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1.
Life Sci ; 243: 117323, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31954160

RESUMO

AIMS: Circular RNAs (circRNAs) have been emerged as novel regulators in multiple tumorigenesis, including melanoma. CircRNA_0084043 was recently demonstrated to be deregulated in human melanoma cells. Nevertheless, its role and mechanism are largely unrevealed in melanoma. MATERIALS AND METHODS: Expression of circ_0084043, miRNA (miR)-429 and tribbles homolog 2 (TRIB2) was detected using reverse transcription-quantitative PCR quantitative PCR (RT-qPCR) and western blotting. Cell proliferation, apoptosis, migration and invasion were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and transwell assays, respectively. The activation of Wnt/ß-catenin pathway was evaluated by western blotting. The target binding among circ_0084043, miR-429 and TRIB2 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. In vivo, mice xenograft model was generated to investigate tumor growth. KEY FINDINGS: Expression of circ_0084043 and TRIB2 was upregulated in human melanoma tissues and cell lines. Both circ_0084043 knockdown and TRIB2 silencing could decrease cell proliferation, migration and invasion, but facilitate apoptosis in A375 and SK-MEL-28 cells. Furthermore, TRIB2 restoration partially abrogated the tumor-suppressive role of circ_0084043 knockdown in melanoma cells in vitro. Then, we verified that circ_0084043 positively and physically controlled TRIB2 expression through sponging miR-429. Besides, expression of ß-catenin, c-Myc and cyclinD1 was inhibited in A375 and SK-MEL-28 cells when circ_0084043 was knocked down, accompanied with increased miR-429 and decreased TRIB2. Notably, circ_0084043 downregulation impeded tumor growth of A375 cells in vivo. SIGNIFICANCE: Knockdown of circ_0084043 suppressed the malignant development of melanoma presumably through modulating miR429/TRIB2 axis and inactivating Wnt/ß-catenin signaling pathway.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Melanoma/patologia , MicroRNAs/metabolismo , RNA Circular/genética , Neoplasias Cutâneas/patologia , Via de Sinalização Wnt , beta Catenina/metabolismo , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Masculino , Melanoma/metabolismo , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Neoplasias Cutâneas/metabolismo
2.
Adv Exp Med Biol ; 1131: 649-679, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646529

RESUMO

Multifunctional calcium/calmodulin-stimulated protein kinases control a broad range of cellular functions in a multitude of cell types. This family of kinases contain several structural similarities and all are regulated by phosphorylation, which either activates, inhibits or modulates their kinase activity. As these protein kinases are widely or ubiquitously expressed, and yet regulate a broad range of different cellular functions, additional levels of regulation exist that control these cell-specific functions. Of particular importance for this specificity of function for multifunctional kinases is the expression of specific binding proteins that mediate molecular targeting. These molecular targeting mechanisms allow pools of kinase in different cells, or parts of a cell, to respond differently to activation and produce different functional outcomes.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Regulação Enzimológica da Expressão Gênica , Terapia de Alvo Molecular , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Fosforilação
3.
Planta ; 250(5): 1773-1779, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31440828

RESUMO

MAIN CONCLUSION: Accumulation of calcium/calmodulin-dependent protein kinase (CCaMK) in root cell nucleus depends on its kinase activity but not on nuclear symbiotic components crucial for nodulation. Plant calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator of symbioses with rhizobia and arbuscular mycorrhizal fungi as it decodes symbiotic calcium signals induced by microsymbionts. CCaMK is expressed mainly in root cells and localizes to the nucleus, where microsymbiont-triggered calcium oscillations occur. The molecular mechanisms that control CCaMK localization are unknown. Here, we analyzed the expression and subcellular localization of mutated CCaMK in the roots of Lotus japonicus and found a clear relation between CCaMK kinase activity and its stability. Kinase-defective CCaMK variants showed lower protein levels than the variants with kinase activity. The levels of transcripts driven by the CaMV 35S promoter were similar among the variants, indicating that stability of CCaMK is regulated post-translationally. We also demonstrated that CCaMK localized to the root cell nucleus in several symbiotic mutants, including cyclops, an interaction partner and phosphorylation target of CCaMK. Our results suggest that kinase activity of CCaMK is required not only for the activation of downstream symbiotic components but also for its stability in root cells.


