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1.
Plant Mol Biol ; 102(3): 323-337, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31900819

RESUMO

KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ferro/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Difosfato de Adenosina/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Clorofila , Cloroplastos/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Homeostase , Mitocôndrias/metabolismo , Mutação , Monoéster Fosfórico Hidrolases/genética , Folhas de Planta/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo
2.
Chemistry ; 26(1): 165-170, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31691395

RESUMO

Phosphorylation is an important post-translational modification on proteins involved in many cellular processes; however, understanding of the regulation and mechanisms of global phosphorylation remains limited. Herein, we utilize self-assembled monolayers on gold for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) with three phosphorylated peptide arrays to profile global phosphatase activity in cell lysates derived from five mammalian cell lines. Our results reveal significant differences in the activities of protein phosphatases on phospho- serine, threonine, and tyrosine substrates and suggest that phosphatases play a much larger role in the regulation of global phosphorylation on proteins than previously understood.


Assuntos
Peptídeos/química , Monoéster Fosfórico Hidrolases/metabolismo , Análise Serial de Proteínas/métodos , Animais , Linhagem Celular , Humanos , Camundongos , Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato
3.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31740531

RESUMO

l-Serine is a nonessential amino acid and a key intermediate in several relevant metabolic pathways. In bacteria, the major source of l-serine is the phosphorylated pathway, which comprises three enzymes: d-3-phosphoglycerate dehydrogenase (PGDH; SerA), phosphoserine amino transferase (PSAT; SerC), and l-phosphoserine phosphatase (PSP; SerB). The Brucella abortus genome encodes two PGDHs (SerA-1 and SerA-2), involved in the first step in l-serine biosynthesis, and one PSAT and one PSP, responsible for the second and third steps, respectively. In this study, we demonstrate that the serA1 serA2 double mutant and the serC and serB single mutants are auxotrophic for l-serine. These auxotrophic mutants can be internalized but are unable to replicate in HeLa cells and in J774A.1 macrophage-like cells. Replication defects of auxotrophic mutants can be reverted by cell medium supplementation with l-serine at early times postinfection. In addition, the serB mutant is attenuated in the murine intraperitoneal infection model and has an altered lipid composition, since the lack of l-serine abrogates phosphatidylethanolamine synthesis in this strain. Taken together, these results reveal that limited availability of l-serine within the host cell impairs proliferation of the auxotrophic strains, highlighting the relevance of this biosynthetic pathway in Brucella pathogenicity.


Assuntos
Brucella abortus/crescimento & desenvolvimento , Brucella abortus/metabolismo , Proliferação de Células/fisiologia , Serina/metabolismo , Animais , Vias Biossintéticas/fisiologia , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Redes e Vias Metabólicas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/fisiologia
4.
Insect Mol Biol ; 29(1): 48-55, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31294881

RESUMO

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori.


Assuntos
Bombyx/enzimologia , Monoéster Fosfórico Hidrolases/química , Serina/biossíntese , Sequência de Aminoácidos , Animais , Bombyx/genética , Bombyx/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Larva/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Seda
5.
J Agric Food Chem ; 67(49): 13518-13525, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31757125

RESUMO

Coordinating unsaturated metal sites (CUS) on the surface of metal-organic frameworks (MOFs) could be used to adsorb His-tagged proteins. The specific adsorption between CUS and His-tagged proteins could reduce preparation steps, shorten preparation time, and could also avoid the binding between the metal ion of metalloenzyme active center and the chelating agent to ensure the enzyme activity. In this study, MIL-88A was synthesized by hydrothermal method and used to purify and immobilize His-tagged organophosphohydrolase (OpdA) in one step for organophosphate bioremediation. Under optimized conditions, OpdA@MIL-88A had a maximal activity of 1554 U/gprotein, which was nearly 5 times higher than free OpdA. Compared with free OpdA, OpdA@MIL-88A exhibited improved organic solvent tolerance, SDS tolerance, thermal stability, and storage stability. OpdA@MIL-88A was used to degrade organophosphorus pesticides on grapes and cucumbers. After reuse 6 times, OpdA@MIL-88A retained more than 66% and 61% of the initial activity, respectively. Therefore, this proposed strategy provided a facile and effective method for degradation of organophosphorus pesticides.


