RESUMO
Protein posttranslational modifications play crucial roles in plant immunity through modulating a complicated signaling network mediated by different hormones. We previously demonstrated that OsATL32, an ATL-type E3 ligase, negatively contributes to rice immunity against Magnaporthe oryzae. Here, we show that OsATL32 forms a loop with OsPPKL2 and OsGSK2 through distinct protein posttranslational modifications to modulate rice immunity. OsATL32 ubiquitinates OsPPKL2, a protein phosphatase with Kelch-like repeat domains that exerts positive roles in regulating rice immunity against M. oryzae and chitin-triggered immune responses, for degradation. The glycogen synthase kinase 2 (OsGSK2), which acts as a negative regulator of rice immunity against M. oryzae and chitin-triggered immune responses, phosphorylates OsATL32 to elevate its protein stability and E3 ligase activity on OsPPKL2. Moreover, OsPPKL2 directly dephosphorylates OsGSK2, affecting its kinase activity on substrates including OsATL32 for phosphorylation. Like OsGSK2 as a BR signaling repressor, OsATL32 negatively regulates BR signaling; conversely, OsPPKL2 plays a positive role in BR signaling. These findings provide a molecular mechanism in which OsATL32 serves as a node connecting BR signaling and immunity by associating with OsPPKL2 and OsGSK2, assembling into a distinct protein posttranslational modifications-linked loop that functions in rice BR signaling and immunity.
Assuntos
Oryza , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas , Processamento de Proteína Pós-Traducional , Oryza/genética , Oryza/imunologia , Oryza/microbiologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Fosforilação , Ubiquitinação , Transdução de Sinais , Magnaporthe/fisiologia , Brassinosteroides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Regulação da Expressão Gênica de Plantas , Quitina/metabolismo , Quinases da Glicogênio Sintase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , AscomicetosRESUMO
The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.
Assuntos
Cromatina , Posicionamento Cromossômico , Cromossomos Humanos , Genoma Humano , Quinases da Glicogênio Sintase , Ensaios de Triagem em Larga Escala , Análise de Célula Única , Humanos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Posicionamento Cromossômico/efeitos dos fármacos , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA/análise , DNA/metabolismo , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/deficiência , Quinases da Glicogênio Sintase/genética , Ensaios de Triagem em Larga Escala/métodos , Interfase , Reprodutibilidade dos Testes , RNA/análise , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única/métodos , CoesinasRESUMO
Molecular characterization of fish muscle proteins are nowadays considered as a key component to understand the role of specific proteins involved in various physiological and metabolic processes including their up and down regulation in the organisms. Coldwater fish specimens including snow trouts hold different types of proteins which help them to survive in highly diversified temperatures fluctuating from 0 to 20 °C. So, in current study, the liquid chromatography mass spectrometry using label free quantification technique has been used to investigate the muscle proteome profile of Schizothorax labiatus. For proteomic study, two weight groups of S. labiatus were taken from river Sindh. The proteomic analysis of group 1 revealed that a total of 235 proteins in male and 238 in female fish were recorded. However, when male and female S. labiatus were compared with each other on the basis of spectral count and abundance of peptides by ProteinLynx Global Server software, a total of 14 down-regulated and 22 up-regulated proteins were noted in this group. The highly down-regulated ones included homeodomain protein HoxA2b, retinol-binding protein 4, MHC class II beta chain and proopiomelanocortin while as the highly expressed up-regulated proteins comprised of gonadotropin I beta subunit, NADH dehydrogenase subunit 4, manganese superoxide dismutase, recombinase-activating protein 2, glycosyltransferase, chymotrypsin and cytochrome b. On the other hand, the proteomic characterisation of group 2 of S. labiatus revealed that a total of 227 proteins in male and 194 in female fish were recorded. When male and female S. labiatus were compared with each other by label free quantification, a total of 20 down-regulated and 18 up-regulated proteins were recorded. The down-regulated protein expression of group 2 comprised hepatic lipase, allograft inflammatory factor-1, NADH dehydrogenase subunit 4 and myostatin 1 while the highly expressed up-regulated proteins included glycogen synthase kinase-3 beta variant 2, glycogen synthase kinase-3 beta variant 5, cholecystokinin, glycogen synthase kinase-3 beta variant 3 and cytochrome b. Significant (P < 0.05) difference in the expression of down-regulated and up-regulated proteins was also noted between the two sexes of S. labiatus in each group. According to MS analysis, the proteins primarily concerned with the growth, skeletal muscle development and metabolism were down-regulated in river Sindh, which indicates that growth of fish during the season of collection i.e., winter was slow owing to less food availability, gonad development and low metabolic activity. While, the proteins related to immune response of fish were also noted to be down-regulated thereby signifying that the ecosystem has less pollution loads, microbial, pathogenic and anthropogenic activities. It was also found that the proteins involved in glycogen metabolism, reproductive and metabolic processes, particularly lipid metabolism were up-regulated in S. labiatus. The significant expression of these proteins may be connected to pre-spawning, gonad development and use of stored food as source of energy. The information generated in this study can be applied to future research aimed at enhancing food traceability, food safety, risk management and authenticity analysis.