Assuntos
Sinalização do Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Lotus/enzimologia , Simbiose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Núcleo Celular/metabolismo , Lotus/genética , Lotus/microbiologia , Lotus/fisiologia , Mutação , Micorrizas/fisiologia , Fosforilação , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética , Estabilidade Proteica , Rhizobium/fisiologia
4.
Med Sci Monit ; 25: 4627-4638, 2019 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-31266934

RESUMO

BACKGROUND Subclinical epileptiform discharges (SEDs) are defined as epileptiform electroencephalographic (EEG) discharges without clinical signs of seizure in patients. The subthreshold convulsant discharge (SCD) is a frequently used model for SEDs. This study aimed to investigate the effect of levetiracetam (LEV), an anti-convulsant drug, on cognitive impairment of SCD model rats and to assess the associated mechanisms. MATERIAL AND METHODS A SCD rat model was established. Rats were divided into an SCD group, an SCD+ sodium valproate (VPA) group, and an SCD+ levetiracetam (LEV) group. The Morris water maze was used to evaluate the capacity of positioning navigation and space exploration. The field excitatory post-synaptic potentials (fEPSPs) were evaluated using a bipolar stimulation electrode. NCAM, GAP43, PS95, and CaMK II levels were detected using Western blot and RT-PCR, respectively. PKC activity was examined by a non-radioactive method. RESULTS LEV shortens the latency of platform seeking in SCD rats in positioning navigation. fEPSP slopes were significantly lower in the SCD group, and LEV treatment significantly enhanced the fEPSP slopes compared to the SCD group (P<0.05). The NCAM and GAP-43 levels were increased and PSD-95 levels were increased in SCD rats (P<0.05), which were improved by LEV treatment. The PKC activity and CaMK II levels were decreased in SCD rats and LEV treatment significantly enhanced PKC activity and increased CaMK II levels. CONCLUSIONS Cognitive impairment in of SCD model rats may be caused by decreased PKC activity, low expression of CaMK II, and inhibition of LTP formation. LEV can improve cognitive function by activating the PKC-GAP-43-CaMK signal transduction pathway.


Assuntos
Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/prevenção & controle , Levetiracetam/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Modelos Animais de Doenças , Eletroencefalografia , Proteína GAP-43/efeitos dos fármacos , Proteína GAP-43/metabolismo , Hipocampo/metabolismo , Levetiracetam/metabolismo , Masculino , Fosforilação , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Convulsões/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ácido Valproico/uso terapêutico
5.
Gerontology ; 65(6): 620-633, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31242498

RESUMO

BACKGROUND: Carbonylation is an oxidative modification of the proteins and a marker of oxidative stress. The accumulation of toxic carbonylated proteins might be one of the onsets of pathogenesis in hippocampal aging or neurodegeneration. Enormous evidence indicates that regular aerobic exercise might alleviate the dysfunction of carbonylated proteins, but the adaptational mechanisms in response to exercise are unclear. OBJECTIVE: This study explored the carbonyl stress mechanism in the hippocampus using proteomics and the role of calmodulin-dependent protein kinases (CAMK)-AMP-activated protein kinase (AMPK)-Beclin1 signaling pathways in alleviating aging or improving function with regular aerobic exercise. METHODS: Twenty-four healthy 13-month-old male Sprague-Dawley rats (average 693.21 ± 68.85 g) were randomly divided into middle-aged sedentary control group (M-SED, n = 12) and middle-aged aerobic exercise runner group (M-EX, n = 12). The M-EX group participated in regular aerobic exercise - treadmill running - with exercise intensity increasing gradually from 50-55% to 65-70% of maximum oxygen consumption (V˙O2max) over 10 weeks. The targeted proteins of oxidative modification were profiled by avidin magnetic beads and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Western blots were used to test for molecular targets. RESULTS: Regular aerobic exercise restores the intersessional habituation and rescues the hippocampus morphological structure in middle-aged rats. -ESI-Q-TOF-MS screened 56 carbonylated proteins only found in M-SED and 16 carbonylated proteins only found in M-EX, indicating aerobic exercise decreased carbonyl stress. Intriguingly, Ca2+/CAMK II alpha (CAMKIIα) was carbonylated only in the M-SED group at the oxidative modification site of 4-hydroxynonenal adducts, while regular aerobic exercise alleviated CAMKIIα carbonylation. Regular aerobic exercise significantly increased the expression and phosphorylated, active levels of CAMKIIα and AMPKα1. It also upregulated the expression of Beclin1 and microtubule-associated protein1-light chain 3 in the hippocampus. CONCLUSION: Quantification of CAMKIIα carbonylation may be a potential biomarker of the hippocampal senescence. Additionally, regular aerobic exercise-induced autophagy via the activation of CAMK-AMPK-Beclin1 signaling pathway may mitigate the hippocampal neurodegeneration or pathological changes by alleviating protein carbonylation (carbonyl stress).


