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1.
Adv Exp Med Biol ; 1164: 225-233, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576552

RESUMO

Immune checkpoint blockade (ICB) has proved successful in the immunotherapeutic treatment of various human cancers. Despite its success, most patients are still not cured while immunogenic cold cancers are still poorly responsive. There is a need for novel clinical interventions in immunotherapy, either alone or in conjunction with ICB. Here, we outline our recent discovery that the intracellular signaling kinase glycogen synthase kinase-3 (GSK-3) is a central regulator of PD-1 in T-cells. We demonstrate the application of small molecule inhibitor (SMI) approaches to down-regulate PD-1 in tumor immunotherapy. GSK-3 SMIs were found as effective as anti-PD-1 in the elimination of melanoma in mouse models. We propose the development of novel SMIs to target co-receptors for the future of immunotherapy.


Assuntos
Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase , Imunoterapia , Melanoma , Animais , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Humanos , Melanoma/terapia , Camundongos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/fisiologia
2.
J Agric Food Chem ; 67(27): 7694-7705, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31250637

RESUMO

Liver plays a central role in modulating blood glucose level. Our most recent findings suggested that supplementation with microbiota metabolite sodium butyrate (NaB) could ameliorate progression of type 2 diabetes mellitus (T2DM) and decrease blood HbA1c in db/db mice. To further investigate the role of butyrate in homeostasis of blood glucose and glycogen metabolism, we carried out the present study. In db/db mice, we found significant hypertrophy and steatosis in hepatic lobules accompanied by reduced glycogen storage, and expression of GPR43 was significantly decreased by 59.38 ± 3.33%; NaB administration significantly increased NaB receptor G-protein coupled receptor 43 (GPR43) level and increased glycogen storage in both mice and HepG2 cells. Glucose transporter 2 (GLUT2) and sodium-glucose cotransporter 1 (SGLT1) on cell membrane were upregulated by NaB. The activation of intracellular signaling Protein kinase B (PKB), also known as AKT, was inhibited while glycogen synthase kinase 3 (GSK3) was activated by NaB in both in vivo and in vitro studies. The present study demonstrated that microbiota metabolite NaB possessed beneficial effects on preserving blood glucose homeostasis by promoting glycogen metabolism in liver cells, and the GPR43-AKT-GSK3 signaling pathway should contribute to this effect.


Assuntos
Ácido Butírico/administração & dosagem , Diabetes Mellitus Tipo 2/metabolismo , Glicogênio Hepático/metabolismo , Animais , Glicemia/análise , Ácido Butírico/metabolismo , Imunofluorescência , Microbioma Gastrointestinal/fisiologia , Transportador de Glucose Tipo 2/análise , Hemoglobina A Glicada/análise , Quinase 3 da Glicogênio Sintase/metabolismo , Células Hep G2 , Homeostase/efeitos dos fármacos , Humanos , Fígado/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/análise , Transdução de Sinais/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/análise
3.
Int J Mol Sci ; 20(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060255

RESUMO

GSK3 (glycogen synthase kinase 3) is a conserved protein kinase governing numerous regulatory pathways. In Drosophila melanogaster, GSK3 is encoded by shaggy (sgg), which forms 17 annotated transcripts corresponding to 10 protein isoforms. Our goal was to demonstrate how differential sgg transcription affects lifespan, which GSK3 isoforms are important for the nervous system, and which changes in the nervous system accompany accelerated aging. Overexpression of three sgg transcripts affected the lifespan in a stage- and tissue-specific way: sgg-RA and sgg-RO affected the lifespan only when overexpressed in muscles and in embryos, respectively; the essential sgg-RB transcript affected lifespan when overexpressed in all tissues tested. In the nervous system, only sgg-RB overexpression affected lifespan, causing accelerated aging in a neuron-specific way, with the strongest effects in dopaminergic neurons and the weakest effects in GABAergic neurons. Pan-neuronal sgg-RB overexpression violated the properties of the nervous system, including the integrity of neuron bodies; the number, distribution, and structure of mitochondria; cytoskeletal characteristics; and synaptic activity. Such changes observed in young individuals indicated premature aging of their nervous system, which paralleled a decline in survival. Our findings demonstrated the key role of GSK3 in ensuring the link between the pathology of neurons and lifespan.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Estágios do Ciclo de Vida/genética , Longevidade/genética , Animais , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Especificidade de Órgãos/genética , Fenótipo
4.
Dev Genes Evol ; 229(4): 89-102, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31041506