Assuntos
Cyprinidae , Ecossistema , Animais , Masculino , Feminino , Truta , Cromatografia Líquida , Proteômica/métodos , Citocromos b , Espectrometria de Massas em Tandem , Proteínas de Peixes/genética , Quinases da Glicogênio SintaseRESUMO
Plant Glycogen Synthase Kinases (GSKs) enable a crosstalk among the brassinosteroid signaling and phytohormonal- and stress-response pathways to regulate various physiological processes. Initial information about regulation of the GSK proteins' activity was obtained, however, mechanisms that modulate expression of the GSK genes during plant development and stress responses remain largely unknown. Taking into account the importance of the GSK proteins, combined with the lack of in-depth knowledge about modulation of their expression, research in this area may provide a significant insight into mechanisms regulating these aspects of plant biology. In the current study, a detailed analysis of the GSK promoters in rice and Arabidopsis was performed, including identification of the CpG/CpNpG islands, tandem repeats, cis-acting regulatory elements, conserved motifs, and transcription factor-binding sites. Moreover, characterization of expression profiles of the GSK genes in different tissues, organs and under various abiotic stress conditions was performed. Additionally, protein-protein interactions between products of the GSK genes were predicted. Results of this study provided intriguing information about these aspects and insight into various regulatory mechanisms that influence non-redundant and diverse functions of the GSK genes during development and stress responses. Therefore, they may constitute a reference for future research in other plant species.
Assuntos
Arabidopsis , Oryza , Arabidopsis/genética , Arabidopsis/metabolismo , Oryza/genética , Oryza/metabolismo , Quinases da Glicogênio Sintase/genética , Quinases da Glicogênio Sintase/metabolismo , Estresse Fisiológico/genética , Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , FilogeniaRESUMO
As drug-binding kinetics has become an important factor to be considered in modern drug discovery, this work evaluated the ability of the Milestoning method in computing the absolute dissociation rate of a ligand from the serine-threonine kinase, glycogen synthase kinase 3ß, which is a target for designing drugs to treat diseases such as neurodegenerative disorders and diabetes. We found that the Milestoning method gave good agreement with experiment with modest computational costs. Although the time scale for dissociation lasted tens of seconds, the collective molecular dynamics simulations total less than 1µs. Computing the committor function helped to identify the transition states (TSs), in which the ligand moved substantially away from the binding pocket. The glycine-rich loop with a serine residue attaching to its tips was found to undergo large movement from the bound to the TSs and might play a role in controlling drug-dissociation kinetics.
Assuntos
Simulação de Dinâmica Molecular , Ligantes , Quinases da Glicogênio Sintase , Glicogênio Sintase Quinase 3 betaRESUMO
Memory impairment and emotional disorder are two common clinical comorbidities in patients with epilepsy. It is imperative to develop a novel therapeutic agent or a strategy. 6-Chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) is a dopamine-1 receptor agonist and sigma-1 receptor allosteric modulator, which displays the neuron-protective and anti-neuroinflammation activity. We examined the effect of SKF83959 on the memory impairment and emotional disorder in the latent period of epilepsy using the mice post-status epilepticus model. We found that SKF83959 ameliorated memory impairment and depressive-like mood, alleviated the neuron damage and the formation of gliosis in hippocampus, suppressed the rise of pro-inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, and induced nitric oxide synthase in the latent period of epilepsy. Additionally, SKF83959 significantly inhibited the activity of calcineurin and glycogen synthase kinase-3ß. All of these protective actions were reversed by BD1047 (a sigma-1 receptor antagonist). In addition, the intra-hippocampus injection of ketoconazole (a dehydroepiandrosterone synthesis inhibitor) also reversed the protective activity of SKF83959. Thus, we concluded that SKF83959 ameliorated the memory impairment and depressive-like mood in epilepsy via allosterically activating the sigma-1 receptor and subsequently inhibiting the calcineurin/glycogen synthase kinase-3ß pathway.
