of CFTR. As a result, 14-3-3 beta/alpha appears to be an important molecular regulator for osmosensory signal transduction in gill of turbot.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Linguados/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Inativação Gênica , Brânquias/metabolismo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Pressão Osmótica/fisiologia , Hipófise/metabolismo , Interferência de RNA , Alinhamento de Sequência , Trocadores de Sódio-Hidrogênio/genética , ATPase Trocadora de Sódio-Potássio/genética , Baço/metabolismo , Distribuição Tecidual
8.
BMC Plant Biol ; 20(1): 147, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32268879

RESUMO

BACKGROUND: Plant Na+/H+ antiporters (NHXs) are membrane-localized proteins that maintain cellular Na+/K+ and pH homeostasis. Considerable evidence highlighted the critical roles of NHX family in plant development and salt response; however, NHXs in cotton are rarely studied. RESULTS: The comprehensive and systematic comparative study of NHXs in three Gossypium species was performed. We identified 12, 12, and 23 putative NHX proteins from G. arboreum, G. raimondii, and G. hirsutum, respectively. Phylogenetic study revealed that repeated polyploidization of Gossypium spp. contributed to the expansion of NHX family. Gene structure analysis showed that cotton NHXs contain many introns, which will lead to alternative splicing and help plants to adapt to high salt concentrations in soil. The expression changes of NHXs indicate the possible differences in the roles of distinct NHXs in salt response. GhNHX1 was proved to be located in the vacuolar system and intensively induced by salt stress in cotton. Silencing of GhNHX1 resulted in enhanced sensitivity of cotton seedlings to high salt concentrations, which suggests that GhNHX1 positively regulates cotton tolerance to salt stress. CONCLUSION: We characterized the gene structure, phylogenetic relationship, chromosomal location, and expression pattern of NHX genes from G. arboreum, G. raimondii, and G. hirsutum. Our findings indicated that the cotton NHX genes are regulated meticulously and differently at the transcription level with possible alternative splicing. The tolerance of plants to salt stress may rely on the expression level of a particular NHX, rather than the number of NHXs in the genome. This study could provide significant insights into the function of plant NHXs, as well as propose promising candidate genes for breeding salt-resistant cotton cultivars.


Assuntos
Gossypium/metabolismo , Tolerância ao Sal , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Gossypium/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Trocadores de Sódio-Hidrogênio/genética
9.
J Biol Chem ; 295(20): 7075-7095, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32277048

RESUMO

Genetic screening has identified numerous variants of the endosomal solute carrier family 9 member A6 (SLC9A6)/(Na+,K+)/H+ exchanger 6 (NHE6) gene that cause Christianson syndrome, a debilitating X-linked developmental disorder associated with a range of neurological, somatic, and behavioral symptoms. Many of these variants cause complete loss of NHE6 expression, but how subtler missense substitutions or nonsense mutations that partially truncate its C-terminal cytoplasmic regulatory domain impair NHE6 activity and endosomal function are poorly understood. Here, we describe the molecular and cellular consequences of six unique mutations located in the N-terminal cytoplasmic segment (A9S), the membrane ion translocation domain (L188P and G383D), and the C-terminal regulatory domain (E547*, R568Q, and W570*) of human NHE6 that purportedly cause disease. Using a heterologous NHE6-deficient cell expression system, we show that the biochemical, catalytic, and cellular properties of the A9S and R568Q variants were largely indistinguishable from those of the WT transporter, which obscured their disease significance. By contrast, the L188P, G383D, E547*, and W570* mutants exhibited variable deficiencies in biosynthetic post-translational maturation, membrane sorting, pH homeostasis in recycling endosomes, and cargo trafficking, and they also triggered apoptosis. These findings broaden our understanding of the molecular dysfunctions of distinct NHE6 variants associated with Christianson syndrome.


