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1.
Chem Biol Interact ; 314: 108848, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610156

RESUMO

Cardiomyocyte injury induced by acute myocardial infarction contributes to myocardial dysfunction. Accumulating evidence has demonstrated that pleckstrin homology domain leucine-rich repeat protein phosphatase 2 (PHLPP2) is a cytoprotective protein that protects against various adverse injuries. However, whether PHLPP2 participates in regulating myocardial-infarction-induced cardiomyocyte injury remains unknown. In the present study, we aimed to investigate the biological role and molecular mechanism of PHLPP2 in regulating hypoxia-induced cardiomyocyte injury. Cardiomyocytes were cultured in an anaerobic chamber for 24 h to induce hypoxic injury in vitro. The expression of PHLPP2 was determined by real-time quantitative PCR and Western blot analysis. Cell viability was measured by MTT assay. Cell apoptosis was assessed by TUNEL and caspase-3 activity assays. Intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA probe. PHLPP2 expression was highly upregulated in hypoxia-injured cardiomyocytes. Inhibition of PHLPP2 by small interfering RNA (siRNA)-mediated gene silencing significantly improved the viability of hypoxia-injured cardiomyocytes and attenuated hypoxia-induced apoptosis and ROS production. In contrast, PHLPP2 overexpression exacerbated hypoxia-induced apoptosis and ROS production in cardiomyocytes. Mechanism research revealed that PHLPP2 silencing increased the phosphorylation of glycogen synthase kinase (GSK)-3ß and promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In addition, PHLPP2 inhibition promoted Nrf2/antioxidant response element (ARE) transcriptional activity. However, Nrf2 silencing markedly reversed PHLPP2-inhibition-mediated cardioprotection, while GSK-3ß inhibition partially blocked the PHLPP2-overexpression-induced adverse effect. Taken together, these findings demonstrate that PHLPP2 inhibition alleviates hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE antioxidant signaling via inactivating GSK-3ß, a pathway that highlights the importance of the PHLPP2/GSK-3ß/Nrf2/ARE signaling axis in regulation of cardiomyocyte injury. Our study suggests a potential relevance for PHLPP2 in acute myocardial infarction, and this protein may serve as a promising target for cardioprotection.


Assuntos
Elementos de Resposta Antioxidante/genética , Hipóxia Celular , Fator 2 Relacionado a NF-E2/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Regulação para Baixo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , Pirimidinas/farmacologia , Pirróis/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Plant Physiol Biochem ; 141: 325-331, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207493

RESUMO

Type 2C protein phosphatases (PP2Cs) counteract protein kinases, thereby inhibiting the abscisic acid (ABA)-mediated response to abiotic stress in Arabidopsis thaliana. In the absence of stress, the promoters of PP2C genes (e.g., ABI1, ABI2, and HAI1) are negatively regulated by repressors that suppress gene transcription in a signal-independent manner. Quantitative reverse transcription PCR (RT-qPCR) and chromatin immunoprecipitation (ChIP) assays revealed that the levels of PP2C gene transcripts and RNA polymerase II (RNAPII) that stalled at the transcription start sites (TSS) of PP2C gene loci were increased under salt stress. The salt-induced increases in RNA polymerase-mediated transcription were reduced in 35S:AtMYB44 plants, confirming that AtMYB44 acts as a repressor of PP2C gene transcription. ChIP assays revealed that AtMYB44 repressors are released and nucleosomes are evicted from the promoter regions in response to salt stress. Under these conditions, histone H3 acetylation (H3ac) and methylation (H3K4me3) around the TSS regions significantly increased. The salt-induced increases in PP2C gene transcription were reduced in abf3 plants, indicating that ABF3 activates PP2C gene transcription. Overall, our data indicate that salt stress converts PP2C gene chromatin from a repressor-associated suppression status to an activator-mediated transcription status. In addition, we observed that the Arabidopsis mutant brm-3, which is moderately defective in SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) activity, produced more PP2C gene transcripts under salt stress conditions, indicating that BRM ATPase contributes to the repression of PP2C gene transcription.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/química , Nucleossomos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Estresse Salino , Trifosfato de Adenosina/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Montagem e Desmontagem da Cromatina , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
3.
J Basic Microbiol ; 59(8): 834-845, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31210376