Assuntos
Autofagia , Hipocampo/metabolismo , Hipocampo/patologia , Condicionamento Físico Animal , Carbonilação Proteica , Animais , Proteína Beclina-1/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Espectrometria de Massas , Proteínas Associadas aos Microtúbulos/metabolismo , Proteômica , Distribuição Aleatória , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para Cima
6.
Virol Sin ; 34(3): 278-286, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953292

RESUMO

MicroRNAs (miRNAs) encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2 (HSV-2) infection. In this study, miRNA-H4-5p and miRNA-H4-3p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions. The results showed that miRNA-H4-5p could reverse apoptosis induced by actinomycin D (Act-D) and promote cell cycle progression, but miRNA-H4-3p had no such obvious functions. Bioinformatics analysis, luciferase report assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blotting demonstrated that miRNA-H4-5p could bind to the 3'-untranslated region (UTR) of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase-like 2 (CDKL2) to negatively regulate their expression. We verified that these two targeted genes were associated with cell apoptosis and cell cycle. Furthermore, in HeLa cells infected with HSV-2, we detected significantly reduced expression of CDKN2A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5p on these two target genes. Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection, providing further information on the roles of encoded herpesvirus miRNAs in pathogen-host interaction.


Assuntos
Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dactinomicina/farmacologia , Herpesvirus Humano 2/genética , MicroRNAs/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular , Biologia Computacional , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células HeLa , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Latência Viral
7.
Cell Commun Signal ; 17(1): 7, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30665402

RESUMO

Through a genome-wide screen we have identified calcium-tolerant deletion mutants for five genes in the budding yeast Saccharomyces cerevisiae. In addition to CNB1 and RCN1 that are known to play a role in the calcium signalling pathway, the protein kinase gene CMK2, the sphingolipid homeostasis-related gene ORM2 and the gene SIF2 encoding the WD40 repeat-containing subunit of Set3C histone deacetylase complex are involved in the calcium sensitivity of yeast cells to extracellular calcium. Cmk2 and the transcription factor Crz1 have opposite functions in the response of yeast cells to calcium stress. Deletion of CMK2 elevates the level of calcium/calcineurin signalling and increases the expression level of PMR1 and PMC1, which is dependent on Crz1. Effects of Cmk2 on calcium sensitivity and calcium/calcineurin signalling are dependent on its kinase activity. Therefore, Cmk2 is a negative feedback controller of the calcium/calcineurin signalling pathway. Furthermore, the cmk2 crz1 double deletion mutant is more resistant than the crz1 deletion mutant, suggesting that Cmk2 has an additional Crz1-independent role in promoting calcium tolerance.


Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biocatálise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Deleção de Genes , Modelos Biológicos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Int J Biol Macromol ; 123: 704-712, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30414416

RESUMO

Calmodulin binding receptor like cytoplasmic kinase 2 (CRCK2) belongs to the family of receptor like kinases (RLKs) which is mainly implicated in pathways associated with the stress responses in plants. The protein from the stem of Oroxylum indicum was isolated and purified using anion-exchange followed by gel filtration chromatography. The purity of protein was checked using SDS-PAGE, which showed a single band of 50 kDa. The purified protein was identified as CRCK2 using MALDI-TOF. Using I-TASSER, a bioinformatics tools, the model of protein was constructed and its secondary structure was predicted using VADAR. The secondary structure content was also determined by far-UV CD, which indicated that the CRCK2 is mainly ß-sheet dominating protein (43% ß-sheet). The secondary structural content predication from computational method is in close agreement with the result obtained by CD spectropolarimeter. This study validates I-TASSER model for determination of structure of a protein. Moreover, stability of CRCK2 was monitored against heat- and guanidinium chloride (GdmCl)-induced denaturation by using circular dichroism (CD) and fluorescence spectroscopy. Denaturation curve analysis gave values of 2.88 ±â€¯0.12 kcal mol-1and 4.11 ±â€¯0.09 M for ∆°GD (Gibbs free energy change at 25 °C) and Cm (midpoint of denaturation), respectively. It has been observed that purified CRCK2 is quite stable protein against both heat-induced as well as GdmCl-induced denaturation. This is very first report of purification and biophysical characterization of CRCK2 protein from medicinal plant O. indicum.


