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1.
Plant Physiol ; 187(4): 1856-1875, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-35235671

RESUMO

Plant plasma membrane H+-ATPases and Ca2+-ATPases maintain low cytoplasmic concentrations of H+ and Ca2+, respectively, and are essential for plant growth and development. These low concentrations allow plasma membrane H+-ATPases to function as electrogenic voltage stats, and Ca2+-ATPases as "off" mechanisms in Ca2+-based signal transduction. Although these pumps are autoregulated by cytoplasmic concentrations of H+ and Ca2+, respectively, they are also subject to exquisite regulation in response to biotic and abiotic events in the environment. A common paradigm for both types of pumps is the presence of terminal regulatory (R) domains that function as autoinhibitors that can be neutralized by multiple means, including phosphorylation. A picture is emerging in which some of the phosphosites in these R domains appear to be highly, nearly constantly phosphorylated, whereas others seem to be subject to dynamic phosphorylation. Thus, some sites might function as major switches, whereas others might simply reduce activity. Here, we provide an overview of the relevant transport systems and discuss recent advances that address their relation to external stimuli and physiological adaptations.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Bombas de Íon/metabolismo , Fenômenos Fisiológicos Vegetais/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Transdução de Sinais/efeitos dos fármacos , Membrana Celular/metabolismo
2.
J Integr Plant Biol ; 64(2): 268-286, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35023285

RESUMO

The plant plasma membrane (PM) H+ -ATPase is an essential enzyme controlling plant growth and development. It is an important factor in response to abiotic and biotic stresses and is subject to tight regulation. We are in demand for new sustainable natural growth regulators and as a key enzyme for regulation of transport into the plant cell the PM H+ -ATPase is a potential target for these. In this review, we have evaluated the known non-protein natural compounds with regulatory effects on the PM H+ -ATPase, focusing on their mechanism of action and their potential as biologicals/growth regulators in plant production of future sustainable agriculture.


Assuntos
Produtos Biológicos , Agricultura , Produtos Biológicos/metabolismo , Membrana Celular/metabolismo , Células Vegetais/metabolismo , ATPases Translocadoras de Prótons/metabolismo
3.
Plant J ; 104(2): 433-446, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32713048

RESUMO

Acidification of the apoplastic space facilitates cell wall loosening and is therefore a key step in cell expansion. PSY1 is a growth-promoting secreted tyrosine-sulfated glycopeptide whose receptor directly phosphorylates and activates the plasma membrane H+ -ATPase, which results in acidification and initiates cellular expansion. Although the mechanism is not clear, the Rapid Alkalinization Factor (RALF) family of small, secreted peptides inhibits the plasma membrane H+ -ATPase, leading to alkalinization of the apoplastic space and reduced growth. Here we show that treating Arabidopsis thaliana roots with PSY1 induced the transcription of genes encoding the RALF peptides RALF33 and RALFL36. A rapid burst of intracellular Ca2+ preceded apoplastic alkalinization in roots triggered by RALFs, with peptide-specific signatures. Ca2+ channel blockers abolished RALF-induced alkalinization, indicating that the Ca2+ signal is an obligatory part of the response and that it precedes alkalinization. As expected, fer mutants deficient in the RALF receptor FERONIA did not respond to RALF33. However, we detected both Ca2+ and H+ signatures in fer mutants upon treatment with RALFL36. Our results suggest that different RALF peptides induce extracellular alkalinization by distinct mechanisms that may involve different receptors.


Assuntos
Arabidopsis/metabolismo , Sinalização do Cálcio/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Glicopeptídeos/farmacologia , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Mutação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Inibidores da Bomba de Prótons/farmacologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Receptores de Peptídeos/genética , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Vanadatos/farmacologia
4.
New Phytol ; 226(3): 770-784, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31880817