RESUMO

A bacterium's ability to thrive in the presence of multiple environmental stressors simultaneously determines its resilience. We showed that activation of the SigB-controlled general stress response by mild environmental or energy stress provided significant cross-protection to subsequent lethal oxidative, disulfide and nitrosative stress in Bacillus subtilis. SigB activation is mediated via the stressosome and RsbP, the main conduits of environmental and energy stress, respectively. Cells exposed to mild environmental stress while lacking the major stressosome components RsbT or RsbRA were highly sensitive to subsequent oxidative stress, whereas rsbRB, rsbRC, rsbRD, and ytvA null mutants showed a spectrum of sensitivity, confirming their redundant roles and suggesting they could modulate the signals generated by environmental or oxidative stress. By contrast, cells encountering stationary phase stress required RsbP but not RsbT to survive subsequent oxidative stress. Interestingly, optimum cross-protection against nitrosative stress caused by sodium nitropruside required SigB but not the known regulators, RsbT and RsbP, suggesting an additional and as yet uncharacterized route of SigB activation independent of the known regulators. Together, these results provide mechanistic information on how B. subtilis promotes enhanced resistance against lethal oxidative stress during mild environmental and energy stress conditions.


Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Estresse Oxidativo/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Fator sigma/metabolismo , Transdução de Sinais , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Viabilidade Microbiana , Estresse Nitrosativo/fisiologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fator sigma/genética , Transdução de Sinais/genética
4.
Nat Commun ; 10(1): 2760, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235787

RESUMO

Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure.


Assuntos
Redes Reguladoras de Genes/genética , Insuficiência Cardíaca/genética , Miócitos Cardíacos/patologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Benzenoacetamidas , Células Cultivadas , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fosfoproteínas Fosfatases/genética , Cultura Primária de Células , Piridinas , Locos de Características Quantitativas/genética , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA/métodos
5.
Nat Plants ; 5(5): 539-550, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31076735

RESUMO

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.


Assuntos
Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Membrana/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Interferência de RNA , RNA Nucleotidiltransferases/fisiologia , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , RNA de Plantas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transgenes/genética
6.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027321

RESUMO

Juniper (Juniperus communis L.) is a northern coniferous plant generally used as a spice and for nutritional purposes in foods and drinks. It was previously reported that juniper extract (JE) affects p53 activity, cellular stress, and gene expression induced cell death in human neuroblastoma cells. Therefore, the effects of juniper on p53 and Akt signaling was examined further in A549 lung, 22RV1 and DU145 prostate, and HepG2 liver cancer cells using Western blot, confocal microscopy, and MTT analysis. We found that juniper simultaneously decreased cell viability, activated the p53 pathway, and inactivated the PI3K/Akt pathway. The p53 activation was associated with increased nuclear p53 level. Akt was dephosphorylated, and its inactivation was associated with increased levels of PHLPP1 and PHLPP2 phosphatases. Parallel increases of PARP suggest that JE decreased cell viability by activating cell death. In adtion, JE potentiated the effects of gemcitabine and 5-fluorouracil anticancer drugs. Thus, JE can activate cell death in different cancer cell lines through p53 and Akt pathways.


Assuntos
Morte Celular/efeitos dos fármacos , Citostáticos/farmacologia , Juniperus/química , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Fluoruracila/farmacologia , Células Hep G2 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Supressora de Tumor p53/genética
7.
Int J Mol Sci ; 20(8)2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027230

RESUMO

Leaf senescence is a highly-programmed developmental process regulated by an array of multiple signaling pathways. Our group previously reported that overexpression of the protein phosphatase-encoding gene SSPP led to delayed leaf senescence and significantly enhanced cytokinin responses. However, it is still unclear how the delayed leaf senescence phenotype is associated with the enhanced cytokinin responses. In this study, we introduced a cytokinin receptor AHK3 knockout into the 35S:SSPP background. The phenotypic analysis of double mutant revealed that AHK3 loss-of-function reversed the delayed leaf senescence induced by SSPP. Moreover, we found the hypersensitivity of 35S:SSPP to exogenous cytokinin treatment disappeared due to the introduction of AHK3 knockout. Collectively, our results demonstrated that AHK3-mediated cytokinin signaling is required for the delayed leaf senescence caused by SSPP overexpression and the detailed mechanism remains to be further elucidated.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Histidina Quinase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Fosfoproteínas Fosfatases/genética , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
J Neuroinflammation ; 16(1): 75, 2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961627