Assuntos
Bignoniaceae/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Conformação Proteica em Folha beta , Estrutura Secundária de Proteína , Bignoniaceae/química , Fenômenos Biofísicos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Dicroísmo Circular , Modelos Químicos , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
9.
Biochim Biophys Acta Mol Cell Res ; 1866(7): 1054-1067, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30582936

RESUMO

ROCO kinases form a family of proteins characterized by kinase activity in addition to the presence of the so-called ROC (Ras of complex proteins)/COR (C-terminal of ROC) domains having a role in their GTPase activity. These are the death-associated protein kinase (DAPK) 1 and the leucine-rich repeat kinases (LRRK) 1 and 2. These kinases all play roles in cellular life and death decisions and in autophagy in particular. Related to the ROCO kinases is DAPK 2 that however cannot be classified as a ROCO protein due to the absence of the ROC/COR domains. This review aims to bring together what is known about the relation between these proteins and intracellular Ca2+ signals in the induction and regulation of autophagy. Interestingly, DAPK 1 and 2 and LRRK2 are all linked to Ca2+ signaling in their effects on autophagy, though in various ways. Present evidence supports an upstream role for LRRK2 that via lysosomal and endoplasmic reticulum Ca2+ release can trigger autophagy induction. In contrast herewith, DAPK1 and 2 react on existing Ca2+ signals to stimulate the autophagic pathway. Further research will be needed for obtaining a full understanding of the role of these various kinases in autophagy and to assess their exact relation with intracellular Ca2+ signaling as this would be helpful in the development of novel therapeutic strategies against neurodegenerative disorders, cancer and auto-immune diseases. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.


Assuntos
Autofagia , Sinalização do Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Humanos , Neoplasias/patologia , Neoplasias/terapia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia
10.
Appl Microbiol Biotechnol ; 103(2): 819-832, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30417308

RESUMO

Ca2+/calmodulin-dependent protein kinases (CaMKs) are unique second-messenger molecules that impact almost all cellular processes in eukaryotes. In this study, five genes encoding different CaMKs were characterized in the nematode-trapping fungus Arthrobotrys oligospora. These CaMKs, which were retrieved from the A. oligospora genome according to their orthologs in fungi such as Aspergillus nidulans and Neurospora crassa, were expressed at a low level in vitro during mycelial growth stages. Five deletion mutants corresponding to these CaMKs led to growth defects in different media and increased sensitivity to several environmental stresses, including H2O2, menadione, SDS, and Congo red; they also reduced the ability to produce conidia and traps, thus causing a deficiency in nematicidal ability as well. In addition, the transcriptional levels of several typical sporulation-related genes, such as MedA, VelB, and VeA, were down-regulated in all ΔCaMK mutants compared with the wild-type (WT) strain. Moreover, these mutants exhibited hypersensitivity to heat shock and ultraviolet-radiation stresses compared with the WT strain. These results suggest that the five CaMKs in A. oligospora are involved in regulating multiple cellular processes, such as growth, environmental stress tolerance, conidiation, trap formation, and virulence.


Assuntos
Ascomicetos/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Animais , Ascomicetos/crescimento & desenvolvimento , Biologia Computacional , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Micélio/enzimologia , Micélio/crescimento & desenvolvimento , Nematoides/microbiologia , Estresse Fisiológico
11.
Gene ; 683: 35-40, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30292871

