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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
Mol Cell ; 74(2): 378-392.e5, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30904392

RESUMO

Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Here, we report that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. Cancer-associated hotspot mutations in the pseudosubstrate of PKCß that impair autoinhibition result in dephosphorylated and unstable enzymes. Protein-level analysis reveals that PKCα is fully phosphorylated at the PHLPP site in over 5,000 patient tumors, with higher PKC levels correlating (1) inversely with PHLPP1 levels and (2) positively with improved survival in pancreatic adenocarcinoma. Thus, PHLPP1 provides a proofreading step that maintains the fidelity of PKC autoinhibition and reveals a prominent loss-of-function mechanism in cancer by suppressing the steady-state levels of PKC.


Assuntos
Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Proteína Quinase C beta/genética , Proteína Quinase C-alfa/genética , Humanos , Isoenzimas/genética , Mutação com Perda de Função/genética , Neoplasias/patologia , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Controle de Qualidade , Transdução de Sinais/genética
11.
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
12.
Mol Med Rep ; 19(4): 2497-2502, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720109

RESUMO

Phosphoglycerate mutase 1 (PGAM1) is reported to be involved in spermatogenic dysfunction. However, the association between PGAM1 and busulfan­induced hypospermatogenesis and spermatogenic cell apoptosis remains unclear. The aim of the current study was to investigate the association between PGAM1 expression and busulfan­induced hypospermatogenesis, and the effect of PGAM1 expression on spermatogenic cell apoptosis. PGAM1 expression was detected in mouse models of busulfan­induced hypospermatogenesis by western blotting, reverse transcription­quantitative polymerase chain reaction and immunohistochemistry. Then, spermatogenic cell apoptosis in mouse models of busulfan­induced hypospermatogenesis was assessed by TUNEL assay. The effect and potential mechanism of PGAM1 downregulation on spermatogenic cells were further investigated. The results indicated that PGAM1 expression was significantly downregulated in the mouse models of busulfan­induced hypospermatogenesis, compared with those with normal spermatogenesis (P<0.05). Furthermore, the TUNEL assay revealed that the apoptosis of spermatogenic cells was accelerated in the mouse model of busulfan­induced hypospermatogenesis. In addition, PGAM1 knockdown promoted the apoptosis of spermatogenic cells in vitro, which was associated with the P53/Caspase 3/Caspase 6/Caspase 9 signaling pathway. In conclusion, these data indicate that PGAM1 knockdown is associated with busulfan­induced hypospermatogenesis and contributes to spermatogenic cell apoptosis by regulating the P53/Caspase 3/Caspase 6/Caspase 9 signaling pathway.


Assuntos
Apoptose/genética , Bussulfano/efeitos adversos , Técnicas de Silenciamento de Genes , Oligospermia/etiologia , Fosfoproteínas Fosfatases/genética , Espermatozoides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Regulação da Expressão Gênica , Masculino , Camundongos , Espermatozoides/efeitos dos fármacos
13.
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
14.
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
15.
Cardiovasc Drugs Ther ; 33(1): 13-23, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30637549