RESUMO

BACKGROUND: Activation of microglial cells plays an important role in neuroinflammation after ischemic stroke. Inhibiting the activation of microglial cells has been suggested as a potential therapeutic approach in the treatment of ischemic stroke. METHODS: Oxygen-glucose deprivation in primary microglial cells and transient middle cerebral artery occlusion (MCAO) in C57BL/6 mice were used as the in vitro and in vivo ischemic stroke models. Microarray analysis was performed to investigate the overall impact of long non-coding RNAs (lncRNAs) on the inflammation status of microglial cells. RT-qPCR was used to evaluate the lncRNA levels and mRNA levels of cytokines and microglial cell markers. ELISA was taken to measure the level of cytokines. Immunofluorescence was used to observe the activation of microglial cells. Western blotting was performed to test the p65 phosphorylation. RESULTS: In this study, we showed that LncRNA-1810034E14Rik was significantly decreased in LPS-treated or oxygen-glucose deprivation-induced microglial cells. Overexpression of 1810034E14Rik decreased the infarct volume and alleviated brain damage in MCAO mice. 1810034E14Rik overexpression reduced the expression of inflammatory cytokines not only in ischemic stroke mice but also in oxygen-glucose deprivation-induced microglial cells. Moreover, 1810034E14Rik overexpression could suppress the activation of microglial cells and inhibit the phosphorylation of p65. CONCLUSIONS: LncRNA-1810034E14Rik plays an anti-inflammatory role in ischemic stroke and regulates p65 phosphorylation, making it a potential target for stroke treatment.


Assuntos
Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Microglia/metabolismo , Fosfoproteínas Fosfatases/metabolismo , RNA Longo não Codificante/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Animais Recém-Nascidos , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glucose/deficiência , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Microglia/efeitos dos fármacos , Fosfoproteínas Fosfatases/genética , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia
9.
Plant Mol Biol ; 100(3): 303-317, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945147

RESUMO

KEY MESSAGE: Overexpression of the poplar PP2C protein phosphatase gene PtrHAB2 resulted in increased tree height and altered leaf morphology and phyllotaxy, implicating PP2C phosphatases as growth regulators functioning under favorable conditions. We identified and studied Populus trichocarpa genes, PtrHAB1 through PtrHAB15, belonging to the clade A PP2C family of protein phosphatases known to regulate abscisic acid (ABA) signaling. PtrHAB1 through PtrHAB3 and PtrHAB12 through PtrHAB15 were the most highly expressed genes under non-stress conditions. The poplar PP2C genes were differentially regulated by drought treatments. Expression of PtrHAB1 through PtrHAB3 was unchanged or downregulated in response to drought, while all other PtrHAB genes were weakly to strongly upregulated in response to drought stress treatments. Yeast two-hybrid assays involving seven ABA receptor proteins (PtrRCAR) against 12 PtrHAB proteins detected 51 interactions involving eight PP2Cs and all PtrRCAR proteins with 22 interactions requiring the addition of ABA. PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 also interacted with the sucrose non-fermenting related kinase 2 proteins PtrSnRK2.10 and PtrSnRK2.11, supporting conservation of a SnRK2 signaling cascade regulated by PP2C in poplar. Additionally, PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 interacted with the mitogen-activated protein kinase protein PtrMPK7. Due to its interactions with PtrSnRK2 and PtrMPK7 proteins, and its reduced expression during drought stress, PtrHAB2 was overexpressed in poplar to test its potential as a growth regulator under non-stress conditions. 35S::PtrHAB2 transgenics exhibited increased growth rate for a majority of transgenic events and alterations in leaf phyllotaxy and morphology. These results indicate that PP2Cs have additional roles which extend beyond canonical ABA signaling, possibly coordinating plant growth and development in response to environmental conditions.


