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1.
Biochemistry (Mosc) ; 84(4): 407-415, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228932

RESUMO

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used site-directed mutagenesis to investigate the role of amino acid residues ß139, ß158, ß189, and ß319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The ßN158L substitution produced no effect on the enzyme activity, while substitutions ßF139Y, ßF189L, and ßV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations ßF189L and ßV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues ß139, ß189, and ß319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Difosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Cinética , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Força Próton-Motriz , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Alinhamento de Sequência
2.
Aquat Toxicol ; 212: 88-97, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31077970

RESUMO

Ionocytes are specialized cells in the epidermis of embryonic zebrafish (Danio rerio) that play important roles in ion homeostasis and have functional similarities to mammalian renal cells. Here, we examined whether these cells might also share another functional similarity with renal cells, which is the presence of efflux transporter activities useful for elimination of toxic small molecules. Xenobiotic transporters (XTs), including the ATP-Binding Cassette (ABC) family, are a major defense mechanism against diffusible toxic molecules in aquatic embryos, including zebrafish, but their activity in the ionocytes has not previously been studied. Using fluorescent small molecule substrates of XT, we observed that specific populations of ionocytes uptake and efflux fluorescent small molecules in a manner consistent with active transport. We specifically identified a P-gp/ABCB1 inhibitor-sensitive efflux activity in the H+-ATPase-rich (HR) ionocytes, and show that these cells exhibit enriched expression of the ABCB gene, abcb5. The results extend our understanding of the functional significance of zebrafish ionocytes and indicate that these cells could play an important role in protection of the fish embryo from harmful small molecules.


Assuntos
Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Xenobióticos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Ânions , Transporte Biológico , Epiderme/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Mitocôndrias/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Peixe-Zebra/genética
3.
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
4.
Analyst ; 144(9): 3030-3037, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30901016

RESUMO

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.


Assuntos
Transporte Biológico/fisiologia , Corantes Fluorescentes/química , Lipossomos/metabolismo , Fosfatidiletanolaminas/química , Rodaminas/química , Animais , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Células COS , Bovinos , Linhagem Celular Tumoral , Cercopithecus aethiops , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal/métodos , Fragmentos de Peptídeos/metabolismo , Fosfatidiletanolaminas/síntese química , Fosfatidiletanolaminas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Rodaminas/síntese química , Rodaminas/metabolismo , Soroalbumina Bovina/química
5.
J Agric Food Chem ; 67(15): 4337-4345, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30865450

RESUMO

Mitochondria play an essential part in fighting against pathogen infection in the defense responses of fruits. In this study, we investigated the reactive oxygen species (ROS) production, energy metabolism, and changes of mitochondrial proteins in harvested muskmelon fruits ( Cucumis melo cv. Yujinxiang) inoculated with Trichothecium roseum. The results indicated that the fungal infection obviously induced the H2O2 accumulation in mitochondria. Enzyme activities were inhibited in the first 6 h postinoculation (hpi), including succinic dehydrogenase, cytochrome c oxidase, H+-ATPase, and Ca2+-ATPase. However, the activities of Ca2+-ATPase and H+-ATPase and the contents of intracellular adenosine triphosphate (ATP) were improved to a higher level at 12 hpi. A total of 42 differentially expressed proteins were identified through tandem mass tags-based proteomic analyses, which are mainly involved in energy metabolism, stress responses and redox homeostasis, glycolysis and tricarboxylic acid cycle, and transporter and mitochondria dysfunction. Taken together, our results suggest that mitochondria play crucial roles in the early defense responses of muskmelons against T. roseum infection through regulation of ROS production and energy metabolism.