Assuntos
Ataxia , Endossomos , Epilepsia , Doenças Genéticas Ligadas ao Cromossomo X , Deficiência Intelectual , Microcefalia , Mutação de Sentido Incorreto , Transtornos da Motilidade Ocular , Trocadores de Sódio-Hidrogênio , Substituição de Aminoácidos , Animais , Ataxia/genética , Ataxia/metabolismo , Cricetinae , Endossomos/química , Endossomos/genética , Endossomos/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Transtornos da Motilidade Ocular/genética , Transtornos da Motilidade Ocular/metabolismo , Domínios Proteicos , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo
10.
Comp Biochem Physiol B Biochem Mol Biol ; 243-244: 110437, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32247057

RESUMO

Salinity is an important abiotic factor for aquatic organisms. In fish, changes in salinity affect physiological responses and alter the immune system. Takifugu rubripes is an important economic marine fish, and mechanisms of T. rubripes adaptation to salinity changes need to be further documented. In this study, a transcriptome sequencing technique was used to analyse genes that were differentially expressed in the T. rubripes gill after low-salinity stress for 30 d, and differential gene expression was further validated by quantitative real-time PCR (qPCR). After assembly, 385 differentially expressed genes (DEGs) were identified, including 182 upregulated genes and 203 downregulated genes. The DEGs were assigned to Gene Ontology (GO) classes with a total of 1647 functional terms. Most DEGs were assigned to biological process (984; 59.8%) followed by molecular function (445; 27.0%) and cellular component (218; 13.2%). Further KEGG analysis allocated 385 DEGs to 95 KEGG pathways. After q-value correction, 7 pathways (Glycolysis/Gluconeogenesis; Biosynthesis of amino acids; Carbon metabolism; Fructose and mannose metabolism; Pentose phosphate pathway; Metabolism of xenobiotics by cytochrome P450; and Glycine, serine and threonine metabolism) remained significant. qPCR results indicated that the transcripts of six selected genes sharply increased after 30 d of low-salinity stress. Low-salinity stress obviously increased SLC39A6, SLC5A9, NKAα1, CYP1A1, CYP1B1, and GSTA expression. In contrast, the genes encoding Aldoaa, GPI, FBP2 and GAPDH exhibited downregulation. In addition, three solute carrier (SLC) genes selected from the DEGs were further studied for differential expression patterns after low-salinity exposure, and the results showed that the SLCs were upregulated in T. rubripes after 72 h of low-salinity exposure. This investigation provides data for understanding the molecular mechanisms of fish responses to low-salinity stress and provides a reference for rationally setting salinity levels in aquaculture.


Assuntos
Estresse Salino/genética , Transdução de Sinais/genética , Takifugu/metabolismo , Transcriptoma/genética , Aclimatação/genética , Aminoácidos/metabolismo , Animais , Frutose/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Ontologia Genética , Gluconeogênese/genética , Glicólise/genética , Manose/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Takifugu/genética
11.
Mol Cell Biochem ; 468(1-2): 13-20, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32130622

RESUMO

Mammalian Na+/H+ exchanger type I isoform (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH (pHi) by removing one intracellular proton in exchange for one extracellular sodium ion. Abnormal activity of the protein occurs in cardiovascular disease and breast cancer. The purpose of this study is to examine the role of negatively charged amino acids of extracellular loop 3 (EL3) in the activity of the NHE protein. We mutated glutamic acid 217 and aspartic acid 226 to alanine, and to glutamine and asparagine, respectively. We examined effects on expression levels, cell surface targeting and activity of NHE1, and also characterized affinity for extracellular sodium and lithium ions. Individual mutation of these amino acids had little effect on protein function. However, mutation of both these amino acids together impaired transport, decreasing the Vmax for both Na+ and Li+ ions. We suggested that amino acids E217 and D226 form part of a negatively charged coordination sphere, which facilitates cation transport in the NHE1 protein.