Assuntos
Ácido Abscísico/metabolismo , Crescimento e Desenvolvimento/fisiologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Reguladores de Crescimento de Planta/fisiologia , Populus/crescimento & desenvolvimento , Populus/metabolismo , Estresse Fisiológico , Secas , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Crescimento e Desenvolvimento/genética , Fenótipo , Reguladores de Crescimento de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Populus/genética , Proteínas Quinases/genética , Transdução de Sinais , Regulação para Cima
10.
Nat Commun ; 10(1): 1858, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015415

RESUMO

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático/patologia , Metiltransferases/metabolismo , MicroRNAs/metabolismo , Neoplasias Pancreáticas/patologia , Fumaça/efeitos adversos , Tabaco/toxicidade , Adenosina/análogos & derivados , Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/etiologia , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Proteínas Correpressoras/metabolismo , Metilação de DNA , Progressão da Doença , Células Epiteliais/patologia , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Metiltransferases/genética , MicroRNAs/sangue , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/mortalidade , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Prognóstico , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fumar/efeitos adversos , Fumar/sangue , Regulação para Cima
11.
Int J Biol Macromol ; 131: 912-924, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30914363

RESUMO

Human pyridoxal 5'-phosphate phosphatase (PLPP), also known as a chronophin, is a phosphatase belonging to subfamily II of the HAD phosphatases, characterized by a large cap domain. As a member of the subfamily, its cap-open conformation is expected for substrate binding. We determined apo and PLP-bound PLPP/chronophin structures showing a cap-closed conformation. The active site, in which a PLP molecule was found, is too small to accommodate a phospho-cofilin peptide, the substrate of chronophin. A conformational change to a cap-open conformation may be required for substrate binding. The core and cap domains are joined through linker peptide hinges that change conformation to open the active site. The crystal structures reveal that a disulphide bond between the cap and core domains restricts the hinge motion. The enzyme displays PLP dephosphorylation activity in the cap-closed conformation with the disulphide bond and even in the crystal state, in which repositioning of the cap and core domains is restricted. Structural analysis suggests that a small substrate such as PLP can bind to the active site through a small movement of a local motif. However, a change to the cap-open conformation is required for binding of larger substrates such as phosphopeptides to the active site.


Assuntos
Modelos Moleculares , Fosfoproteínas Fosfatases/química , Monoéster Fosfórico Hidrolases/química , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Análise Espectral , Especificidade por Substrato
12.
Med Hypotheses ; 125: 8-9, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30902157

RESUMO

To date, there is no clear evidence for memory formation. In this article, we provide a framework to understand how memory is formed. The information collected by sensory organs is converted to a digital current that enters the presynaptic neuron through axonal conductance. Digital waves are converted to analog waves in the synapses. The analog current of information flows into the postsynapse. The degree of Ca2+ influx in the postsynapse is proportional to the voltage of each wave of analog current. The activation (via dephosphorylation) of the phosphorylated phosphatase, Slingshot, is regulated by Ca2+ concentration in the spine. After dephosphorylation by Slingshot, activated cofilin binds the parallel actin bundle. The wide helical twist angle of an actin filament that has been decorated with cofilin confers high electric potential to the filament. Phosphorylation results in the deactivation of the actin filament bound to cofilin, which in turn results in the cleavage of cofilin and actin filament, followed by a decrease in the twist angle of the actin filament. Next, the electric potential energy is discharged by the actin filament as it returns to its non-cofilin bound state, resulting in the formation of additional analog waves in the postsynapse.


Assuntos
Hipocampo/fisiopatologia , Memória , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios , Cálcio/metabolismo , Calmodulina/metabolismo , Cofilina 1/metabolismo , Hipocampo/fisiologia , Humanos , Memória de Longo Prazo , Modelos Neurológicos , Neurônios/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Eletricidade Estática
13.
Clin Lab ; 65(3)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30868840