RESUMO

BACKGROUND AND AIMS: Aberrant DNA methylation of cyclin-dependent kinase-like 2 (CDKL2) had been observed in several types of tumors. Herein, the present study was aimed to explore the epigenetic and expression status of CDKL2 and evaluate the diagnostic potential of CDKL2 methylation in hepatocellular carcinoma (HCC). METHODS: The methylation status of CDKL2 was detected by methylation-sensitive restriction enzyme based quantitative PCR (MSRE-qPCR) and bisulfite genomic sequencing (BGS). The mRNA expression of CDKL2 was measured using real-time quantitative PCR (qPCR). The correlations between the methylation of CDKL2 and mRNA expression, clinicopathological features were evaluated. RESULTS: Compared with normal liver tissues, the methylation levels of CDKL2 were significantly increased in the HCC tissues and cell lines (All p < 0.05). And the receiver operating characteristic (ROC) analysis showed that the hypermethylation of CDKL2 had a high specificity and sensitivity to distinguish adjacent non-tumor tissues from HCC tissues. Additionally, the mRNA expression levels of CDKL2 were decreased both in HCC tissues and cell lines than those in normal liver tissues (All p < 0.05), and the expression could be upregulated by 5-aza-2'-deoxycytidine treatment in HCC cell lines. Furthermore, the methylation of CDKL2 was negatively correlated with its mRNA expression (p < 0.001, rs = -0.513), and was associated with gender (p = 0.023), age (p = 0.001) and tumor size (p = 0.016). CONCLUSIONS: Our results showed that CDKL2 promoter hypermethylation played an important role in hepatocarcinogenesis and might be a valuable biomarker for HCC diagnosis.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Carcinoma Hepatocelular/genética , Metilação de DNA , Regulação para Baixo , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Regiões Promotoras Genéticas , Análise de Sequência de DNA/métodos , Fatores Sexuais , Carga Tumoral
12.
Plant Mol Biol ; 99(1-2): 113-122, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30536042

RESUMO

KEY MESSAGE: In this manuscript, we demonstrated the negative role of CPK9 in stomatal ABA signaling, and both CPK9 and CPK33 for accurate guard cell function was explored via cpk9/cpk33 double mutants' phenotype. Abscisic acid (ABA) can inhibit stomatal opening and promote stomatal closure by regulating ion channel activity in guard cell membranes. As an important second messenger, calcium (Ca2+) is essentially needed in ABA regulation of stomatal movement. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central Ca2+ signal transduction in plants. Here, we report the functional characterization of CPK9 in Arabidopsis stomatal ABA signaling. CPK9 had high expression in guard cells and the protein was subcellularly located in the cell membrane. A loss-of-function mutant cpk9 showed a much more sensitive phenotype to ABA regulation of stomatal movement and ion channel activity, while CPK9 overexpression lines had opposite phonotypes. These findings demonstrated the negative role of CPK9 in stomatal ABA signaling. As the closest homolog of CPK33, we also proved that stomatal movement of the cpk9/cpk33 double mutants was more sensitive to ABA than either single mutants. These results revealed the role of CPK9 in guard cells, and the need of both CPK9 and CPK33 for accurate guard cell function.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Canais Iônicos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Membrana Celular/metabolismo , Genes Reporter , Canais Iônicos/genética , Transporte de Íons , Mutação , Reguladores de Crescimento de Planta/metabolismo , Estômatos de Plantas/genética , Proteínas Quinases/genética
13.
J Tissue Eng Regen Med ; 13(3): 369-384, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30550638

RESUMO

Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Técnicas de Cultura de Células/métodos , Channelrhodopsins/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Optogenética , Regiões Promotoras Genéticas/genética , Sinapsinas/genética , Alginatos/farmacologia , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Reologia , Sinapsinas/metabolismo
14.
Biomed Res Int ; 2019: 2523032, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31930114

RESUMO

Osteosarcoma (OS) is one of the most common malignant bone tumors in adolescents with a poor prognosis. Though miR-509-5p has been reported as a tumor suppressor in several human cancers, the role of miR-509-5p in OS remains unclear. In this study, our result of real-time PCR (RT-PCR) showed that the expression of miR-509-5p was significantly decreased in OS tissues and cell lines. Overexpression of miR-509-5p significantly suppressed cell proliferation and invasion in OS cell lines. Moreover, we identified tribbles homolog 2 (TRIB2) as the direct target of miR-509-5p. Knockdown of TRIB2 could inhibit the malignant capacity of OS cells. At last, we reported that TRIB2 could inhibit the bioactivity of the tumor suppressor gene p21 via blocking its transcriptional activity. Collectively, our study revealed that miR-509-5p functions as a tumor suppressor by targeting TRIB2 in OS and thus could affect the activity of p21, suggesting that miR-509-5p is a novel preventive intervention for OS patients.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proliferação de Células/genética , MicroRNAs/genética , Invasividade Neoplásica/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Invasividade Neoplásica/patologia , Transcrição Genética/genética
15.
Int J Mol Sci ; 19(12)2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558245