Assuntos
Aminoácidos Acídicos/química , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Aminoácidos Acídicos/genética , Animais , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Cricetulus , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Domínios Proteicos/genética , Trocador 1 de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética
12.
BMC Cancer ; 20(1): 202, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164629

RESUMO

BACKGROUND: Na+/H+ exchanger regulatory factor 1 (NHERF1) is an important scaffold protein participates in the modulation of a variety of intracellular signal pathways. NHERF1 was able to enhance the effects of chemo-drugs in breast and cervical cancer cells. Anaplastic lymphoma kinase (ALK) fusion mutations are validated molecules targeted therapy in lung cancers, where crizotinib can be used as the specific inhibitor to suppress tumor progression. However, due to the less frequent occurrence of ALK mutations and the complexity for factors to determine drug responses, the genes that could alter crizotinib sensitivity are unclear. METHODS: Both ALK-translocated and ALK-negative lung adenocarcinoma specimens in tissue sections were collected for immunohistochemistry. The possible mechanisms of NHERF1 and its role in the cell sensitivity to crizotinib were investigated using an ALK-positive and crizotinib-sensitive lung adenocarcinoma cell line H3122. Either a NHERF1 overexpression vector or agents for NHERF1 knockdown was used for crizotinib sensitivity measures, in association with cell viability and apoptosis assays. RESULTS: The expression level of NHERF1 in ALK-translocated NSCLC was significantly higher than that in other lung cancer tissues. NHERF1 expression in ALK positive lung cancer cells was regulated by ALK activities, and was in return able to alter the sensitivity to crizotinib. The function of NHERF1 to influence crizotinib sensitivity was depending on its subcellular distribution in cytosol instead of its nucleus localized form. CONCLUSION: Ectopically overexpressed NHERF1 could be a functional protein for consideration to suppress lung cancers. The determination of NHERF1 levels in ALK positive NSCLC tissues might be useful to predict crizotinib resistance, especially by distinguishing cytosolic or nuclear localized NHERF1 for the overexpressed molecules.


Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Crizotinibe/farmacologia , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Regulação para Cima , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Fosfoproteínas/genética , Trocadores de Sódio-Hidrogênio/genética
13.
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
14.
BMC Genomics ; 21(1): 131, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32033524

RESUMO

BACKGROUND: Seashore paspalum (Paspalum vaginatum), a halophytic warm-seasoned perennial grass, is tolerant of many environmental stresses, especially salt stress. To investigate molecular mechanisms underlying salinity tolerance in seashore paspalum, physiological characteristics and global transcription profiles of highly (Supreme) and moderately (Parish) salinity-tolerant cultivars under normal and salt stressed conditions were analyzed. RESULTS: Physiological characterization comparing highly (Supreme) and moderately (Parish) salinity-tolerant cultivars revealed that Supreme's higher salinity tolerance is associated with higher Na+ and Ca2+ accumulation under normal conditions and further increase of Na+ under salt-treated conditions (400 mM NaCl), possibly by vacuolar sequestration. Moreover, K+ retention under salt treatment occurs in both cultivars, suggesting that it may be a conserved mechanism for prevention of Na+ toxicity. We sequenced the transcriptome of the two cultivars under both normal and salt-treated conditions (400 mM NaCl) using RNA-seq. De novo assembly of about 153 million high-quality reads and identification of Open Reading Frames (ORFs) uncovered a total of 82,608 non-redundant unigenes, of which 3250 genes were identified as transcription factors (TFs). Gene Ontology (GO) annotation revealed the presence of genes involved in diverse cellular processes in seashore paspalum's transcriptome. Differential expression analysis identified a total of 828 and 2222 genes that are responsive to high salinity for Supreme and Parish, respectively. "Oxidation-reduction process" and "nucleic acid binding" are significantly enriched GOs among differentially expressed genes in both cultivars under salt treatment. Interestingly, compared to Parish, a number of salt stress induced transcription factors are enriched and show higher abundance in Supreme under normal conditions, possibly due to enhanced Ca2+ signaling transduction out of Na+ accumulation, which may be another contributor to Supreme's higher salinity tolerance. CONCLUSION: Physiological and transcriptome analyses of seashore paspalum reveal major molecular underpinnings contributing to plant response to salt stress in this halophytic warm-seasoned perennial grass. The data obtained provide valuable molecular resources for functional studies and developing strategies to engineer plant salinity tolerance.


