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1.
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
2.
Plant Mol Biol ; 103(4-5): 545-560, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32504260

RESUMO

KEY MESSAGE: OsGTγ-2, a trihelix transcription factor, is a positive regulator of rice responses to salt stress by regulating the expression of ion transporters. Salinity stress seriously restricts rice growth and yield. Trihelix transcription factors (GT factors) specifically bind to GT elements and play a diverse role in plant morphological development and responses to abiotic stresses. In our previous study, we found that the GT-1 element (GAAAAA) is a key element in the salinity-induced OsRAV2 promoter. Here, we identified a rice OsGTγ family member, OsGTγ-2, which directly interacted with the GT-1 element in the OsRAV2 promoter. OsGTγ-2 specifically targeted the nucleus, was mainly expressed in roots, sheathes, stems and seeds, and was induced by salinity, osmotic and oxidative stresses and abscisic acid (ABA). The seed germination rate, seedling growth and survival rate under salinity stress was improved in OsGTγ-2 overexpressing lines (PZmUbi::OsGTγ-2). In contrast, CRISPR/Cas9-mediated OsGTγ-2 knockout lines (osgtγ-2) showed salt-hypersensitive phenotypes. In response to salt stress, different Na+ and K+ acclamation patterns were observed in PZmUbi::OsGTγ-2 lines and osgtγ-2 plants were observed. The molecular mechanism of OsGTγ-2 in rice salt adaptation was also investigated. Several major genes responsible for ion transporting, such as the OsHKT2; 1, OsHKT1; 3 and OsNHX1 were transcriptionally regulated by OsGTγ-2. A subsequent yeast one-hybrid assay and EMSA indicated that OsGTγ-2 directly interacted with the promoters of OsHKT2; 1, OsNHX1 and OsHKT1; 3. Taken together, these results suggest that OsGTγ-2 is an important positive regulator involved in rice responses to salt stress and suggest a potential role for OsGTγ-2 in regulating salinity adaptation in rice.


Assuntos
Aclimatação/fisiologia , Proteínas de Ligação a DNA/metabolismo , Oryza/fisiologia , Estresse Salino/fisiologia , Tolerância ao Sal/genética , Fatores de Transcrição/metabolismo , Ácido Abscísico/metabolismo , Aclimatação/genética , Adaptação Fisiológica , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/crescimento & desenvolvimento , Desenvolvimento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Salinidade , Plântula/genética , Sementes/metabolismo , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico/genética , Simportadores/metabolismo , Fatores de Transcrição/genética
3.
J Neurosci ; 40(26): 4972-4980, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32430294

RESUMO

Synaptic vesicles need to be recycled and refilled rapidly to maintain high-frequency synaptic transmission. However, little is known about the control of neurotransmitter transport into synaptic vesicles, which determines the contents of synaptic vesicles and the strength of synaptic transmission. Here, we report that Na+ substantially accumulated in the calyx of Held terminals of juvenile mice of either sex during high-frequency spiking. The activity-induced elevation of cytosolic Na+ activated vesicular Na+/H+ exchanger, facilitated glutamate loading into synaptic vesicles, and increased quantal size of asynchronous released vesicles but did not affect the vesicle pool size or release probability. Consequently, presynaptic Na+ increased the EPSCs and was required to maintain the reliable high-frequency signal transmission from the presynaptic calyces to the postsynaptic medial nucleus of the trapezoid body (MNTB) neurons. Blocking Na+/H+ exchange activity decreased the postsynaptic current and caused failures in postsynaptic firing. Therefore, during high-frequency synaptic transmission, when large amounts of glutamate are released, Na+ accumulated in the terminals, activated vesicular Na+/H+ exchanger, and regulated glutamate loading as a function of the level of vesicle release.SIGNIFICANCE STATEMENT Auditory information is encoded by action potentials (APs) phase-locked to sound frequency at high rates. A large number of synaptic vesicles are released during high-frequency synaptic transmission; accordingly, synaptic vesicles need to be recycled and refilled rapidly. We have recently found that a Na+/H+ exchanger expressed on synaptic vesicles promotes vesicle filling with glutamate. Here, we showed that when a large number of synaptic vesicles are released during high-frequency synaptic transmission, Na+ accumulates in axon terminals and facilitates glutamate uptake into synaptic vesicles. Na+ thus accelerates vesicle replenishment and sustains reliable synaptic transmission.