Assuntos
Cucumis melo/metabolismo , Cucumis melo/microbiologia , Metabolismo Energético , Hypocreales/fisiologia , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Cucumis melo/enzimologia , Cucumis melo/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo
6.
Plant Sci ; 280: 408-415, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824019

RESUMO

Using various inhibitors and scavengers we took advantage of the size of sunflower (Helianthus annuus) seeds to investigate in vivo the effects of hormones, namely abscisic acid (ABA) and ethylene (ET), and reactive oxygen species (ROS) on the polarization of dormant (D) and non-dormant (ND) embryonic seed cells using microelectrodes. Our data show that D and ND seed cells present different polarization likely due to the regulation of plasma membrane (PM) H+-ATPase activity. The data obtained after addition of hormones or ROS scavengers further suggest that ABA dependent inhibition of PM H+-ATPases could participate in dormancy maintenance and that ET-and ROS-dependent PM H+-ATPase stimulation could participate in dormancy release in sunflower seeds.


Assuntos
Helianthus/enzimologia , Dormência de Plantas , Reguladores de Crescimento de Planta/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Abscísico/metabolismo , Membrana Celular/enzimologia , Etilenos/metabolismo , Germinação , Helianthus/genética , Helianthus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia
7.
Molecules ; 24(5)2019 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857224

RESUMO

The plasma membrane H⁺-ATPase was purified from the yeast K. lactis. The oligomeric state of the H⁺-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H⁺-ATPase hexamer in both MASs as the sole/main oligomeric state-in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H⁺-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H⁺-ATPase. It is concluded that the quaternary structure of the H⁺-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer.


Assuntos
Membrana Celular/enzimologia , Kluyveromyces/enzimologia , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Western Blotting , Cromatografia em Gel , Substâncias Macromoleculares/metabolismo
8.
Cell Stress Chaperones ; 24(2): 469-473, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30737613

RESUMO

The cellular stress response (CSR) is a universal inducible reaction modulated, among others, by heat, drugs, and hormones. We aimed to investigate the role of L-thyroxine (T4) on the heat shock (HS) response in Saccharomyces cerevisiae. The CSR was evaluated by determining growth and viability of post-logarithmic phase grown yeast cultures after HS at 53 °C for 30 min. We found that long-term T4 exposure can induce a dose-dependent and Hsp90 and H+ trafficking-related thermotolerance in yeast.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Termotolerância , Tiroxina/farmacologia , Sobrevivência Celular
9.
Nat Commun ; 10(1): 626, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733444

RESUMO

Many Gram-negative bacteria, including causative agents of dysentery, plague, and typhoid fever, rely on a type III secretion system - a multi-membrane spanning syringe-like apparatus - for their pathogenicity. The cytosolic ATPase complex of this injectisome is proposed to play an important role in energizing secretion events and substrate recognition. We present the 3.3 Å resolution cryo-EM structure of the enteropathogenic Escherichia coli ATPase EscN in complex with its central stalk EscO. The structure shows an asymmetric pore with different functional states captured in its six catalytic sites, details directly supporting a rotary catalytic mechanism analogous to that of the heterohexameric F1/V1-ATPases despite its homohexameric nature. Situated at the C-terminal opening of the EscN pore is one molecule of EscO, with primary interaction mediated through an electrostatic interface. The EscN-EscO structure provides significant atomic insights into how the ATPase contributes to type III secretion, including torque generation and binding of chaperone/substrate complexes.


Assuntos
Microscopia Crioeletrônica/métodos , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/ultraestrutura , Sistemas de Secreção Tipo III/metabolismo , Sistemas de Secreção Tipo III/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Estrutura Secundária de Proteína
10.
BMC Plant Biol ; 19(1): 74, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30764771