Assuntos
Paspalum/genética , Tolerância ao Sal/genética , Cálcio/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Paspalum/metabolismo , Bombas de Próton/genética , Bombas de Próton/metabolismo , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
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
16.
Sci Rep ; 10(1): 3609, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32107410

RESUMO

Acid-base homeostasis is critical for normal growth, development, and hearing function. The sodium-hydrogen exchanger 6 (NHE6), a protein mainly expressed in early and recycling endosomes, plays an important role in regulating organellar pH. Mutations in NHE6 cause complex, slowly progressive neurodegeneration. Little is known about NHE6 function in the mouse cochlea. Here, we found that all NHE isoforms were expressed in wild-type (WT) mouse cochlea. Nhe6 knockout (KO) mice showed significant hearing loss compared to WT littermates. Immunohistochemistry in WT mouse cochlea showed that Nhe6 was localized in the organ of Corti (OC), spiral ganglion (SG), stria vascularis (SV), and afferent nerve fibres. The middle and the inner ears of WT and Nhe6 KO mice were not different morphologically. Given the putative role of NHE6 in early endosomal function, we examined Rab GTPase expression in early and late endosomes. We found no change in Rab5, significantly lower Rab7, and higher Rab11 levels in the Nhe6 KO OC, compared to WT littermates. Because Rabs mediate TrkB endosomal signalling, we evaluated TrkB phosphorylation in the OCs of both strains. Nhe6 KO mice showed significant reductions in TrkB and Akt phosphorylation in the OC. In addition, we examined genes used as markers of SG type I (Slc17a7, Calb1, Pou4f1, Cal2) and type II neurons (Prph, Plk5, Cacna1g). We found that all marker gene expression levels were significantly elevated in the SG of Nhe6 KO mice, compared to WT littermates. Anti-neurofilament factor staining showed axon loss in the cochlear nerves of Nhe6 KO mice compared to WT mice. These findings indicated that BDNF/TrkB signalling was disrupted in the OC of Nhe6 KO mice, probably due to TrkB reduction, caused by over acidification in the absence of NHE6. Thus, our findings demonstrated that NHEs play important roles in normal hearing in the mammalian cochlea.


Assuntos
Endossomos/metabolismo , Perda Auditiva/metabolismo , Neurônios/fisiologia , Órgão Espiral/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Gânglio Espiral da Cóclea/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Perda Auditiva/genética , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Órgão Espiral/patologia , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/genética
17.
Life Sci ; 243: 117226, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31904366

RESUMO

Hypertension is a risk factor for premature death and roughly 50% of hypertensive patients are salt-sensitive. The incidence of salt-sensitive hypertension increases with age. However, the mechanisms of salt-sensitive hypertension are not well understood. We had demonstrated decreased renal sodium­hydrogen exchanger regulatory factor 1 (NHERF1) expression in old salt-resistant F344 rats. Based on those studies we hypothesized that NHERF1 expression is required for the development of some forms of salt-sensitive hypertension. To address this hypothesis, we measured blood pressure in NHERF1 expressing salt-sensitive 4-mo and 24-mo-old male and female Fischer Brown Norway (FBN) rats male and female 18-mo-old NHERF1 knock-out (NHERF1-/-) mice and wild-type (WT) littermates on C57BL/6J background after feeding high salt (8% NaCl) diet for 7 days. Our data demonstrate that 8% salt diet increased blood pressure in both male and female 24-mo-old FBN rats but not in 4-mo-old FBN rats and in 18-mo-old male and female WT mice but not in NHERF1-/- mice. Renal dopamine 1 receptor (D1R) expression was decreased in 24-mo-old rats, compared with 4-mo-old FBN rats. However, sodium chloride cotransporter (NCC) expression increased in 24-mo-old FBN rats. In FBN rats, age had no effect on NaK ATPase α1 and NKCC2 expression. By contrast, high salt diet increased the renal expressions of NKCC2, and NCC in 24-mo-old FBN rats. High salt diet also increased NKCC2 and NCC expression in WT mice but not NHERF1-/- mice. Our data suggest that renal NHERF1 expression confers salt sensitivity with aging, associated with increased expression of sodium transporters.