Assuntos
Ácido Glutâmico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/fisiologia , Sódio/metabolismo
5.
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
6.
Cardiovasc Diabetol ; 19(1): 46, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32264868

RESUMO

BACKGROUND: The clear evidence of cardiovascular benefits in cardiovascular outcome trials of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in type 2 diabetes might suggest an effect on atherosclerotic plaque vulnerability and/or thrombosis, in which myeloid angiogenic cells (MAC) and platelets (PLT) are implicated. We tested the effects of SGLT2i on inflammation and oxidant stress in a model of stearic acid (SA)-induced lipotoxicity in MAC and on PLT activation. The possible involvement of the Na+/H+ exchanger (NHE) was also explored. METHOD: MAC and PLT were isolated from peripheral blood of healthy subjects and incubated with/without SGLT2i [empagliflozin (EMPA) and dapagliflozin (DAPA) 1-100 µM] to assess their effects on SA (100 µM)-induced readouts of inflammation, oxidant stress and apoptosis in MAC and on expression of PLT activation markers by flow-cytometry after ADP-stimulation. Potential NHE involvement was tested with amiloride (aspecific NHE inhibitor) or cariporide (NHE1 inhibitor). Differences among culture conditions were identified using one-way ANOVA or Friedman test. RESULTS: NHE isoforms (1,5-9), but not SGLT2 expression, were expressed in MAC and PLT. EMPA and DAPA (100 µM) significantly reduced SA-induced inflammation (IL1ß, TNFα, MCP1), oxidant stress (SOD2, TXN, HO1), but not apoptosis in MAC. EMPA and DAPA (both 1 µM) reduced PLT activation (CD62p and PAC1 expression). SGLT2i effects were mimicked by amiloride, and only partially by cariporide, in MAC, and by both inhibitors in PLT. CONCLUSIONS: EMPA and DAPA ameliorated lipotoxic damage in stearate-treated MAC, and reduced ADP-stimulated PLT activation, potentially via NHE-inhibition, thereby pointing to plaque stabilization and/or thrombosis inhibition as potential mechanism(s) involved in SGLT2i-mediated cardiovascular protection.


Assuntos
Difosfato de Adenosina/farmacologia , Compostos Benzidrílicos/farmacologia , Plaquetas/efeitos dos fármacos , Células Progenitoras Endoteliais/efeitos dos fármacos , Glucosídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Transportador 2 de Glucose-Sódio/metabolismo , Ácidos Esteáricos/toxicidade , Apoptose/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/patologia , Células Cultivadas , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Humanos , Mediadores da Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo
7.
Am J Physiol Renal Physiol ; 318(6): F1377-F1390, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308020

RESUMO

Ste20-like kinase SLK is critical for embryonic development and may play an important role in wound healing, muscle homeostasis, cell migration, and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria and damage to podocytes as they age. The present study addressed the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3- to 4-mo-old control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin, and podocalyxin. Glomeruli of adriamycin-treated SLK KO mice showed diffuse increases in the matrix and sclerosis as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, Na+/H+ exchanger regulatory factor 2, and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation as well as disruption of the cytoskeleton and podocyte apical membrane structure.


Assuntos
Citoesqueleto de Actina/enzimologia , Doxorrubicina , Glomerulosclerose Segmentar e Focal/enzimologia , Nefrose/enzimologia , Podócitos/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Citoesqueleto de Actina/patologia , Actinas/metabolismo , Albuminúria/induzido quimicamente , Albuminúria/enzimologia , Albuminúria/genética , Animais , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Nefrose/induzido quimicamente , Nefrose/genética , Nefrose/patologia , Fosfoproteínas/metabolismo , Fosforilação , Podócitos/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
8.
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
9.
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
10.
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
11.
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
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.
Circ Res ; 126(5): 644-659, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31997705