Assuntos
Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/metabolismo , Estruturas Metalorgânicas/química , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Monoéster Fosfórico Hidrolases/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Meios de Cultura/química , Meios de Cultura/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Compostos Organofosforados/química , Praguicidas/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
6.
J Ind Microbiol Biotechnol ; 46(12): 1725-1731, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31501960

RESUMO

Pentose sugars are increasingly being used in industrial applications of Saccharomyces cerevisiae. Although L-arabinose is a highlighted pentose that has been identified as next-generation biomass, arabinose fermentation has not yet undergone extensive development for industrial utilization. In this study, we integrated a heterologous fungal arabinose pathway with a deletion of PHO13 phosphatase gene. PHO13 deletion increased arabinose consumption rate and specific ethanol productivity under aerobic conditions and consequently depleted sedoheptulose by activation of the TAL1 gene. Global metabolite profiling indicated upregulation of the pentose phosphate pathway and downstream effects such as trehalose accumulation and downregulation of the TCA cycle. Our results suggest that engineering of PHO13 has ample potential for arabinose conversion to ethanol as an industrial source for biofuels.


Assuntos
Arabinose/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aerobiose , Etanol/metabolismo , Fermentação , Heptoses/metabolismo , Via de Pentose Fosfato , Monoéster Fosfórico Hidrolases/genética , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência
7.
Nat Commun ; 10(1): 4424, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562313

RESUMO

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5'-3' RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5'-3' exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Exorribonucleases/genética , Genes de Plantas , Mutagênese , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Estabilidade de RNA
8.
Cell Biochem Biophys ; 77(4): 357-366, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31562588

RESUMO

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.


Assuntos
Corrente Citoplasmática/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Physarum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
9.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31527126

RESUMO

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Clindamicina/farmacologia , Histidina Quinase/metabolismo , Proteínas Repressoras/metabolismo , Streptococcus pyogenes/metabolismo , Estreptolisinas/biossíntese , Proteínas de Bactérias/biossíntese , Histidina Quinase/antagonistas & inibidores , Histidina Quinase/genética , Magnésio/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Streptococcus pyogenes/patogenicidade
10.
Chem Commun (Camb) ; 55(82): 12388-12391, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31559988

RESUMO

Antibodies are widely used both in clinical practice and in research. However, the development of methods to increase the ratio of antibodies to recognize phosphorylated proteins remains challenging. In this study, we report a novel and useful method for the efficient production of antibodies for phosphorylated proteins. Based on our previously developed vaccine adjuvant Nap-GDFDFDY, we prepared hydrogels by the Ca2+-induced self-assembly of a phosphorylated peptide gelator Nap-GDFDFpDY. The hydrogel could protect phosphorylated antigens from being dephosphorylated by endogenous phosphatase, thus selectively increasing the ratio of the antibodies for phosphorylated proteins. Our study provides a useful strategy for the production of antibodies to recognize proteins with specific posttranslational modifications.


Assuntos
Anticorpos/química , Anticorpos/imunologia , Formação de Anticorpos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Monoéster Fosfórico Hidrolases/análise , Monoéster Fosfórico Hidrolases/imunologia , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação
11.
BMC Plant Biol ; 19(1): 381, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477017

RESUMO

BACKGROUND: Trehalose-6-phosphate phosphatases (TPPs), which are encoded by members of the TPP gene family, can improve the drought tolerance of plants. However, the molecular mechanisms underlying the dynamic regulation of TPP genes during drought stress remain unclear. In this study, we explored the function of an Arabidopsis TPP gene by conducting comparative analyses of a loss-of-function mutant and overexpression lines. RESULTS: The loss-of-function mutation of Arabidopsis thaliana TPPF, a member of the TPP gene family, resulted in a drought-sensitive phenotype, while a line overexpressing TPPF showed significantly increased drought tolerance and trehalose accumulation. Compared with wild-type plants, tppf1 mutants accumulated more H2O2 under drought, while AtTPPF-overexpressing plants accumulated less H2O2 under drought. Overexpression of AtTPPF led to increased contents of trehalose, sucrose, and total soluble sugars under drought conditions; these compounds may play a role in scavenging reactive oxygen species. Yeast one-hybrid and luciferase activity assays revealed that DREB1A could bind to the DRE/CRT element within the AtTPPF promoter and activate the expression of AtTPPF. A transcriptome analysis of the TPPF-overexpressing plants revealed that the expression levels of drought-repressed genes involved in electron transport activity and cell wall modification were upregulated, while those of stress-related transcription factors related to water deprivation were downregulated. These results indicate that, as well as its involvement in regulating trehalose and soluble sugars, AtTPPF is involved in regulating the transcription of stress-responsive genes. CONCLUSION: AtTPPF functions in regulating levels of trehalose, reactive oxygen species, and sucrose levels during drought stress, and the expression of AtTPPF is activated by DREB1A in Arabidopsis. These findings shed light on the molecular mechanism by which AtTPPF regulates the response to drought stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Secas , Regulação da Expressão Gênica de Plantas/genética , Mutação com Perda de Função/genética , Monoéster Fosfórico Hidrolases/genética , Estresse Fisiológico/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo
12.
PLoS Pathog ; 15(9): e1007972, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31487328