RESUMO

CDPKs (Ca2+-Dependent Protein Kinases) are very important regulators in plant response to abiotic stress. The molecular regulatory mechanism of CDPKs involved in salt stress tolerance remains unclear, although some CDPKs have been identified in salt-stress signaling. Here, we investigated the function of an Arabidopsis CDPK, CPK12, in salt-stress signaling. The CPK12-RNA interference (RNAi) mutant was much more sensitive to salt stress than the wild-type plant GL1 in terms of seedling growth. Under NaCl treatment, Na⁺ levels in the roots of CPK12-RNAi plants increased and were higher than levels in GL1 plants. In addition, the level of salt-elicited H2O2 production was higher in CPK12-RNAi mutants than in wild-type GL1 plants after NaCl treatment. Collectively, our results suggest that CPK12 is required for plant adaptation to salt stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Estresse Salino , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , Peróxido de Hidrogênio/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Interferência de RNA , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Sódio/metabolismo
16.
BMC Genomics ; 19(1): 937, 2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30558527

RESUMO

BACKGROUND: Nicotiana attenuata is an ecological model plant whose 2.57 Gb genome has recently been sequenced and assembled and for which miRNAs and their genomic locations have been identified. To understand how this plant's miRNAs are reconfigured during plant-arbuscular mycorrhizal fungal (AMF) interactions and whether hostplant calcium- and calmodulin dependent protein kinase (CCaMK) expression which regulates the AMF interaction also modulates miRNAs levels and regulation, we performed a large-scale miRNA analysis of this plant-AMF interaction. RESULTS: Next generation sequencing of miRNAs in roots of empty vector (EV) N. attenuata plants and an isogenic line silenced in CCaMK expression (irCCaMK) impaired in AMF-interactions grown under competitive conditions with and without AMF inoculum revealed a total of 149 unique miRNAs: 67 conserved and 82 novel ones. The majority of the miRNAs had a length of 21 nucleotides. MiRNA abundances were highly variable ranging from 400 to more than 25,000 reads per million. The miRNA profile of irCCaMK plants impaired in AMF colonization was distinct from fully AMF-functional EV plants grown in the same pot. Six conserved miRNAs were present in all conditions and accumulated differentially depending on treatment and genotype; five (miR6153, miR403a-3p, miR7122a, miR167-5p and miR482d, but not miR399a-3p) showed the highest accumulation in AMF inoculated EV plants compared to inoculated irCCaMK plants. Furthermore, the accumulation patterns of sequence variants of selected conserved miRNAs showed a very distinct pattern related to AMF colonization - one variant of miR473-5p specifically accumulated in AMF-inoculated plants. Also abundances of miR403a-3p, miR171a-3p and one of the sequence variants of miR172a-3p increased in AMF-inoculated EV compared to inoculated irCCaMK plants and to non-inoculated EV plants, while miR399a-3p was most strongly enriched in AMF inoculated irCCaMK plants grown in competition with EV. The analysis of putative targets of selected miRNAs revealed an involvement in P starvation (miR399), phytohormone signaling (Nat-R-PN59, miR172, miR393) and defense (e.g. miR482, miR8667, Nat-R-PN-47). CONCLUSIONS: Our study demonstrates (1) a large-scale reprograming of miRNAs induced by AMF colonization and (2) that the impaired AMF signaling due to CCaMK silencing and the resulting reduced competitive ability of irCCaMK plants play a role in modulating signal-dependent miRNA accumulation.


Assuntos
MicroRNAs/metabolismo , Micorrizas/fisiologia , Tabaco/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Genótipo , MicroRNAs/genética , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Simbiose , Tabaco/metabolismo , Tabaco/microbiologia , Transcriptoma
17.
Mol Cancer ; 17(1): 172, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30541550