Assuntos
Calcineurina , Epilepsia , Ratos , Animais , Camundongos , Regulação Alostérica , Ratos Sprague-Dawley , Agonistas de Dopamina/farmacologia , Epilepsia/tratamento farmacológico , Quinases da Glicogênio Sintase , Receptor Sigma-1RESUMO
Brain microvascular endothelial cells (BMECs) are essential component of the blood-brain barrier (BBB). BMECs strictly regulate the entry of various molecules into the central nervous system from the peripheral circulation by forming tight junctions and expressing various influx/efflux transporters and receptors. In vitro BBB models have been widely reported with primary BMECs isolated from animals, although it is known that the expression patterns and levels of transporters and receptors in BMECs differ between humans and animals. Recently, several methods to differentiate BMECs from human induced pluripotent stem (hiPS) cell have been developed. However, the expression of P-glycoprotein (P-gp), which is a key efflux transporter, in hiPS cell-derived BMECs was detected at a relatively low level compared with primary human BMECs. In this study, we examined the involvement of the canonical Wnt signaling pathway, which contributes to the development of BBB formation, in the regulation of P-gp expression in hiPS cell-derived BMECs. We found that the barrier integrity was significantly enhanced in hiPS cell-derived BMECs treated with glycogen synthase kinase-3ß (GSK-3ß) inhibitors, which are known to positively regulate the canonical Wnt signaling pathway. In addition, our data also showed P-gp expression level was increased by treatment with GSK-3ß inhibitors. In conclusion, physiological barrier function and P-gp expression in BMECs can be enhanced by the canonical Wnt signaling pathway. Our results may be useful for promoting the development of drugs for central nervous system diseases using in vitro BBB model.
Assuntos
Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Diferenciação Celular/fisiologia , Células Endoteliais/metabolismo , Quinases da Glicogênio Sintase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Extra-proliferation and increased migration of vascular smooth cells con-tribute to the formation of atherosclerosis. Ras small G proteins play a critical role in the prolif-eration and migration of a wide range of cells. Mulberry, an economic fruit in Asia, exhibits anti-inflammation, anti-migration, and anti-oxidant properties. The mechanisms of action of mulberry extracts on K-Ras small G protein-induced proliferation and migration of vascular smooth muscle cell have not been extensively investigated. In this study, we explored the effects of mulberry polyphenol extracts (MPE) on the proliferation and migration of K-Ras-overexpressing A7r5 smooth muscle cells. The overexpression of K-Ras enhanced the ex-pression and activity of matrix metalloproteinase (MMP)-2, promoted vascular endothelial growth factor (VEGF) production, and eventually triggered the migration of A7r5 cells. Treatment with MPE attenuated K-Ras-induced phenomenon. In addition, MPE blocked K-Ras-induced actin fibril stress. MPE dose-dependently diminished K-Ras-induced Rho A, Rac1, CDC42, and phosphorylated focal adhesion kinase (FAK) expression. MPE elevated Rho B ex-pression. Phosphorylated AKT and glycogen synthase kinase (GSK) induced by K-Ras were also repressed by MPE treatment. MPE enhanced the interaction of IκB with NFκB. MPE restored the G0/G1 population and p21 and p27 expressions, which were repressed by K-Ras. Finally, MPE triggered the degradation of K-Ras by ubiquitination. MPE inhibited the migration and proliferation of vascular smooth cell through K-Ras-induced pathways and eventually pre-vented atherosclerosis.