RESUMO

The biogenesis of bacterial cell-envelope polysaccharides requires the translocation, across the plasma membrane, of sugar sub-units that are produced inside the cytoplasm. To this end, the hydrophilic sugars are anchored to a lipid phosphate carrier (undecaprenyl phosphate (C55-P)), yielding membrane intermediates which are translocated to the outer face of the membrane. Finally, the glycan moiety is transferred to a nascent acceptor polymer, releasing the carrier in the "inactive" undecaprenyl pyrophosphate (C55-PP) form. Thus, C55-P is generated through the dephosphorylation of C55-PP, itself arising from either de novo synthesis or recycling. Two types of integral membrane C55-PP phosphatases were described: BacA enzymes and a sub-group of PAP2 enzymes (type 2 phosphatidic acid phosphatases). The human pathogen Helicobacter pylori does not contain BacA homologue but has four membrane PAP2 proteins: LpxE, LpxF, HP0350 and HP0851. Here, we report the physiological role of HP0851, renamed HupA, via multiple and complementary approaches ranging from a detailed biochemical characterization to the assessment of its effect on cell envelope metabolism and microbe-host interactions. HupA displays a dual function as being the main C55-PP pyrophosphatase (UppP) and phosphatidylglycerol phosphate phosphatase (PGPase). Although not essential in vitro, HupA was essential in vivo for stomach colonization. In vitro, the remaining UppP activity was carried out by LpxE in addition to its lipid A 1-phosphate phosphatase activity. Both HupA and LpxE have crucial roles in the biosynthesis of several cell wall polysaccharides and thus constitute potential targets for new therapeutic strategies.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Helicobacter pylori/metabolismo , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação a DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Helicobacter pylori/patogenicidade , Camundongos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Fosfatidato Fosfatase , Monoéster Fosfórico Hidrolases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Polimixina B/farmacologia , Pirofosfatases/metabolismo , Estômago
13.
Environ Sci Pollut Res Int ; 26(29): 29649-29659, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401803

RESUMO

This study was aiming to treat the chlorpyrifos (CPF), an organophosphate (OP) pesticide with microbial enzyme extract, and assess the toxicity effects of CPF before/after its treatment on the integrity of DNA (deoxyribonucleic acid) and the activities of enzymes AChE (acetylcholinestrase), GST (glutathione S-transferase), SOD (superoxide dismutase), CAT (catalase), and MDA (malondialdehyde) in different organs of rat. The untreated CPF in rat significantly increased the DNA damage and decreased the activities of all these enzymes. Among all the organs studied, the liver was the most affected organ. Further, CPF was treated with an OPH (organophosphate hydrolase) enzyme obtained from CPF degrading bacterial laboratory isolate Pseudomonas sp. (ChlD) to neutralize the toxicity of CPF. The crude intracellular enzyme extract degraded > 90% of added CPF and > 80% of its toxic intermediate 3,5,6-trichloropyridinol (TCP) which resulted in > 80% reduction of CPF toxicity in different organs of rat. Thus, this study not only illustrated the adverse effect of OPs on mammalian system but also suggested a highly efficient and eco-friendly way to remove the harmful pesticide from the environment and agricultural food products which may help to reduce the exposure of humans to such lethal toxicants.


Assuntos
Clorpirifos/metabolismo , Clorpirifos/toxicidade , Enzimas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Ensaio Cometa , Inseticidas/metabolismo , Inseticidas/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Malondialdeído/metabolismo , Pseudomonas/enzimologia , Ratos Wistar , Testes de Toxicidade
14.
Mol Cells ; 42(8): 604-616, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31446747