RESUMO

BACKGROUND: Na+ extrusion from cells is important for plant growth in high saline environments. SOS1 (salt overly sensitive 1), an Na+/H+ antiporter located in the plasma membrane (PM), functions in toxic Na+ extrusion from cells using energy from an electrochemical proton gradient produced by a PM-localized H+-ATPase (AHA). Therefore, SOS1 and AHA are involved in plant adaption to salt stress. RESULTS: In this study, the genes encoding SOS1 and AHA from the halophyte Sesuvium portulacastrum (SpSOS1 and SpAHA1, respectively) were introduced together or singly into Arabidopsis plants. The results indicated that either SpSOS1 or SpAHA1 conferred salt tolerance to transgenic plants and, as expected, Arabidopsis plants expressing both SpSOS1 and SpAHA1 grew better under salt stress than plants expressing only SpSOS1 or SpAHA1. In response to NaCl treatment, Na+ and H+ in the roots of plants transformed with SpSOS1 or SpAHA1 effluxed faster than wild-type (WT) plant roots. Furthermore, roots co-expressing SpSOS1 and SpAHA1 had higher Na+ and H+ efflux rates than single SpSOS1/SpAHA1-expressing transgenic plants, resulting in the former amassing less Na+ than the latter. As seen from comparative analyses of plants exposed to salinity stress, the malondialdehyde (MDA) content was lowest in the co-transgenic SpSOS1 and SpAHA1 plants, but the K+ level was the highest. CONCLUSION: These results suggest SpSOS1 and SpAHA1 coordinate to alleviate salt toxicity by increasing the efficiency of Na+ extrusion to maintain K+ homeostasis and protect the PM from oxidative damage induced by salt stress.


Assuntos
Aizoaceae/genética , Arabidopsis/genética , ATPases Translocadoras de Prótons/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Aizoaceae/fisiologia , Arabidopsis/fisiologia , Membrana Celular/metabolismo , Expressão Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , ATPases Translocadoras de Prótons/genética , Tolerância ao Sal , Plantas Tolerantes a Sal , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética
11.
Molecules ; 24(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704145

RESUMO

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αß)3δab2. Subunits c10 or (αß)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αß)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme's rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ouro , Cinética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Nanotubos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
12.
Molecules ; 24(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717122

RESUMO

This study aimed to test the effectiveness of ethyl gallate (EG) against S. mutans biofilm formation on solid surfaces (polystyrene, glass) and acidogenicity, and to examine the effect on expression of related genes. The biofilm that is formed by S. mutans bacteria was evaluated using colorimetric assay and optical profilometry, while the pH of the biofilm growth medium was measured with microelectrode. The expression of genes encoding glucan binding protein B (gbpB), glucosyltranferases B, -C, -D (gtfB, -C, -D) and F-ATPase (atpD, atpF) was assessed using a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). It was revealed that all of the EG concentrations significantly suppressed S. mutans biofilm build-up on polystyrene and glass surfaces, and inhibited acidogenicity, in a dose-dependent manner, compared to the activity of untreated bacteria (p < 0.05). The highest concentration of EG (3.53 mM) reduced biofilm formation on polystyrene and glass surfaces by 68% and more than 91%, respectively, and prevented a decrease in pH levels by 95%. The RT-qPCR data demonstrate that the biofilm-producing bacteria treated with EG underwent significant gene expression changes involving the gtfC (a 98.6 increase in fold change), gtfB gene (a 47.5 increase in fold change) and the gbpB gene (a 13.8 increase in fold change). However, for the other genes tested (gtfD, atpD and atpF), the EG treatments did not produce significant expression change compared to the control. EG produced significant gene expression change in three genes-gtfC, gtfB, and gbpB; it has the capacity to inhibit S. mutans biofilm formation on solid surfaces (polystyrene, glass), as well as acidogenicity. Therefore, EG might be used as an antibiofilm and/or anticaries agent for oral formulations in order to reduce the prevalence of dental caries.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Ácido Gálico/análogos & derivados , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Meios de Cultura/química , Cárie Dentária/microbiologia , Cárie Dentária/prevenção & controle , Relação Dose-Resposta a Droga , Ácido Gálico/farmacologia , Vidro/química , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lectinas/genética , Lectinas/metabolismo , Testes de Sensibilidade Microbiana , Poliestirenos/química , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Streptococcus mutans/genética , Streptococcus mutans/crescimento & desenvolvimento , Streptococcus mutans/metabolismo
13.
Environ Sci Pollut Res Int ; 26(5): 4975-4986, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30604360