RESUMO

Background: MicroRNA-493 (miR-493) was upregulated in prostate cancer (PCa). This study was designed to investigate the mechanism underlying miR-493 mediated pro-proliferation in PCa cells. Methods: Expression of miR-493 in PCa cell lines (DU145 and PC3) and control cells was determined using qRT-PCR. PCa cells were transfected with miR-493 mimics, inhibitor, negative control (NC), PH domain leucine-rich-repeats protein phosphatase 2 (PHLPP2), and Akt expressing plasmids and Akt inhibitor MK-2206. Cell proliferation, quantitative expression of miRNA and mRNA were detected. Protein expression was determined using western blotting analysis. Results: Results showed that miR-493 in PCa cells was upregulated compared with RWPE-1 cells. Cells transfected with miR-493 mimics or inhibitor significantly reduced or enhanced expression of PHLPP2 (p < 0.05), respectively. Cell proliferation was significantly enhanced by miR-493 overexpression, or inhibited by PHLPP2 overexpression. The administration of Akt inhibitor MK 2206 attenuated miR-493-enhanced cell proliferation. PCa cells transfected with Akt express vectors partially enhanced PHLPP2-reduced cell proliferation. Conclusions: These results demonstrated that miR-493 acted as a onco-miR in PCa cells and promoted PCa cell proliferation via inhibiting tumor suppresser PHLPP2 expression and activating Akt signaling pathway.


Assuntos
Adenocarcinoma/metabolismo , MicroRNAs/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proliferação de Células , Humanos , Masculino , Células PC-3 , Transdução de Sinais , Regulação para Cima
14.
Int J Mol Sci ; 20(6)2019 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-30897702

RESUMO

The protein phosphatase (PP2C) gene family, known to participate in cellular processes, is one of the momentous and conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. Recently, PP2Cs were identified in Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to be reported. In the current research, we found 87 (Gossypium arboreum), 147 (Gossypium barbadense), 181 (Gossypium hirsutum), and 99 (Gossypium raimondii) PP2C-encoding genes in total from the cotton genome. Herein, we provide a comprehensive analysis of the PP2C gene family in cotton, such as gene structure organization, gene duplications, expression profiling, chromosomal mapping, protein motif organization, and phylogenetic relationships of each species. Phylogenetic analysis further categorized PP2C genes into 12 subgroups based on conserved domain composition analysis. Moreover, we observed a strong signature of purifying selection among duplicated pairs (i.e., segmental and dispersed) of Gossypium hirsutum. We also observed the tissue-specific response of GhPP2C genes in organ and fiber development by comparing the RNA-sequence (RNA-seq) data reported on different organs. The qRT-PCR validation of 30 GhPP2C genes suggested their critical role in cotton by exposure to heat, cold, drought, and salt stress treatments. Hence, our findings provide an overview of the PP2C gene family in cotton based on various bioinformatic tools that demonstrated their critical role in organ and fiber development, and abiotic stress tolerance, thereby contributing to the genetic improvement of cotton for the resistant cultivar.


Assuntos
Gossypium/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gossypium/genética , Fosfoproteínas Fosfatases/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Nat Commun ; 10(1): 531, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705304

RESUMO

PGAM5 is a mitochondrial protein phosphatase whose genetic ablation in mice results in mitochondria-related disorders, including neurodegeneration. Functions of PGAM5 include regulation of mitophagy, cell death, metabolism and aging. However, mechanisms regulating PGAM5 activation and signaling are poorly understood. Using electron cryo-microscopy, we show that PGAM5 forms dodecamers in solution. We also present a crystal structure of PGAM5 that reveals the determinants of dodecamer formation. Furthermore, we observe PGAM5 dodecamer assembly into filaments both in vitro and in cells. We find that PGAM5 oligomerization into a dodecamer is not only essential for catalytic activation, but this form also plays a structural role on mitochondrial membranes, which is independent of phosphatase activity. Together, these findings suggest that modulation of the oligomerization of PGAM5 may be a regulatory switch of potential therapeutic interest.


Assuntos
Microscopia Crioeletrônica/métodos , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/ultraestrutura , Animais , Morte Celular/genética , Morte Celular/fisiologia , Camundongos , Degradação Mitocondrial/genética , Degradação Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Polimerização
16.
PLoS One ; 14(2): e0211426, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707732

RESUMO

The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen, Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1-16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1-16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/química , Fosfoproteínas Fosfatases/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cloreto de Lítio/farmacologia , Mutagênese , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
Acta Pharmacol Sin ; 40(6): 850-858, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30796354