Assuntos
Aterosclerose , Proteínas Monoméricas de Ligação ao GTP , Morus , Actinas/metabolismo , Antioxidantes/farmacologia , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Regulação para Baixo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Frutas/metabolismo , Quinases da Glicogênio Sintase/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Músculo Liso Vascular , Miócitos de Músculo Liso , Polifenóis/metabolismo , Polifenóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Alzheimer's disease (AD) has pathological hallmarks including amyloid beta (Aß) plaque formation. Currently approved single-target drugs cannot effectively ameliorate AD. Medicinal herbs and their derived ingredients (MHDIs) have multitarget and multichannel properties, engendering exceptional AD treatment outcomes. This review delineates how in in vivo models MHDIs suppress Aß deposition by downregulating ß- and γ-secretase activities; inhibit oxidative stress by enhancing the antioxidant activities and reducing lipid peroxidation; prevent tau hyperphosphorylation by upregulating protein phosphatase 2A expression and downregulating glycogen synthase kinase-3ß expression; reduce inflammatory mediators partly by upregulating brain-derived neurotrophic factor/extracellular signal-regulated protein kinase 1/2-mediated signaling and downregulating p38 mitogen-activated protein kinase (p38 MAPK)/c-Jun N-terminal kinase (JNK)-mediated signaling; attenuate synaptic dysfunction by increasing presynaptic protein, postsynaptic protein, and acetylcholine levels and preventing acetylcholinesterase activity; and protect against neuronal apoptosis mainly by upregulating Akt/cyclic AMP response element-binding protein/B-cell lymphoma 2 (Bcl-2)-mediated anti-apoptotic signaling and downregulating p38 MAPK/JNK/Bcl-2-associated x protein (Bax)/caspase-3-, Bax/apoptosis-inducing factor-, C/EBP homologous protein/glucose-regulated protein 78-, and autophagy-mediated apoptotic signaling. Therefore, MHDIs listed in this review protect against Aß-induced cognitive decline by inhibiting Aß accumulation, oxidative stress, tau hyperphosphorylation, inflammation, synaptic damage, and neuronal apoptosis in the cortex and hippocampus during the early and late AD phases.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Plantas Medicinais , Acetilcolina , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/uso terapêutico , Fator de Indução de Apoptose/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Caspase 3/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Glucose/efeitos adversos , Quinases da Glicogênio Sintase , Humanos , Mediadores da Inflamação/uso terapêutico , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Plantas Medicinais/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3ß, is a protein kinase that phosphorylates Pah1 on serine (Ser12, Ser602, and Ser818) and threonine (Thr163, Thr164, Thr522) residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 µM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 µM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased â¼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites (Thr163, Thr164, and Ser602) indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfatidato Fosfatase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Animais , Quinases da Glicogênio Sintase/metabolismo , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Saccharomyces cerevisiae/metabolismoRESUMO
The incidence of Alzheimer's disease (AD) is significantly higher in people with diabetes. Insulin and insulin receptor (IR) signaling intermediates are expressed in the brain. Insulin exerts multiple function in the brain. The role of compromised IR signaling in AD pathogenesis and the therapeutic value of insulin attract broad attention. This review summarizes the collective insulin action in the brain related to key factors of AD pathogenesis, updates the key features of insulin resistance in the AD brain and assesses the therapeutic potential of insulin and insulin-sensitizing drugs. Insulin stimulates neural growth and survival, suppresses amyloidogenic processing of the amyloid precursor protein (AßPP) and inhibits the Tau phosphorylation kinase, glycogen synthase kinase 3ß. Central nervous IR signaling regulates systemic metabolism and increases glucose availability to neurons. The expression of IR and its downstream effectors is reduced in AD brain tissues. Insulin and insulin-sensitizing drugs can improve cognitive function in AD patients and AD animal models. Systemic insulin delivery is less effective than intranasal insulin treatment. The penetrance of insulin-sensitizing drugs to the blood brain barrier is problematic and new brain-prone drugs need be developed. Insulin resistance manifested by the degradation and the altered phosphorylation of IR intermediates precedes overt AD syndrome. Type 3 diabetes as a pure form of brain insulin resistance without systemic insulin resistance is proposed as a causal factor in AD. Further research is needed for the identification of critical factors leading to impaired IR signaling and the development of new molecules to stimulate brain IR signaling.