RESUMO

PURPOSE: Necroptosis is an important form of cell death following myocardial ischemia/reperfusion (I/R) and phosphoglycerate mutase 5 (PGAM5) functions as the convergent point for multiple necrosis pathways. This study aims to investigate whether inhibition of PGAM5 could reduce I/R-induced myocardial necroptosis and the underlying mechanisms. METHODS: The SD rat hearts (or H9c2 cells) were subjected to 1-h ischemia (or 10-h hypoxia) plus 3-h reperfusion (or 4-h reoxygenation) to establish the I/R (or H/R) injury model. The myocardial injury was assessed by the methods of biochemistry, H&E (hematoxylin and eosin), and PI/DAPI (propidium iodide/4',6-diamidino-2-phenylindole) staining, respectively. Drug interventions or gene knockdown was used to verify the role of PGAM5 in I/R (or H/R)-induced myocardial necroptosis and possible mechanisms. RESULTS: The I/R-treated heart showed the injuries (increase in infarct size and creatine kinase release), upregulation of PGAM5, dynamin-related protein 1 (Drp1), p-Drp1-S616, and necroptosis-relevant proteins (RIPK1/RIPK3, receptor-interacting protein kinase 1/3; MLKL, mixed lineage kinase domain-like); these phenomena were attenuated by inhibition of PGAM5 or RIPK1. In H9c2 cells, H/R treatment elevated the levels of PGAM5, RIPK1, RIPK3, MLKL, Drp1, and p-Drp1-S616 and induced mitochondrial dysfunctions (elevation in mitochondrial membrane potential and ROS level) and cellular necrosis (increase in LDH release and the ratio of PI+/DAPI+ cells); these effects were blocked by inhibition or knockdown of PGAM5. CONCLUSIONS: Inhibition of PGAM5 can reduce necroptosis in I/R-treated rat hearts through suppression of Drp1; there is a positive feedback between RIPK1 and PGAM5, and PGAM5 might serve as a novel therapeutic target for prevention of myocardial I/R injury.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Inibidores Enzimáticos/farmacologia , Glicolatos/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fosfoglicerato Mutase/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo , Masculino , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
16.
PLoS Genet ; 15(1): e1007904, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615605

RESUMO

Brassinosteroids (BRs) are steroid hormones essential for plant growth and development. The BR signaling pathway has been studied in some detail, however, the functions of the BRASSINOSTEROID-SIGNALING KINASE (BSK) family proteins in the pathway have remained elusive. Through forward genetics, we identified five semi-dominant mutations in the BSK3 gene causing BSK3 loss-of-function and decreased BR responses. We therefore investigated the function of BSK3, a receptor-like cytoplasmic kinase, in BR signaling and plant growth and development. We find that BSK3 is anchored to the plasma membrane via N-myristoylation, which is required for its function in BR signaling. The N-terminal kinase domain is crucial for BSK3 function, and the C-terminal three tandem TPR motifs contribute to BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation. Interestingly, the effects of BSK3 on BR responses are dose-dependent, depending on its protein levels. Our genetic studies indicate that kinase dead BSK3K86R protein partially rescues the bsk3-1 mutant phenotypes. BSK3 directly interacts with the BSK family proteins (BSK3 and BSK1), BRI1 receptor kinase, BSU1 phosphatase, and BIN2 kinase. BIN2 phosphorylation of BSK3 enhances BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation, BSK3/BRI1 interaction, and BSK3/BSU1 interaction. Furthermore, we find that BSK3 upregulates BSU1 transcript and protein levels to activate BR signaling. BSK3 is broadly expressed and plays an important role in BR-mediated root growth, shoot growth, and organ separation. Together, our findings suggest that BSK3 may function as a scaffold protein to regulate BR signaling. The results of our studies provide new insights into early BR signaling mechanisms.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brassinosteroides/metabolismo , Fosfoproteínas Fosfatases/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação com Perda de Função/genética , Fenótipo , Fosforilação , Plantas Geneticamente Modificadas/genética , Transdução de Sinais
17.
Appl Microbiol Biotechnol ; 103(3): 1351-1362, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30610282

RESUMO

The Ser/Thr protein phosphatase Ppt1 (yeast)/PP5 (humans) has been implicated in signal transduction-mediated growth and differentiation, DNA damage/repair, cell cycle progression, and heat shock responses. Little, however, is known concerning the functions of Ppt1/PP5 in filamentous fungi. In this study, the Ppt1 gene MaPpt1 was characterized in the insect pathogenic fungus, Metarhizium acridum. The MaPpt1 protein features a three-tandem tetratricopeptide repeat (TPR) domain and a peptidyl-prolyl cis-trans isomerase-like (PP2Ac) domain. Subcellular localization using an MaPpt1::eGFP fusion protein revealed that MaPpt1 was localized in the cytoplasm of spores, but gathered at the septa in growing hyphae. Targeted gene inactivation of MaPpt1 in M. acridum resulted in unexpected reprogramming of normal aerial conidiation to microcycle conidiation. Although overall vegetative growth was unaffected, a significant increase in conidial yield was noted in ΔMaPpt1. Stress-responsive phenotypes and virulence were largely unaffected in ΔMaPpt1. Exceptionally, ΔMaPpt1 displayed increased UV tolerance compared to wild type. Digital gene expression data revealed that MaPpt1 mediates transcription of sets of genes involved in conidiation, polarized growth, cell cycle, cell proliferation, DNA replication and repair, and some important signaling pathways. These data indicate a unique role for Ppt1 in filamentous fungal development and differentiation.