RESUMO

Serine/threonine phosphatase (Stp1) is a member of the bacterial Mg2+- or Mn2+- dependent protein phosphatase/protein phosphatase 2C family, which is involved in the regulation of Staphylococcus aureus virulence. Aurintricarboxylic acid (ATA) is a known Stp1 inhibitor with an IC50 of 1.03 µM, but its inhibitory mechanism has not been elucidated in detail because the Stp1-ATA cocrystal structure has not been determined thus far. In this study, we performed 400 ns molecular dynamics (MD) simulations of the apo-Stp1 and Stp1-ATA complex models. During MD simulations, the flap subdomain of the Stp1-ATA complex experienced a clear conformational transition from an open state to a closed state, whereas the flap domain of apo-Stp1 changed from an open state to a semi-open state. In the Stp1-ATA complex model, the hydrogen bond (H-bond) between D137 and N142 disappeared, whereas critical H-bond interactions were formed between Q160 and H13, Q160/R161 and ATA, as well as N162 and D198. Finally, four residues (D137, N142, Q160, and R161) in Stp1 were mutated to alanine and the mutant enzymes were assessed using phosphate enzyme activity assays, which confirmed their important roles in maintaining Stp1 activity. This study indicated the inhibitory mechanism of ATA targeting Stp1 using MD simulations and sheds light on the future design of allosteric Stp1 inhibitors.


Assuntos
Ácido Aurintricarboxílico/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Ácido Aurintricarboxílico/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Inibidores Enzimáticos/química , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Mutação , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência
18.
Cell Commun Signal ; 17(1): 14, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30786936

RESUMO

BACKGROUND: Glucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation. RESULTS: Our kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism. CONCLUSIONS: Our results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Mitocôndrias/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Cinética , Camundongos , Mitocôndrias/efeitos dos fármacos , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteômica , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Fatores de Tempo
19.
Nat Plants ; 5(3): 258-262, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804511

RESUMO

Striga parasitizes major crops in arid regions, depriving the host crop of nutrients through the transpiration stream and causing vast agricultural damage. Here, we report on the mechanism underlying how Striga maintains high transpiration under drought conditions. We found that Striga did not respond to abscisic acid, the phytohormone responsible for controlling stomatal closure. Protein phosphatase 2C of Striga (ShPP2C1) is not regulated by abscisic acid receptors, and this feature is attributable to specific mutations in its amino acid sequence. Moreover, Arabidopsis transformed with ShPP2C1 showed an abscisic acid-insensitive phenotype, indicating that ShPP2C1 functions as a dominant negative regulator of abscisic acid signal transduction. These findings suggest that ShPP2C1 interrupts abscisic acid signalling in Striga, resulting in high transpiration and subsequent efficient absorption of host nutrients under drought conditions.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Plantas/metabolismo , Transpiração Vegetal/fisiologia , Proteína Fosfatase 2C/metabolismo , Striga/fisiologia , Ácido Abscísico/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Secas , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteína Fosfatase 2C/genética , Transdução de Sinais , Sorghum/parasitologia , Striga/efeitos dos fármacos
20.
Int J Mol Sci ; 20(3)2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704032

RESUMO

As a nutritional active protein in foods, multiple studies of the biological activities of lactoferrin had been undertaken, including antioxidant, antiviral, anti-inflammatory, antitumor, antibiosis, and antiparasitic effects, while the mechanism related with its protection of cardiovascular system remained elusive. In the present work, the effect of lactoferrin on the viability of HUVECs (human umbilical vein endothelial cells) was detected to select the proper doses. Moreover, transcriptomics detection and data analysis were performed to screen out the special genes and the related pathways. Meanwhile, the regulation of lactoferrin in the functional factors thromboxane A2 (TXA2) and prostacyclin (PGI2) was detected. Then, the small interfering RNA (SiRNA) fragment of the selected gene pyridoxal phosphatase (PDXP) was transfected into HUVECs to validate its role in protecting HUVECs function. Results showed that lactoferrin inhibited the expression of TXA2 and activated expression of PGI2, as well as activated expression of PDXP, which significantly up-regulated the synthesis of vitamin B6 (VB6) and the phosphoinositide 3-kinase (PI3K)/ serine/threonine-protein kinase (AKT)/ extracellular regulated protein kinases (ERK) 1/2 pathway. For the first time, we revealed that lactoferrin could induce the synthesis of VB6 and protect HUVECs function through activating PDXP gene and the related pathway.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Lactoferrina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Vitamina B 6/metabolismo , Ensaio de Imunoadsorção Enzimática , Epoprostenol/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Tromboxano A2/metabolismo
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