RESUMO

Pathogenic fungi often target the plant plasma membrane (PM) H+ -ATPase during infection. To identify pathogenic compounds targeting plant H+ -ATPases, we screened extracts from 10 Stemphylium species for their effect on H+ -ATPase activity. We identified Stemphylium loti extracts as potential H+ -ATPase inhibitors, and through chemical separation and analysis, tenuazonic acid (TeA) as a potent H+ -ATPase inhibitor. By assaying ATP hydrolysis and H+ pumping, we confirmed TeA as a H+ -ATPase inhibitor both in vitro and in vivo. To visualize in planta inhibition of the H+ -ATPase, we treated pH-sensing Arabidopsis thaliana seedlings with TeA and quantified apoplastic alkalization. TeA affected both ATPase hydrolysis and H+ pumping, supporting a direct effect on the H+ -ATPase. We demonstrated apoplastic alkalization of A. thaliana seedlings after short-term TeA treatment, indicating that TeA effectively inhibits plant PM H+ -ATPase in planta. TeA-induced inhibition was highly dependent on the regulatory C-terminal domain of the plant H+ -ATPase. Stemphylium loti is a phytopathogenic fungus. Inhibiting the plant PM H+ -ATPase results in membrane potential depolarization and eventually necrosis. The corresponding fungal H+ -ATPase, PMA1, is less affected by TeA when comparing native preparations. Fungi are thus able to target an essential plant enzyme without causing self-toxicity.


Assuntos
Arabidopsis , Ácido Tenuazônico , Arabidopsis/metabolismo , Ascomicetos , Membrana Celular/metabolismo , ATPases Translocadoras de Prótons/metabolismo
6.
Plant J ; 80(6): 951-64, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25267325

RESUMO

Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2 and PSY1R observed might provide a general paradigm for regulation of plasma membrane proton transport by receptor kinases.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Receptores de Peptídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Citoplasma/metabolismo , Fosforilação , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , ATPases Translocadoras de Prótons/genética , Receptores de Peptídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plântula/genética , Plântula/metabolismo
7.
BMC Genomics ; 15: 441, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24906416

RESUMO

BACKGROUND: Small-secreted peptides are emerging as important components in cell-cell communication during basic developmental stages of plant cell growth and development. Plant peptide containing sulfated tyrosine 1 (PSY1) has been reported to promote cell expansion and differentiation in the elongation zone of roots. PSY1 action is dependent on a receptor PSY1R that triggers a signaling cascade leading to cell elongation. However little is known about cellular functions and the components involved in PSY1-based signaling cascade. RESULTS: Differentially expressed genes were identified in a wild type plant line and in a psy1r receptor mutant line of Arabidopsis thaliana after treatment with PSY1. Seventy-seven genes were found to be responsive to the PSY1 peptide in wild type plants while 154 genes were responsive in the receptor mutant plants. PSY1 activates the transcripts of genes involved in cell wall modification. Gene enrichment analysis revealed that PSY1-responsive genes are involved in responses to stimuli, metabolic processes and biosynthetic processes. The significant enrichment terms of PSY1-responsive genes were higher in psy1r mutant plants compared to in wild type plants. Two parallel responses to PSY1 were identified, differing in their dependency on the PSY1R receptor. Promoter analysis of the differentially expressed genes identified a light regulatory motif in some of these. CONCLUSION: PSY1-responsive genes are involved in cellular functions and stimuli responses suggesting a crosstalk between developmental cues and environmental stimuli. Possibly, two parallel responses to PSY1 exist. A motif involved in light regulation was identified in the promoter region of the differentially expressed genes. Reduced hypocotyl growth was observed in etiolated receptor mutant seedlings.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Peptídeos/metabolismo , Receptores de Peptídeos/genética , Transdução de Sinais , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas
8.
J Exp Bot ; 65(9): 2463-72, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24723394