RESUMO

RATIONALE: Previous studies identified a defect in Ang III (angiotensin III [des-aspartyl1-angiotensin II])-elicited AT2R (Ang type-2 receptor)-mediated natriuresis in renal proximal tubule cells of spontaneously hypertensive rats (SHR). OBJECTIVE: This study aimed to delineate in prehypertensive SHR kidneys the receptor or postreceptor defect causing impaired AT2R signaling and renal sodium (Na+) retention by utilizing the selective AT2R agonist compound-21 (C-21). METHODS AND RESULTS: Female 4-week-old Wistar Kyoto and SHR rats were studied after 24-hour systemic AT1R (Ang II type-1 receptor) blockade. Left kidneys received 30-minute renal interstitial infusions of vehicle followed by C-21 (20, 40, and 60 ng/[kg·min], each dose 30 minutes). Right kidneys received vehicle infusions. In Wistar Kyoto, C-21 dose-dependently increased urine Na+ excretion from 0.023±0.01 to 0.064±0.02, 0.087±0.01, and 0.089±0.01 µmol/min (P=0.008, P<0.0001, and P<0.0001, respectively) and renal interstitial fluid levels of AT2R downstream signaling molecule cGMP (cyclic guanosine 3',5' monophosphate) from 0.91±0.3 to 3.1±1.0, 5.9±1.2 and 5.3±0.5 fmol/mL (P=nonsignificant, P<0.0001, and P<0.0001, respectively). In contrast, C-21 did not increase urine Na+ excretion or renal interstitial cGMP in SHR. Mean arterial pressure was slightly higher in SHR but within the normotensive range and unaffected by C-21. In Wistar Kyoto, but not SHR, C-21 induced AT2R translocation to apical plasma membranes of renal proximal tubule cells, internalization/inactivation of NHE-3 (sodium-hydrogen exchanger-3) and Na+/K+ATPase (sodium-potassium-atpase) and phosphorylation of AT2R-cGMP downstream signaling molecules Src (Src family kinase), ERK (extracellular signal-related kinase), and VASP (vasodilator-stimulated phosphoprotein). To test whether cGMP could bypass the natriuretic defect in SHR, we infused 8-bromo-cGMP. This restored natriuresis, Na+ transporter internalization/inactivation, and Src and VASP phosphorylation, but not apical plasma membrane AT2R recruitment. In contrast, 8-bromo-cAMP administration had no effect on natriuresis or AT2R recruitment in SHR. CONCLUSIONS: The results demonstrate a primary renal proximal tubule cell AT2R natriuretic defect in SHR that may contribute to the development of hypertension. Since the defect is abrogated by exogenous intrarenal cGMP, the renal cGMP pathway may represent a viable target for the treatment of hypertension. Visual Overview: An online visual overview is available for this article.


Assuntos
Hipertensão/metabolismo , Túbulos Renais Proximais/metabolismo , Natriurese , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Moléculas de Adesão Celular/metabolismo , GMP Cíclico/metabolismo , Líquido Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Hipertensão/genética , Túbulos Renais Proximais/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Transporte Proteico , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Quinases da Família src/metabolismo
14.
Nat Commun ; 11(1): 186, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924762

RESUMO

Sodium (Na+) toxicity is one of the major damages imposed on crops by saline-alkaline stress. Here we show that natural maize inbred lines display substantial variations in shoot Na+ contents and saline-alkaline (NaHCO3) tolerance, and reveal that ZmNSA1 (Na+ Content under Saline-Alkaline Condition) confers shoot Na+ variations under NaHCO3 condition by a genome-wide association study. Lacking of ZmNSA1 promotes shoot Na+ homeostasis by increasing root Na+ efflux. A naturally occurred 4-bp deletion decreases the translation efficiency of ZmNSA1 mRNA, thus promotes Na+ homeostasis. We further show that, under saline-alkaline condition, Ca2+ binds to the EF-hand domain of ZmNSA1 then triggers its degradation via 26S proteasome, which in turn increases the transcripts levels of PM-H+-ATPases (MHA2 and MHA4), and consequently enhances SOS1 Na+/H+ antiporter-mediated root Na+ efflux. Our studies reveal the mechanism of Ca2+-triggered saline-alkaline tolerance and provide an important gene target for breeding saline-alkaline tolerant maize varieties.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Tolerância ao Sal/fisiologia , Sódio/metabolismo , Zea mays/fisiologia , Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica de Plantas , Estudo de Associação Genômica Ampla , Homeostase , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Tolerância ao Sal/genética , Cloreto de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Zea mays/genética
15.
PLoS Pathog ; 16(1): e1008295, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31971989