RESUMO

BACKGROUND: Cellular senescence is a state of irreversible cell growth arrest and senescence cells permanently lose proliferation potential. Induction of cellular senescence might be a novel therapy for cancer cells. TRIB2 has been reported to participate in regulating proliferation and drug resistance of various cancer cells. However, the role of TRIB2 in cellular senescence of colorectal cancer (CRC) and its molecular mechanism remains unclear. METHODS: The expression of TRIB2 in colorectal cancer tissues and adjacent tissues was detected by immunohistochemistry and RT-PCR. The growth, cell cycle distribution and cellular senescence of colorectal cancer cells were evaluated by Cell Counting Kit-8 (CCK8) assay, flow cytometry detection and senescence-associated ß-galactosidase staining, respectively. Western blot, RT-PCR and luciferase assay were performed to determine how TRIB2 regulates p21. Immunoprecipitation (IP) and chromatin-immunoprecipitation (ChIP) were used to investigate the molecular mechanisms. RESULTS: We found that TRIB2 expression was elevated in CRC tissues compared to normal adjacent tissues and high TRIB2 expression indicated poor prognosis of CRC patients. Functionally, depletion of TRIB2 inhibited cancer cells proliferation, induced cell cycle arrest and promoted cellular senescence, whereas overexpression of TRIB2 accelerated cell growth, cell cycle progression and blocked cellular senescence. Further studies showed that TRIB2 physically interacted with AP4 and inhibited p21 expression through enhancing transcription activities of AP4. The rescue experiments indicated that silencing of AP4 abrogated the inhibition of cellular senescence induced by TRIB2 overexpression. CONCLUSION: These data demonstrate that TRIB2 suppresses cellular senescence through interaction with AP4 to down-regulate p21 expression. Therefore, TRIB2 could be a potential target for CRC treatment.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Neoplasias Colorretais/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oncogenes/genética , Transdução de Sinais/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Senescência Celular/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo
18.
J Mol Med (Berl) ; 96(11): 1267-1277, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30324339

RESUMO

The function and mechanism of action of MLL-TET1 (MT1) fusion protein in hematological cells are unclear and require further investigation. In the present study, we found that the MT1 fusion protein attenuated the expression of Cebpa, Csf1r, and Cd11b and inhibited the differentiation of myeloid progenitor cells. Increased binding of the MT1 fusion protein to the Trib2 promoter upregulated Trib2 mRNA and protein expression and downregulated Cebpa expression. Trib2 knockdown relieved the inhibition of myeloid cell differentiation induced by the MT1 fusion protein. Thus, TRIB2 is important for the survival of leukemia cells during MT1-related leukemogenesis and is important in maintaining differentiation blockade of leukemic cells. KEY MESSAGES: • MLL-TET1 fusion decreases the 5-hmC levels in the myeloid progenitor cells. • MLL-TET1 fusion inhibits myeloid differentiation through decreased expression of Cebpa. • MLL-TET1 fusion blocks the differentiation of the myeloid progenitor cells by overexpressing Trib2. • Knockdown of Trib2 in MLL-TET1 transduced cells induces myeloid differentiation.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oxigenases de Função Mista/metabolismo , Células Progenitoras Mieloides/fisiologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7
19.
Sci Signal ; 11(549)2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254057

RESUMO

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein down-regulation through drug "off-targets" might be relevant for other inhibitors that serendipitously target pseudokinases.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/enzimologia , Afatinib/farmacologia , Alelos , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Células HeLa , Humanos , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , Células U937
20.
Biochemistry (Mosc) ; 83(6): 613-628, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30195320

RESUMO

A large body of experimental data have shown that aerobic exercise of different duration, intensity, and pattern affect molecular mechanisms regulating mitochondrial biogenesis in skeletal muscles. This review focuses on the effects of exercise duration and intensity on the molecular mechanisms of mitochondrial biogenesis regulation in skeletal muscles, namely PGC-1α-dependent signaling. Studies of the effects of acute exercise and exercise training showed that an increase in the duration of aerobic exercise from 30 to 90 min does not provide additional stimuli to activate signaling pathways regulating post-translational modification of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and expression of the PGC-1α gene (PPARGC1A). Conversely, exercise intensity substantially affects mitochondrial biogenesis due to the increase in the recruitment of type II muscle fibers with accompanying pronounced metabolic shift leading to the activation of signaling cascades and expression of genes regulating mitochondrial biogenesis. Therefore, intermittent exercise, which recruits type II muscle fibers, is more efficient in the activation of mitochondrial biogenesis than work-matched continuous exercise. In skeletal muscle adapted to aerobic training, intensity-dependent activation of mitochondrial biogenesis after acute exercise is associated primarily with the AMP-activated protein kinase/PGC-1α pathway, expression of PGC-1α-regulated genes, and expression of PPARGC1A from the alternative (distal) inducible promoter regulated by the cAMP response element-binding protein 1-related transcription factors and their coactivators. Elucidation of the effects of duration and intensity of aerobic exercise on the PGC-1α-dependent and -independent mechanisms of mitochondrial biogenesis is important for treatment of patients with various metabolic disorders, as well as for optimization of training in athletes.


Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Exercício Físico , Humanos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética
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