RESUMO

Acid rain is a global environmental problem that threatens agricultural production. Calcium (Ca), as a signal substance for physiological activities, has been known to regulate plant growth under abiotic stresses. To clarify whether calcium could be one of possible ways to alleviate the reduction caused by acid rain in agricultural production and investigate its regulating mechanism on adaptation of plants under acid rain stress, we studied the effect of exogenous Ca2+ (5 mM CaCl2) on growth of soybean at different growth stages (seedling, flowering-podding, and filling stages) as well as yield and grain quality of soybean under simulated acid rain (pH 4.5 or pH 3.0) stress. We found that the application of Ca2+ could regulate the activity of plasma membrane H+-ATPase, for mitigating the increase of ammonium and the decrease of nitrate and phosphorus in soybean roots, which mitigated the inhibition on growth and improved the yield and grain quality of soybean under simulated acid rain stress. In addition, the alleviating effect of exogenous Ca2+ on soybean was the most significant at seedling stage. The results indicate that the exogenous Ca2+ could enhance the adaptation of soybean and facilitate the recovery of soybean productivity and grain quality under simulated acid rain stress by maintaining the uptake of nitrate, ammonium, and phosphorus.


Assuntos
Chuva Ácida/análise , Cálcio/farmacologia , Soja/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Adaptação Fisiológica , Transporte Biológico , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Modelos Teóricos , Nitratos/metabolismo , Fósforo/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Soja/efeitos dos fármacos , Soja/metabolismo
14.
Plant Cell Physiol ; 60(5): 935-944, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649552

RESUMO

Brassinosteroids (BRs) are steroid phytohormones that regulate plant growth and development, and promote cell elongation at least in part via the acid-growth process. BRs have been suggested to induce cell elongation by the activating plasma membrane (PM) H+-ATPase. However, the mechanism by which BRs activate PM H+-ATPase has not been clarified. In this study, we investigated the effects of BR on hypocotyl elongation and the phosphorylation status of a penultimate residue, threonine, of PM H+-ATPase, which affects the activation, in the etiolated seedlings of Arabidopsis thaliana. Brassinolide (BL), an active endogenous BR, induced hypocotyl elongation, phosphorylation of the penultimate, threonine residue of PM H+-ATPase, and binding of the 14-3-3 protein to PM H+-ATPase in the endogenous BR-depleted seedlings. Changes in both BL-induced elongation and phosphorylation of PM H+-ATPase showed similar concentration dependency. BL did not induce phosphorylation of PM H+-ATPase in the BR receptor mutant bri1-6. In contrast, bikinin, a specific inhibitor of BIN2 that acts as a negative regulator of BR signaling, induced its phosphorylation. Furthermore, BL accumulated the transcripts of SMALL AUXIN UP RNA 9 (SAUR9) and SAUR19, which suppress dephosphorylation of the PM H+-ATPase penultimate residue by inhibiting D-clade type 2C protein phosphatase in the hypocotyls of etiolated seedlings. From these results, we conclude that BL-induced phosphorylation of PM H+-ATPase penultimate residue is mediated via the BRI1-BIN2 signaling pathway, together with the accumulation of SAURs during hypocotyl elongation.


Assuntos
Brassinosteroides/farmacologia , Membrana Celular/enzimologia , Hipocótilo/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos
15.
Nat Methods ; 16(1): 95-102, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30532082

RESUMO

It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because all Ca2+ indicators are pH sensitive, and Ca2+ transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca2+ simultaneously, CalipHluor functions as a pH-correctable Ca2+ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca2+ changes during endosomal maturation and found a surge in lumenal Ca2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca2+ accumulation-an example of a lysosome-specific Ca2+ importer in animals. By enabling the facile quantification of compartmentalized Ca2+, CalipHluor can expand the understanding of subcellular Ca2+ importers.