Assuntos
Doença de Alzheimer , Diabetes Mellitus , Resistência à Insulina , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Glucose/metabolismo , Quinases da Glicogênio Sintase/metabolismo , Humanos , Insulina/uso terapêutico , Resistência à Insulina/fisiologia , Preparações Farmacêuticas/metabolismo , Receptor de Insulina/metabolismoRESUMO
The upregulation of the adaptor protein NUMB triggers melanocytic differentiation from multipotent skin stem cells, which share many properties with aggressive melanoma cells. Although NUMB acts as a tumor suppressor in various human cancer types, little is known about its role in melanoma. In this study, we investigated the role of NUMB in melanoma progression and its regulatory mechanism. Analysis of The Cancer Genome Atlas melanoma datasets revealed that high NUMB expression in melanoma tissues correlates with improved patient survival. Moreover, NUMB expression is downregulated in metastatic melanoma cells. NUMB knockdown significantly increased the invasion potential of melanoma cells in a three-dimensional collagen matrix in vitro and in the lungs of a mouse model in vivo; it also significantly upregulated the expression of the NOTCH target gene CCNE. Previous studies suggested that Wnt signaling increases NUMB expression. By mimicking Wnt stimulation through glycogen synthase kinase-3 inhibition, we increased NUMB expression in melanoma cells. Furthermore, a glycogen synthase kinase-3 inhibitor reduced the invasion of melanoma cells in a NUMB-dependent manner. Together, our results suggest that NUMB suppresses invasion and metastasis in melanoma, potentially through its regulation of the NOTCHâCCNE axis and that the inhibitors that upregulate NUMB can exert therapeutic effects in melanoma.
Assuntos
Melanoma , Proteínas de Membrana , Proteínas do Tecido Nervoso , Animais , Linhagem Celular Tumoral , Quinases da Glicogênio Sintase/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Via de Sinalização WntRESUMO
In rice (Oryza sativa) and other plants, plant architecture and seed size are closely related to yield. Brassinosteroid (BR) signaling and the mitogen-activated protein kinase (MAPK) pathway (MAPK kinase kinase 10 [MAPKKK10]-MAPK kinase 4 [MAPKK4]-MAPK6) are two major regulatory pathways that control rice architecture and seed size. However, their possible relationship and crosstalk remain elusive. Here, we show that WRKY53 mediated the crosstalk between BR signaling and the MAPK pathway. Biochemical and genetic assays demonstrated that glycogen synthase kinase-2 (GSK2) phosphorylates WRKY53 and lowers its stability, indicating that WRKY53 is a substrate of GSK2 in BR signaling. WRKY53 interacted with BRASSINAZOLE-RESISTANT 1(BZR1); they function synergistically to regulate BR-related developmental processes. We also provide genetic evidence showing that WRKY53 functions in a common pathway with the MAPKKK10-MAPKK4-MAPK6 cascade in leaf angle and seed size control, suggesting that WRKY53 is a direct substrate of this pathway. Moreover, GSK2 phosphorylated MAPKK4 to suppress MAPK6 activity, suggesting that GSK2-mediated BR signaling might also regulated MAPK pathway. Together, our results revealed a critical role for WRKY53 and uncovered sophisticated levels of interplay between BR signaling and the MAPK pathway in regulating rice architecture and seed size.
Assuntos
Brassinosteroides/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Sementes/fisiologia , Regulação da Expressão Gênica de Plantas , Quinases da Glicogênio Sintase/genética , Quinases da Glicogênio Sintase/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Oryza/genética , Fosforilação , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Estabilidade Proteica , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Primary cell therapy continues to face significant hurdles to therapeutic translation including the inherent variations that exist from donor to donor, batch to batch, and scale-up driven modifications to the manufacturing process. Cardiosphere-derived cells (CDCs) are stromal/progenitor cells with clinically demonstrated tissue reparative capabilities. Mechanistic investigations have identified canonical Wnt/ß-catenin signaling as a therapeutic potency marker, and THY1 (CD90) expression as inversely correlated with potency. Here we demonstrate that the cardiosphere formation process increases ß-catenin levels and enriches for therapeutic miR content in the extracellular vesicles of these cells, namely miR-146a and miR-22. We further find that loss of potency is correlated with impaired cardiosphere formation. Finally, our data show that small GSK3ß inhibitors including CHIR, and BIO and "pro-canonical Wnt" culturing conditions can rescue ß-catenin signaling and reduce CD90 expression. These findings identify strategies that could be used to maintain CDC potency and therapeutic consistency.