Assuntos
Envelhecimento/metabolismo , Hipertensão/metabolismo , Fosfoproteínas/fisiologia , Cloreto de Sódio na Dieta/administração & dosagem , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Feminino , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Ratos , Ratos Endogâmicos F344 , Trocadores de Sódio-Hidrogênio/genética
18.
Genes (Basel) ; 11(1)2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906484

RESUMO

X-linked intellectual disability (XLID) is known to contribute up to 10% of intellectual disability (ID) in males and could explain the increased ratio of affected males observed in patients with ID. Over the past decade, next-generation sequencing has clearly stimulated the gene discovery process and has become part of the diagnostic procedure. We have performed targeted next-generation sequencing of 82 XLID genes on 61 non-related male patients with suggestive non-syndromic XLID. These patients were initially referred to the molecular genetics laboratory to exclude Fragile X Syndrome. The cohort includes 47 male patients with suggestive X-linked family history of ID meaning that they had half-brothers or maternal cousins or uncles affected; and 14 male patients with ID and affected brothers whose mothers show skewed X-inactivation. Sequencing data analysis identified 17 candidate variants in 16 patients. Seven families could be re-contacted and variant segregation analysis of the respective eight candidate variants was performed: HUWE1, IQSEC2, MAOA, MED12, PHF8, SLC6A8, SLC9A6, and SYN1. Our results show the utility of targeted next-generation sequencing in unravelling the genetic origin of XLID, especially in retrospective cases. Variant segregation and additional studies like RNA sequencing and biochemical assays also helped in re-evaluating and further classifying the genetic variants found.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Retardo Mental Ligado ao Cromossomo X/diagnóstico , Retardo Mental Ligado ao Cromossomo X/genética , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Exoma/genética , Síndrome do Cromossomo X Frágil , Genes Ligados ao Cromossomo X/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Complexo Mediador/genética , Monoaminoxidase/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Linhagem , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Estudos Retrospectivos , Trocadores de Sódio-Hidrogênio/genética , Fatores de Transcrição , Sequenciamento Completo do Exoma/métodos
19.
Artigo em Inglês | MEDLINE | ID: mdl-31928685

RESUMO

Salt stress is one of the major environmental factors limiting crop productivity. Although physiological and molecular characterization of salt stress response in plants has been the focus for many years, research on transporters for sodium ion (Na+) uptake, translocation and accumulation in plants, particularly in food crops like rice is limited. In this study, we functionally identified an uncharacterized sodium ion transporter named OsNHAD which encodes a putative Na+ / H+ antiporter in rice. Homology search shows its close relation to the Arabidopsis Na+/H+ antiporter AtNHD1 with 72.74% identity of amino acids. OsNHAD transcripts mainly express in leaves and are induced by Na+ stress. Confocal laser scanning microscopy analysis of OsNHAD::GFP fusion in tobacco leaves shows that OsNHAD resides in the chloroplast envelop. Knock-down of OsNHAD by RNA interference led to increased rice sensitivity to Na+, manifested by stunted plant growth, enhanced cellular damage, reduced PSII activity and changed chloroplast morphology. Mutation of OsNHAD also resulted in accumulation of more Na+ in chloroplasts and in shoots as well, suggesting that OsNHAD is involved in mediating efflux and detoxification of Na+ but does not affect K+ accumulation in plant cells. Complementation test reveals that OsNHAD was able to functionally restore the Arabidopsis mutant atnhd1-1 growth phenotype. These results suggest that OsNHAD possibly mediates homeostasis of sodium ions in the subcellular compartments and tissues of the plants when challenged to salt stress.


Assuntos
Cloroplastos/metabolismo , Oryza/fisiologia , Proteínas de Plantas/genética , Estresse Salino/genética , Trocadores de Sódio-Hidrogênio/genética , Oryza/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
20.
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
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