Assuntos
Cálcio/metabolismo , DNA/química , Endossomos/metabolismo , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Transporte de Íons , ATPases Translocadoras de Prótons/metabolismo , Transdução de Sinais
16.
Life Sci ; 218: 81-88, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30550884

RESUMO

AIMS: Idiopathic nature of male infertility disorder needs to be investigated by different horizons of molecular biology for its treatment and to device male contraceptive. Further, it can also aid in advancement of assisted reproductive technology (ART), as nowadays the failure and disquiets of ART are consistent. Herein, we have attempted to find out proteins responsible for male infertility by comparing proteome profile of sperms collected from normal control and asthenozoospermic (AS) males. MAIN METHODS: Differential proteome profiles were studied by 2-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry. The confirmation of proteome profiling results was done by western blotting and ELISA. Quantitative reverse-transcription-PCR was also performed in an independent cohort of AS and normal individuals to investigate the transcriptional regulation of proteins. KEY FINDINGS: Although seven differentially regulated proteins were identified, highpoints of the study were two proteins, TEX40 and ATP6V0A2. Lower expression of a crucial sperm motility related protein, TEX40 is reported for the first time in clinically diagnosed AS males in the present investigation. Most likely with reference to previous findings the down regulation of TEX40 leads to fewer entries of calcium ions in the sperm and lower expression of ATP6V0A2 is responsible for acrosomal de-acidification. SIGNIFICANCE: Conclusively, the down regulation of these two proteins in AS males might result in diminished sperm motility. The findings can be worthwhile for male contraception and ART management besides their use for male infertility therapy.


Assuntos
Acrossomo/metabolismo , Astenozoospermia/fisiopatologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteômica/métodos , ATPases Translocadoras de Prótons/metabolismo , Espermatozoides/metabolismo , Adulto , Astenozoospermia/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Humanos , Masculino , Prognóstico
17.
Aquat Toxicol ; 207: 72-82, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30530206

RESUMO

Waterborne ammonia is an environmental pollutant that is toxic to all aquatic animals. However, ammonia induced toxicity as well as compensatory mechanisms to defend against high environmental ammonia (HEA) are not well documented at present for largemouth bass (Micropterus salmoides), a high value fish for culture and sport fisheries in the United States. To provide primary information on the sensitivity of this species to ammonia toxicity, a 96 h-LC50 test was conducted. Thereafter, responses at physiological, ion-regulatory and transcript levels were determined to get insights into the underlying adaptive strategies to ammonia toxicity. For this purpose, fish were progressively exposed to HEA (8.31 mg/L representing 25% of 96 h-LC50) for 3, 7, 14, 21 and 28 days. Temporal effects of HEA on oxygen consumption rate (MO2), ammonia and urea dynamics, plasma ions (Na+, Cl- and K+), branchial Na+/K+-ATPase (NKA) and H+-ATPase activity, muscle water content (MWC), energy store (glycogen, lipid and protein) as well as branchial mRNA expression of Rhesus (Rh) glycoproteins were assessed. Probit analysis showed that 96 h-LC50 of (total) ammonia (as NH4HCO3) at 25 °C and pH 7.8 was 33.24 mg/L. Results from sub-lethal end-points shows that ammonia excretion rate (Jamm) was strongly inhibited after 7 days of HEA, but was unaffected at 3, 14 and 21 days. At 28 days fish were able to increase Jamm efficiently and concurrently, plasma ammonia re-established to the basal level. Urea production was increased as evidenced by a considerable elevation of plasma urea, but urea excretion rate remained unaltered. Expression of Rhcg isoform (Rhcg2) mRNA was up-regulated in parallel with restored or increased Jamm, suggesting its ammonia excreting role in largemouth bass. Exposure to HEA also displayed pronounced augmentations in NKA activity, exemplified by a rise in plasma [Na+]. Furthermore, [K+], [Cl-] and MWC homeostasis were disrupted followed by recovery to the control levels. H+-ATPase activity was elevated but NKA did not appear to function preferentially as a Na+/NH4+-ATPase. From 14 days onwards MO2 was depressed, potentially an attempt towards minimizing catabolism. Glycogen content in liver and muscle were temporarily depleted, whereas a remarkable increment in protein was evident at the last exposure period. Overall, these data suggest that ammonia induced toxicity can disturb several biological processes in largemouth bass, however, it can adapt to the long-term sub-lethal ammonia concentrations by activating various components of ammonia excretory, ion-regulatory and metabolic pathways.