Assuntos
Benzamidas/química , Biomarcadores/metabolismo , Difenilamina/análogos & derivados , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Antígenos Thy-1/genética , beta Catenina/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Difenilamina/química , Difenilamina/farmacologia , Vesículas Extracelulares , Fibronectinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Coração , Humanos , Camundongos , MicroRNAs , Antígenos Thy-1/metabolismo , Via de Sinalização WntRESUMO
Glycogen synthase kinase 3 (GSK3) is a highly conserved kinase present in all eukaryotes and functions as a key regulator of a wide range of physiological and developmental processes. The kinase, known in land plants as GSK3/SHAGGY-like kinase (GSK), is a key player in the brassinosteroid (BR) signaling pathway. The GSK genes, through the BRs, affect diverse developmental processes and modulate responses to environmental factors. In this work, we describe functional analysis of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. The RNAi-mediated silencing of the target HvGSK1.1 gene was associated with modified expression of its paralogs HvGSK1.2, HvGSK2.1, HvGSK3.1, and HvGSK4.1 in plants grown in normal and in salt stress conditions. Low nucleotide similarity between the silencing fragment and barley GSK genes and the presence of BR-dependent transcription factors' binding sites in promoter regions of barley and rice GSK genes imply an innate mechanism responsible for co-regulation of the genes. The results of the leaf inclination assay indicated that silencing of HvGSK1.1 and the changes of GSK paralogs enhanced the BR-dependent signaling in the plants. The strongest phenotype of transgenic lines with downregulated HvGSK1.1 and GSK paralogs had greater biomass of the seedlings grown in normal conditions and salt stress as well as elevated kernel weight of plants grown in normal conditions. Both traits showed a strong negative correlation with the transcript level of the target gene and the paralogs. The characteristics of barley lines with silenced expression of HvGSK1.1 are compatible with the expected phenotypes of plants with enhanced BR signaling. The results show that manipulation of the GSK-encoding genes provides data to explore their biological functions and confirm it as a feasible strategy to generate plants with improved agricultural traits.
Assuntos
Quinases da Glicogênio Sintase/fisiologia , Hordeum/fisiologia , Tolerância ao Sal/genética , Sementes/crescimento & desenvolvimento , Biomassa , Brassinosteroides/metabolismo , Inativação Gênica , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimentoRESUMO
The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Glucose/metabolismo , Quinases da Glicogênio Sintase/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Aspergillus nidulans/enzimologia , Repressão Catabólica/genética , Fungos/genética , Fungos/metabolismo , Glicerol/metabolismo , Concentração Osmolar , Fosforilação/genética , Mapas de Interação de Proteínas/genética , Proteínas Repressoras/genética , Xilose/metabolismoRESUMO
The plant extract piperine is used as a traditional Chinese medicine due to its antiinflammatory effects and efficacy against numerous types of cancer. The aim of the present study was to investigate the antitumor mechanism of piperine in human osteosarcoma U2OS and 143B cell lines. The effects of piperine on cell apoptosis and invasion of human osteosarcoma cells were assessed using flow cytometry and Transwell assays. Moreover, western blotting was used to measure the effects of piperine on the protein expression levels of the metastasis markers matrix metalloproteinase2 (MMP2) and vascular endothelial growth factor (VEGF). In addition, the involvement of the Wnt/ßcatenin signaling pathway in modulating the effects of piperine was examined via western blot analysis. The results of MTT and Transwell invasion assays indicated that piperine treatment dosedependently reduced U2OS and 143B cell viability and invasion. Furthermore, a significant reduction was identified in MMP2, VEGF, glycogen synthase kinase3ß and ßcatenin protein expression levels, as well as the expression levels of their target proteins cyclooxygenase2, cyclin D1 and cmyc, in U2OS cells after piperine treatment. In addition, similar results were observed in 143B cells. Therefore, the present study demonstrated the efficacy of piperine in osteosarcoma, and identified that the Wnt/ßcatenin signaling pathway may modulate the antitumor effects of piperine on human U2OS and 143B cells.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzodioxóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclina D1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citometria de Fluxo , Quinases da Glicogênio Sintase/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3ß, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3ß during Dengue virus-2 (DENV-2) infection was investigated. METHODS: GSK3ß participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS: Phosphorylation of GSK3ß-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3ß reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3ß. CONCLUSIONS: The results suggest that GSK3ß participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.
Assuntos
Vírus da Dengue/enzimologia , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/fisiologia , Replicação Viral/fisiologia , Aedes/citologia , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Microscopia de Fluorescência , Fosforilação/fisiologia , Transdução de SinaisRESUMO
Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.