Assuntos
Metarhizium/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Proliferação de Células/genética , Reparo do DNA/genética , Replicação do DNA/genética , Deleção de Genes , Metarhizium/metabolismo , Transdução de Sinais/genética , Raios Ultravioleta
18.
FEBS Lett ; 593(3): 339-351, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30556127

RESUMO

Abscisic acid (ABA) regulates growth and developmental processes in response to limiting water conditions. ABA functions through a core signaling pathway consisting of PYR1/PYL/RCAR ABA receptors, type 2C protein phosphatases (PP2Cs), and SnRK2-type protein kinases. Other signaling modules might converge with ABA signals through the modulation of core ABA signaling components. We have investigated the role of the protein kinase WNK8 in ABA signaling. WNK8 interacted with PP2CA and PYR1, phosphorylated PYR1 in vitro, and was dephosphorylated by PP2CA. A hypermorphic wnk8-ct Arabidopsis mutant allele suppressed ABA and glucose hypersensitivities of pp2ca-1 mutants during young seedling development, and WNK8 expression in protoplasts suppressed ABA-induced reporter gene expression. We conclude that WNK8 functions as a negative modulator of ABA signaling.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Ácido Abscísico/genética , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Protoplastos/enzimologia , Tabaco/genética , Tabaco/metabolismo
19.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 74-82, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30036567

RESUMO

This review presents the accumulating evidence for the roles of protein phosphatase 6 (PP6) in cell cycle, DNA damage repair, inflammatory signaling, lymphocyte development, virus infection, tumor formation/progression, cell/tissue size, and non-coding RNA-mediated regulation. PP6 is an evolutionarily conserved and ubiquitously expressed Ser/Thr protein phosphatase most closely related to protein phosphatase 2A (PP2A) and protein phosphatase 4 (PP4). Although abundantly expressed in cells with multiple roles in cellular signaling, PP6 has received less attention than its close relative PP2A. Many studies used okadaic acid as "PP2A" inhibitor, even though these toxins also inhibit PP6 activity, so effects of the inhibitor could have been due to inhibition of both phosphatases. PP6 has its own dedicated subunits that assemble into heterotrimers that presumably fulfill its discrete functions in cells.


Assuntos
Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Ciclo Celular/fisiologia , Tamanho Celular , Reparo do DNA/fisiologia , Regulação da Expressão Gênica , Inflamação/metabolismo , Linfócitos/metabolismo , Neoplasias/metabolismo , RNA não Traduzido/metabolismo , Transdução de Sinais , Viroses/metabolismo
20.
J Genet ; 97(5): 1155-1168, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30555065

RESUMO

To analyse the mechanism of tumourigenic transformation of NIH3T3 cells at the transcriptional level, we used cancerogen 3-methylcholanthrene (3-MCA) and cancerogenic substance phorbol-12-myristate-13-acetate (PMA) to transform NIH3T3 cells and the assessment of transformation was performed using Giemsa staining and methylcellulose colony formation assay. Changes in gene expression profile were detected by Mouse Genome 430 2.0 microarray; and quantitative real-time polymerase chain reaction and Western blotting were used to verify the expression changes of mRNAs and proteins, respectively. With the aid of bioinformatics method, five signalling pathways were identified to participate in different stages of NIH3T3 cell transformation. Further, our study suggested that oncogenes Cyclin A, Myc, Jun and the tumour suppressor gene Ppm1l may play important roles in these pathways.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Ciclina A1/genética , Fosfoproteínas Fosfatases/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Camundongos , Células NIH 3T3
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