RESUMO

Polyamines regulate a variety of cation and K(+) channels, but their potential effects on cation-transporting ATPases are underexplored. In this work, noninvasive microelectrode ion flux estimation and conventional microelectrode techniques were applied to study the effects of polyamines on Ca(2+) and H(+) transport and membrane potential in pea roots. Externally applied spermine or putrescine (1mM) equally activated eosin yellow (EY)-sensitive Ca(2+) pumping across the root epidermis and caused net H(+) influx or efflux. Proton influx induced by spermine was suppressed by EY, supporting the mechanism in which Ca(2+) pump imports 2 H(+) per each exported Ca(2+). Suppression of the Ca(2+) pump by EY diminished putrescine-induced net H(+) efflux instead of increasing it. Thus, activities of Ca(2+) and H(+) pumps were coupled, likely due to the H(+)-pump inhibition by intracellular Ca(2+). Additionally, spermine but not putrescine caused a direct inhibition of H(+) pumping in isolated plasma membrane vesicles. Spermine, spermidine, and putrescine (1mM) induced membrane depolarization by 70, 50, and 35 mV, respectively. Spermine-induced depolarization was abolished by cation transport blocker Gd(3+), was insensitive to anion channels' blocker niflumate, and was dependent on external Ca(2+). Further analysis showed that uptake of polyamines but not polyamine-induced cationic (K(+)+Ca(2+)+H(+)) fluxes were a main cause of membrane depolarization. Polyamine increase is a common component of plant stress responses. Activation of Ca(2+) efflux by polyamines and contrasting effects of polyamines on net H(+) fluxes and membrane potential can contribute to Ca(2+) signalling and modulate a variety of transport processes across the plasma membrane under stress.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Pisum sativum/metabolismo , Proteínas de Plantas/metabolismo , Poliaminas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Transporte Biológico , Membrana Celular/química , Potenciais da Membrana , Pisum sativum/química , Pisum sativum/enzimologia , Pisum sativum/genética , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética
9.
J Biol Chem ; 287(7): 4904-13, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22174420

RESUMO

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase action.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Membrana Celular/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Fosforilação/fisiologia , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
10.
Plant Cell ; 22(4): 1313-32, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20418496

RESUMO

The plasma membrane H(+)-ATPase (PM H(+)-ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H(+)-ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H(+)-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H(+)-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H(+)-ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H(+)-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H(+)-ATPase activity by J3 takes place via inactivation of the PKS5 kinase.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico HSP40/genética , Concentração de Íons de Hidrogênio , Microscopia Confocal , Mutação , Raízes de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , ATPases Translocadoras de Prótons/genética , RNA de Plantas/genética , Cloreto de Sódio/farmacologia
11.
Plant Physiol ; 156(4): 2235-43, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21685179

RESUMO

The Arabidopsis (Arabidopsis thaliana) genome encodes nine Salt Overly Sensitive3 (SOS3)-like calcium-binding proteins (SCaBPs; also named calcineurin B-like protein [CBL]) and 24 SOS2-like protein kinases (PKSs; also named as CBL-interacting protein kinases [CIPKs]). A general regulatory mechanism between these two families is that SCaBP calcium sensors activate PKS kinases by interacting with their FISL motif. In this study, we demonstrated that phosphorylation of SCaBPs by their functional interacting PKSs is another common regulatory mechanism. The phosphorylation site serine-216 at the C terminus of SCaBP1 by PKS24 was identified by liquid chromatography-quadrupole mass spectrometry analysis. This serine residue is conserved within the PFPF motif at the C terminus of SCaBP proteins. Phosphorylation of this site of SCaBP8 by SOS2 has been determined previously. We further showed that CIPK23/PKS17 phosphorylated CBL1/SCaBP5 and CBL9/SCaBP7 and PKS5 phosphorylated SCaBP1 at the same site in vitro and in vivo. Furthermore, the phosphorylation stabilized the interaction between SCaBP and PKS proteins. This tight interaction neutralized the inhibitory effect of PKS5 on plasma membrane H(+)-ATPase activity. These data indicate that SCaBP phosphorylation by their interacting PKS kinases is a critical component of the SCaBP-PKS regulatory pathway in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Sequência Conservada/genética , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
12.
PLoS Biol ; 7(6): e1000139, 2009 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-19564897

RESUMO

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H(+)-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H(+)-ATPase activity. PM H(+)-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H(+)-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H(+)-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Proteínas de Transporte/metabolismo , Estômatos de Plantas/fisiologia , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/fisiologia , Pseudomonas syringae/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Western Blotting , Proteínas de Transporte/genética , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Espectrometria de Massas , Microscopia Confocal , Mutação , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Ligação Proteica , ATPases Translocadoras de Prótons/genética , Pseudomonas syringae/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido , Virulência
13.
Cancers (Basel) ; 13(2)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466582