RESUMO

Phosphoserine phosphatase (PSPH) is one of the key enzymes of the L-serine synthesis pathway. PSPH is reported to affect the progression and survival of several cancers in an L-serine synthesis-independent manner, but the mechanism remains elusive. We demonstrate that PSPH promotes lung cancer progression through a noncanonical L-serine-independent pathway. PSPH was significantly associated with the prognosis of lung cancer patients and regulated the invasion and colony formation of lung cancer cells. Interestingly, L-serine had no effect on the altered invasion and colony formation by PSPH. Upon measuring the phosphatase activity of PSPH on a serine-phosphorylated peptide, we found that PSPH dephosphorylated phospho-serine in peptide sequences. To identify the target proteins of PSPH, we analyzed the protein phosphorylation profile and the PSPH-interacting protein profile using proteomic analyses and found one putative target protein, IRS-1. Immunoprecipitation and immunoblot assays validated a specific interaction between PSPH and IRS1 and the dephosphorylation of phospho-IRS-1 by PSPH in lung cancer cells. We suggest that the specific interaction and dephosphorylation activity of PSPH have novel therapeutic potential for lung cancer treatment, while the metabolic activity of PSPH, as a therapeutic target, is controversial.


Assuntos
Progressão da Doença , Proteínas Substratos do Receptor de Insulina/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Monoéster Fosfórico Hidrolases/metabolismo , Serina/metabolismo , Células A549 , Animais , Humanos , Camundongos , Invasividade Neoplásica , Fosforilação , Transdução de Sinais , Análise de Sobrevida , Ensaio Tumoral de Célula-Tronco
15.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426386

RESUMO

Inositol signaling is believed to play a crucial role in various aspects of plant growth and adaptation. As an important component in biosynthesis and degradation of myo-inositol and its derivatives, inositol phosphatases could hydrolyze the phosphate of the inositol ring, thus affecting inositol signaling. Until now, more than 30 members of inositol phosphatases have been identified in plants, which are classified intofive families, including inositol polyphosphate 5-phosphatases (5PTases), suppressor of actin (SAC) phosphatases, SAL1 phosphatases, inositol monophosphatase (IMP), and phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-related phosphatases. The current knowledge was revised here in relation to their substrates and function in response to abiotic stress. The potential mechanisms were also concluded with the focus on their activities of inositol phosphatases. The general working model might be that inositol phosphatases would degrade the Ins(1,4,5)P3 or phosphoinositides, subsequently resulting in altering Ca2+ release, abscisic acid (ABA) signaling, vesicle trafficking or other cellular processes.


Assuntos
Inositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transdução de Sinais , Aclimatação , Inositol Polifosfato 5-Fosfatases/metabolismo , Fosfatidilinositóis/metabolismo , Fenômenos Fisiológicos Vegetais , Estresse Fisiológico
16.
Cells ; 8(7)2019 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-31284660

RESUMO

The role of kinases in the regulation of cell cycle transitions is very well established, however, over the past decade, studies have identified the ever-growing importance of phosphatases in these processes. It is well-known that an intact or otherwise non-deformed nuclear envelope (NE) is essential for maintaining healthy cells and any deviation from this can result in pathological conditions. This review aims at assessing the current understanding of how phosphatases contribute to the remodelling of the nuclear envelope during its disassembling and reformation after cell division and how errors in this process may lead to the development of diseases.


Assuntos
Pontos de Checagem do Ciclo Celular , Mitose , Membrana Nuclear/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Cromatina/metabolismo , Humanos , Membrana Nuclear/patologia , Mapas de Interação de Proteínas/fisiologia
17.
Molecules ; 24(13)2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31284667

RESUMO

Salinity stress limited the production in over 30% of irrigated crops and 7% of dryland agriculture worldwide. The objective was to evaluate the effects of NaCl-stress on the enzymatic activity in tomato. Two experiments were carried out in germination and early vegetative growth stages. The activity of proline and peroxidase of eight varieties (Missouri, Yaqui, Vita, Feroz, Rio Grande, Tropic, Ace, and Floradade) submitted to NaCl concentrations (0, 50, 100, 150 and 200 mM de NaCl) and the semi-quantitative activity of 19 enzymes APY ZYM® were measured under a completely randomized design with four replications. Data were analyzed using univariate-multivariate analysis of variance, Tukey's HSD (p = 0.05), canonical discriminant and cluster analysis. The results showed significant differences between varieties and NaCl in proline content. Proline increased as the NaCl concentration increased. Peroxidase did no show significant differences. Eight enzymes were included within the model to properly classify the varieties and NaCl. In shoots, varieties and NaCl showed that enzymatic activity was higher in the order of alkaline-phosphatase > leucine arylamidase > acid phosphatase > naphthol-AS-BI-phosphohydrolase > n-acetyl-ß-glucosaminidase > ß-galactosidase, while in roots was higher in the order of alkaline-phosphatase > naphthol-AS-BI-phosphohydrolase > acid phosphatase > n-acetyl-ß-glucosaminidase. Acid and alkali phosphatase, lipase, esterase, ß-galactosidase, and trypsin can be a potential biomarker for NaCl-stress tolerance in tomato.