RESUMO

The HECT domain E3 ubiquitin ligase E6AP (UBE3A) is critical for the development of human papillomavirus (HPV) associated cancers, the neurodevelopment disorder Angelman Syndrome, and some cases of autism spectrum disorders. How E6AP recognizes its cellular targets and how its ubiquitin ligase activity is triggered remain poorly understood, and HPV E6 proteins are models for these processes. We examined diverse E6 proteins from human and non-human papillomaviruses and identified two different modes of interaction between E6 and E6AP. In Type I interactions, E6 can interact directly with the LXXLL peptide motif alone of E6AP (isolated from the rest of E6AP), and then recruit cellular substrates such as p53. In Type II interactions, E6 proteins require additional auxiliary regions of E6AP in either the amino terminus or in the carboxy-terminal HECT domain to interact with the LXXLL peptide motif of E6AP. A region of E6AP amino-terminal to the LXXLL peptide motif both augments association with E6 proteins and is required for E6 proteins to trigger ubiquitin ligase activity in the carboxy-terminal HECT ubiquitin ligase domain of E6AP. In Type I interactions, E6 can associate with E6AP and recruit p53, but a Type II interaction is required for the degradation of p53 or NHERF1. Interestingly, different E6 proteins varied in E6AP auxiliary regions that contributed to enhanced association, indicating evolutionary drift in the formation of Type II interactions. This classification of E6-E6AP interaction types and identification of a region in the E6AP amino terminus that is important for both E6 association and stimulation of ubiquitin ligase activity will inform future structural data of the E6-E6AP complex and future studies aiming to interfere with the activity of the E6-E6AP complex.


Assuntos
Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/metabolismo , Infecções por Papillomavirus/enzimologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Humanos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética
16.
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
17.
Am J Physiol Renal Physiol ; 318(3): F804-F808, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984791

RESUMO

Na+/H+ exchange regulatory factor 1 (NHERF1), a member of a PDZ scaffolding protein family, was first identified as an organizer of membrane-bound protein complexes composed of hormone receptors, signal transduction pathways, and electrolyte and mineral transporters and channels. NHERF1 is involved in the regulation of Na+/H+ exchanger 3, Na+-dependent phosphate transporter 2a, and Na+-K+-ATPase through its ability to scaffold these transporters to the plasma membrane, allowing regulation of these protein complexes with their associated hormone receptors. Recently, NHERF1 has received increased interest in its involvement in a variety of functions, including cell structure and trafficking, tumorigenesis and tumor behavior, inflammatory responses, and tissue injury. In this review, we highlight the evidence for the expansive role of NHERF1 in cell biology and speculate on the implications for renal physiology and pathophysiology.


Assuntos
Regulação da Expressão Gênica/fisiologia , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Humanos , Neoplasias/metabolismo
18.
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
19.
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
20.
J Cell Physiol ; 235(2): 658-665, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31250444

RESUMO

Glioma is the oneof the most prevalent primarybrain tumors. There is a variety of oxidative stresses, inflammatory pathways, apoptosis signaling, and Na+ /H + exchangers (NHEs) involved in the pathophysiology of glioma. Previous studies have indicated a relationship between NHEs and some molecular pathways in glioma. NHEs, including NHE1, NHE5, and NHE9 affect apoptosis, tumor-associated macrophage inflammatory pathways, matrix metalloproteinases, cancer-cell growth, invasion, and migration of glioma. Also, inhibition of NHEs contributes to increased survival in animal models of glioma. Limited studies, however, have assessed the relationship between NHEs and molecular pathways in glioma. This review summarizes current knowledge and evidence regarding the relationship between NHEs and glioma, and the mechanisms involved.


Assuntos
Antineoplásicos/farmacologia , Glioma/tratamento farmacológico , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trocadores de Sódio-Hidrogênio/genética
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