RESUMO

Perturbation in JAK-STAT signaling has been reported in the pathogenesis of cutaneous T cell lymphoma (CTCL). JAK3 is predominantly associated with the intra-cytoplasmic part of IL-2Rγc located in the plasma membrane of hematopoietic cells. Here we demonstrate that JAK3 is also ectopically expressed in the nucleus of malignant T cells. We detected nuclear JAK3 in various CTCL cell lines and primary malignant T cells from patients with Sézary syndrome, a leukemic variant of CTCL. Nuclear localization of JAK3 was independent of its kinase activity whereas STAT3 had a modest effect on nuclear JAK3 expression. Moreover, JAK3 nuclear localization was only weakly affected by blockage of nuclear export. An inhibitor of the nuclear export protein CRM1, Leptomycin B, induced an increased expression of SOCS3 in the nucleus, but only a weak increase in nuclear JAK3. Importantly, immunoprecipitation experiments indicated that JAK3 interacts with the nuclear protein POLR2A, the catalytic subunit of RNA Polymerase II. Kinase assays showed tyrosine phosphorylation of recombinant human Histone H3 by JAK3 in vitro-an effect which was blocked by the JAK inhibitor (Tofacitinib citrate). In conclusion, we provide the first evidence of nuclear localization of JAK3 in malignant T cells. Our findings suggest that JAK3 may have a cytokine-receptor independent function in the nucleus of malignant T cells, and thus a novel non-canonical role in CTCL.

14.
Plants (Basel) ; 9(10)2020 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-33022978

RESUMO

Phosphorous (P) is an essential macronutrient in all organisms serving various fundamental biological processes, and is one of the least available plant nutrients in the soil. The application of inorganic phosphate (Pi) fertilizers is frequent, but it has a high environmental and financial cost. Breeding crops for improved Pi use-efficiency is a promising plant-based solution to pursue a reduction of fertilizer dependency. Availability of tools for monitoring changes of plant cellular Pi concentration in real-time can contribute to advancing knowledge on the molecular basis of Pi transport and homeostasis in plants. Genetically encoded fluorescent sensors have provided new insight on cellular processes. Here, we show that two Pi Fluorescence Resonance Energy Transfer (FRET)-based sensors from the FLIPPi family, the low-affinity FLIPPi-30m and the high-affinity FLIPPi-4µ, can be expressed and analyzed in Arabidopsis thaliana with wild-type background. These FLIPPi sensors had not been tested in plants, but only in mammalian cell lines. We show FRET response and live imaging of Pi levels in seedling roots of Arabidopsis FLIPPi-30m and FLIPPi-4µ lines. Our results reinforce that sensors from the FLIPPi family are valuable tools for studying mechanisms of Pi transport and homeostasis in plants, and for research towards a more sustainable use of Pi fertilization.

15.
Front Microbiol ; 9: 502, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29619016

RESUMO

Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this "zinc flux" could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen.

16.
ChemMedChem ; 13(1): 37-47, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29139202

RESUMO

The fungal plasma membrane H+ -ATPase (Pma1p) is a potential target for the discovery of new antifungal agents. Surprisingly, no structure-activity relationship studies for small molecules targeting Pma1p have been reported. Herein, we disclose a LEGO-inspired fragment assembly strategy for the design, synthesis, and discovery of benzo[d]thiazoles containing a 3,4-dihydroxyphenyl moiety as potential Pma1p inhibitors. A series of 2-(benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones was found to inhibit Pma1p, with the most potent IC50 value of 8 µm in an in vitro plasma membrane H+ -ATPase assay. These compounds were also found to strongly inhibit the action of proton pumping when Pma1p was reconstituted into liposomes. 1-(3,4-Dihydroxyphenyl)-2-((6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)ethan-1-one (compound 38) showed inhibitory activities on the growth of Candida albicans and Saccharomyces cerevisiae, which could be correlated and substantiated with the ability to inhibit Pma1p in vitro.