Assuntos
Esterases/metabolismo , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/fisiologia , N-Glicosil Hidrolases/metabolismo , Peptídeo Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Biomarcadores , Análise por Conglomerados , Ativação Enzimática , Brotos de Planta/fisiologia , Prolina/metabolismo , Proteoma , Proteômica , Plântula/fisiologia
18.
Mol Plant Microbe Interact ; 32(11): 1547-1556, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31287368

RESUMO

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata. The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.


Assuntos
Azorhizobium caulinodans , Sesbania , Simbiose , Azorhizobium caulinodans/enzimologia , Azorhizobium caulinodans/genética , Ativação Enzimática , Mutação , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Sesbania/microbiologia , Propriedades de Superfície , Simbiose/genética
19.
J Appl Microbiol ; 127(4): 1113-1124, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31287935

RESUMO

AIMS: Isolation and identification of genes encoding putative phosphatases from Pseudomonas putida strain P13 DSM 23335. METHODS AND RESULTS: By functional screening of a P. putida P13 genomic library, a number of Pho+ clones were identified. Two genes were identified that encoded proteins exhibiting both phytase and sugar phosphatase activities. The proteins were 249 and 462 amino acids, with molecular masses of 26 and 50 kDa respectively. Sequence alignments revealed no significant similarities to representatives of known phosphatase or phytase gene families. However, the genes were found to have a high similarity to members of the major facilitator superfamily (MFS). Both genes were overexpressed in Escherichia coli and the corresponding partially purified recombinant enzymes were found to have significant phytate-dephosphorylating activity. The protein designated P. putida phytase 1 (Ppp1) displayed the highest activity among potential substrates studied on Na phytate, whereas Ppp2 more likely represents a sugar phosphatase than a phytase. The optimal conditions for phytate dephosphorylation were determined as 60°C and pH 4·5 (Ppp1) or pH 5·0 (Ppp2). CONCLUSIONS: Two novel bacterial phosphatase-encoding genes, named ppp1 and ppp2, were isolated from P. putida P13 DSM 23335 by a functional screening procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphatase-encoding genes are of great importance for industrial applications, particularly in agriculture. The identified phosphatase genes represent a new class of acid phosphatases.


Assuntos
Proteínas de Bactérias , Genes Bacterianos/genética , Monoéster Fosfórico Hidrolases , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo
20.
Clin Lab ; 65(7)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31307179

RESUMO

BACKGROUND: Clinical Chemistry is the backbone of medical treatment, diagnostics, and prevention. The laborato-ries are trying to improve the quality and to reduce diagnostic errors and processing time and safeguard trace-ability of all laboratory procedures to ensure patient safety. Six sigma belongs to statistical quality control and provides a new methodology for measuring and improving process performance in laboratory. METHODS: Activities of AST, ALT, CK, LDH, Amy, and γ-GT were determined by standard kinetic methods on a Vitros 5600 biochemistry analyzer. Two daily quality controls (Verifier I and Verifier II) were run over 60 days. Total percent CV was calculated from routine daily QC. Between-instrument bias was also calculated from daily QC. RESULTS: The calculated sigma metrics for AST were 6.9 and 3.8; for ALT 9.3 and 5.6; for CK 6.6 and 5.3; LDH 5.2 and 5.2; for γ-GT 4.9 and 2.7; and for amylase 8.7 and 7.1. Analytical performance for AST, ALT, CK, LDH, and Amylase is world class. On the other hand, γ-GT analytical performance is poor. CONCLUSIONS: Six Sigma benefits from earlier quality management approaches that creates new challenges for medical laboratories.


Assuntos
Química Clínica/normas , Enzimas/sangue , Laboratórios/normas , Controle de Qualidade , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Amilases/sangue , Amilases/metabolismo , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Química Clínica/métodos , Creatinina , Erros de Diagnóstico/prevenção & controle , Enzimas/metabolismo , Humanos , Cinética , L-Lactato Desidrogenase/sangue , L-Lactato Desidrogenase/metabolismo , Monoéster Fosfórico Hidrolases/sangue , Monoéster Fosfórico Hidrolases/metabolismo , gama-Glutamiltransferase/sangue , gama-Glutamiltransferase/metabolismo
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