Assuntos
Amônia/toxicidade , Bass/fisiologia , Exposição Ambiental/análise , Adenosina Trifosfatases/metabolismo , Animais , Bass/genética , Perfilação da Expressão Gênica , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Glicoproteínas/metabolismo , Íons/sangue , Metaboloma , Músculos/metabolismo , Nitrogênio/metabolismo , Análise de Componente Principal , ATPases Translocadoras de Prótons/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Análise de Sobrevida , Fatores de Tempo , Ureia/metabolismo , Poluentes Químicos da Água/toxicidade
18.
Artigo em Inglês | MEDLINE | ID: mdl-30196154

RESUMO

The mitochondrial F1FO-ATPase, the key enzyme in cell bioenergetics, apparently works in the same way in mollusks and in mammals. We previously pointed out a raft-like arrangement in mussel gill mitochondrial membranes, which apparently distinguishes bivalve mollusks from mammals. To explore the relationship between the microenvironmental features and the enzyme activity, the physico-chemical features of mitochondrial membranes and the F1FO-ATPase activity temperature-dependence are here explored in the Manila clam (Ruditapes philippinarum). Similarly to the mussel, clam gill mitochondrial membrane lipids exhibit a high sterol content (42 mg/g protein), mainly due to phytosterols (cholesterol only attains 42% of total sterols), and abundant polyunsaturated fatty acids (PUFA) (70% of total fatty acids), especially of the n-3 family. However, the F1FO-ATPase activation energies above and below the break in the Arrhenius plot (22.1 °C) are lower than in mussel and mammalian mitochondria. Laurdan fluorescence spectroscopy analyses carried out at 10 °C, 20 °C and 30 °C on mitochondrial membranes and on lipid vesicles obtained from total lipid extracts of mitochondria, indicate a physical state without coexisting domains. This mitochondrial membrane constitution, allowed by lipid-lipid and lipidprotein interactions and involving PUFA-rich phospholipids, phytosterols (much more diversified in clams than in mussels) and proteins, enables the maintenance of a homogeneous physical state in the range 10-30 °C. Consistently, this molecular interaction network would somehow extend the temperature range of the F1FO-ATPase activity and may contribute to clam resilience to temperature changes.


Assuntos
Bivalves/fisiologia , Mudança Climática , Metabolismo dos Lipídeos , Membranas Mitocondriais/metabolismo , Modelos Biológicos , ATPases Translocadoras de Prótons/metabolismo , Animais , Bivalves/enzimologia , Bivalves/crescimento & desenvolvimento , Ativação Enzimática , Estabilidade Enzimática , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-3/química , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/química , Feminino , Temperatura Alta/efeitos adversos , Itália , Bicamadas Lipídicas , Lipossomos , Masculino , Mar Mediterrâneo , Microdomínios da Membrana/química , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Membranas Mitocondriais/química , Fitosteróis/análise , Fitosteróis/metabolismo , ATPases Translocadoras de Prótons/química , Especificidade da Espécie , Esteróis/análise , Esteróis/metabolismo
19.
J Plant Physiol ; 232: 248-256, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30537611