Assuntos
Antifúngicos/química , Membrana Celular/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas Fúngicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tiazóis/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Concentração Inibidora 50 , Cinética , ATPases Translocadoras de Prótons/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
17.
Front Plant Sci ; 8: 2005, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29230231

RESUMO

PSY1R is a leucine-rich repeat (LRR) receptor-like kinase (RLK) previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1) and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H+-ATPases. While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.

18.
Front Plant Sci ; 4: 234, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23874345

RESUMO

Genetically encoded fluorescent biosensors have long proven to be excellent tools for quantitative live imaging, but sensor applications in plants have been lacking behind those in mammalian systems with respect to the variety of sensors and tissue types used. How can this be improved, and what can be expected for the use of genetically encoded fluorescent biosensors in plants in the future? In this review, we present a table of successful physiological experiments in plant tissue using fluorescent biosensors, and draw some conclusions about the specific challenges plant cell biologists are faced with and some of the ways they have been overcome so far.

19.
Integr Biol (Camb) ; 4(1): 99-107, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22134619

RESUMO

Glycolysis in the yeast Saccharomyces cerevisiae exhibits temporal oscillation under anaerobic or semianaerobic conditions. Previous evidence indicated that at least two membrane-bound ATPases, the mitochondrial F(0)F(1) ATPase and the plasma membrane P-type ATPase (Pma1p), were important in regulating the glycolytic oscillation. Measurements of intracellular ATP provide a unique tool to understand the role of these membrane ATPases and how their activities are regulated. We have constructed a new nanobiosensor that can perform time-resolved measurements of intracellular ATP in intact cells. Measurements of the temporal behaviour of intracellular ATP in a yeast strain with oscillating glycolysis showed that, in addition to oscillation in intracellular ATP, there is an overall slow decrease in intracellular ATP because the ATP consumption rate exceeds the ATP production in glycolysis. Measurements of the temporal behaviour of intracellular ATP in yeast strains lacking either of the two membrane bound ATPases have confirmed that F(0)F(1) ATPase and Pma1p contribute significantly to the ATP consumption in the cell and to the regulation of glycolytic oscillation. Furthermore, our measurements also demonstrate that ATPase activity is under strict control. In the absence of glucose ATPase activity is switched off, and the intracellular ATP concentration is high. When glucose is added to the cells the ATP concentration starts to decrease, because ATP consumption exceeds ATP production by glycolysis. Finally, when glucose is used up, the ATP consumption stops immediately. Thus, glucose or some compound derived from glucose must be involved in controlling the activity of these two ATPases.


Assuntos
Trifosfato de Adenosina/metabolismo , Relógios Biológicos/fisiologia , Glicólise/fisiologia , ATPases Translocadoras de Prótons/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/métodos , Microscopia de Fluorescência , Nanopartículas , Saccharomyces cerevisiae/enzimologia
20.
Mol Plant Pathol ; 13(9): 1110-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22863200

RESUMO

A large number of effector candidates have been identified recently in powdery mildew fungi. However, their roles and how they perform their functions remain unresolved. In this study, we made use of host-induced gene silencing and confirmed that the secreted barley powdery mildew effector candidate, CSEP0055, contributes to the aggressiveness of the fungus. This result suggests that CSEP0055 is involved in the suppression of plant defence. A yeast two-hybrid screen indicated that CSEP0055 interacts with members of the barley pathogenesis-related protein families, PR1 and PR17. Interaction with PR17c was confirmed by bimolecular fluorescence complementation analyses. Down-regulation and over-expression of PR17c in epidermal cells of barley confirmed that this protein is important for penetration resistance against the powdery mildew fungus. In line with this, PR17c was found to be apoplastic, localizing to the papillae formed in response to this fungus. The CSEP0055 transcript did not start to accumulate until 24 h after inoculation. This suggests that this gene is expressed too late to influence primary penetration events, but rather sustains the fungus at sites of secondary penetration, where PR17c appears to be able to accumulate.


Assuntos
Ascomicetos/fisiologia , Proteínas Fúngicas/metabolismo , Hordeum/microbiologia , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Fluorescência , Inativação Gênica , Hordeum/citologia , Hordeum/metabolismo , Transporte Proteico , Técnicas do Sistema de Duplo-Híbrido
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