RESUMO

Post-germination plant growth depends on the regulation of reactive oxygen species (ROS) metabolism, spatiotemporal pH changes and Ca+2 homeostasis, whose potential integration has been studied during Vigna radiata (L.) Wilczek root growth. The dissipation of proton (H+) gradients across plasma membrane (PM) by CCCP (protonophore) and the inhibition of PM H+-ATPase by sodium orthovanadate repressed SOD (superoxide dismutase; EC 1.15.1.1) activity as revealed by spectrophotometric and native PAGE assay results. Similar results derived from treatment with DPI (NADPH oxidase inhibitor) and Tiron (O2- scavenger) denote a functional synchronization of SOD, PM H+-ATPase and NOX, as the latter two enzymes are substrate sources for SOD (H+ and O2-, respectively) and are involved in a feed-forward loop. After SOD inactivation, a decline in apoplastic H2O2 content was observed in each treatment group, emerging as a possible cause of the diminution of class III peroxidase (Prx; EC 1.11.1.7), which utilizes H2O2 as a substrate. In agreement with the pivotal role of Ca+2 in PM H+-ATPase and NOX activation, Ca+2 homeostasis antagonists, i.e., LaCl3 (Ca+2 channel inhibitor), EGTA (Ca+2 chelator) and LiCl (endosomal Ca+2 release blocker), inhibited both SOD and Prx. Finally, a drastic reduction in apoplastic OH (hydroxyl radical) concentrations (induced by each treatment, leading to Prx inhibition) was observed via fluorometric analysis. A consequential inhibition of root growth observed under each treatment denotes the importance of the orchestrated functioning of PM H+-ATPase, NOX, Cu-Zn SOD and Prx during root growth. A working model demonstrating postulated enzymatic synchronization with an intervening role of Ca+2 is proposed.


Assuntos
NADPH Oxidases/metabolismo , Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , ATPases Translocadoras de Prótons/metabolismo , Superóxido Dismutase-1/metabolismo , Vigna/enzimologia , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/fisiologia , Peroxidases/fisiologia , Proteínas de Plantas/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Superóxido Dismutase-1/fisiologia , Superóxidos/metabolismo , Vigna/crescimento & desenvolvimento
20.
Pest Manag Sci ; 75(1): 180-186, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29797423

RESUMO

BACKGROUND: Metarhizium acridum is a host-specific fungal pathogen with great potential for locust control. However, the slow killing action of M. acridum has impeded its widespread application. To enhance fungal virulence, we constructed transgenic M. acridum strains that express double-stranded (ds)RNAs targeting the genes of the F1 F0 -ATP synthase α and ß subunits in Locusta migratoria. RESULTS: The two host genes were transcriptionally suppressed in L. migratoria nymphs (instar V) infected by RNA interference (RNAi) strains targeting one or two subunit genes of the host ATP synthase, followed by reduced ATPase activity and ATP synthesis. Consequently, the RNAi strain targeting both subunit genes displayed high virulence that was 3.7-fold that in the wild-type strain. CONCLUSION: Our results demonstrate that dsRNA expression in M. acridum can cause host RNA silencing during infection and greatly enhances the fungal virulence through interference with critical host genes, highlighting a new strategy for augmentation of fungal virulence against insect pests. © 2018 Society of Chemical Industry.


Assuntos
Proteínas de Insetos/genética , Locusta migratoria/microbiologia , Metarhizium/fisiologia , Controle Biológico de Vetores , ATPases Translocadoras de Prótons/genética , RNA de Cadeia Dupla/genética , RNA Fúngico/genética , Animais , Expressão Gênica , Interações Hospedeiro-Patógeno , Controle de Insetos , Proteínas de Insetos/metabolismo , Locusta migratoria/genética , Metarhizium/genética , ATPases Translocadoras de Prótons/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Fúngico/metabolismo
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