RESUMO

The Wnt/beta-catenin pathway has many key roles in the development of animals, including a conserved and central role in the specification of the primary (antero-posterior) body axis. The posterior expression of Wnt ligands and the anterior expression of secreted Wnt inhibitors are known to be conserved during the larval metamorphosis of tapeworms. However, their downstream signaling components for Wnt/beta-catenin signaling have not been characterized. In this work, we have studied the core components of the beta-catenin destruction complex of the human pathogen Echinococcus multilocularis, the causative agent of alveolar echinococcosis. We focused on two Axin paralogs that are conserved in tapeworms and other flatworm parasites. Despite their divergent sequences, both Axins could robustly interact with one E. multilocularis beta-catenin paralog and limited its accumulation in a heterologous mammalian expression system. Similarly to what has been described in planarians (free-living flatworms), other beta-catenin paralogs showed limited or no interaction with either Axin and are unlikely to function as effectors in Wnt signaling. Additionally, both Axins interacted with three divergent GSK-3 paralogs that are conserved in free-living and parasitic flatworms. Axin paralogs have highly segregated expression patterns along the antero-posterior axis in the tapeworms E. multilocularis and Hymenolepis microstoma, indicating that different beta-catenin destruction complexes may operate in different regions during their larval metamorphosis.


Assuntos
Proteína Axina/genética , Complexo de Sinalização da Axina/genética , Echinococcus multilocularis/genética , Quinase 3 da Glicogênio Sintase/genética , Proteínas de Helminto/genética , Hymenolepis/genética , beta Catenina/genética , Sequência de Aminoácidos , Animais , Proteína Axina/química , Proteína Axina/metabolismo , Complexo de Sinalização da Axina/química , Echinococcus multilocularis/crescimento & desenvolvimento , Echinococcus multilocularis/metabolismo , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Helminto/química , Humanos , Hymenolepis/crescimento & desenvolvimento , Hymenolepis/metabolismo , Larva/metabolismo , Filogenia , Alinhamento de Sequência , beta Catenina/metabolismo
5.
Plant Sci ; 283: 290-300, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128699

RESUMO

Moso bamboo (Phyllostachys edulis) is one of the fastest growing species with a maximum growth rate of 1 m/day. However, the regulator genes for this explosive growth phenomenon have not been functionally studied. Here, we found that Moso bamboo GSK3/shaggy-like kinase 1 (PeGSK1) acts as a negative regulator of cell growth. Over-expression of PeGSK1 in Arabidopsis showed significant growth arrest phenotypes, including dwarfism, small leaves, reduced cell length, and disturbed cell elongation of petiole. Furthermore, Overexpression of PeGSK1 fully inhibited the longer hypocotyl phenotype of Arabidopsis atgsk1 mutants. In addition, PeGSK1-overexpressing lines were resistant to exogenous BR treatment and PeGSK1 interacted with the brassinosteroid signal transduction key regulator BZR1. The BZR1-dependent cell growth genes were down-regulated in PeGSK1-overexpressing lines. These results indicated that PeGSK1 is functionally similar to AtGSK1 and inhibited cell growth via the brassinosteroid signaling pathway. Importantly, PeGSK1 also interacted with PeBZR1, and the expression pattern of PeGSK1 was negatively correlated with the internode elongation of bamboo, indicating that PeGSK1 is involved in the cell growth of bamboo. In summary, our results provide insight into the role of brassinosteroids in the rapid-growth of bamboo culms and identifying target genes for the genetic manipulation of plant height.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Proteínas de Plantas/fisiologia , Sasa/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Clonagem Molecular , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Nucleares/metabolismo , Filogenia , Proteínas de Plantas/genética , Sasa/genética , Sasa/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de DNA
6.
Turk Neurosurg ; 29(4): 513-521, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984989

RESUMO

AIM: To investigate the apoptotic and molecular effects of glycogen synthase kinase-3 (GSK-3) in glioblastoma multiforme (GBM). MATERIAL AND METHODS: Human primary glioblastoma cell line (U-87 MG) and the human fetal glial cell line (SVGp12) were used. The cells were exposed to the different doses of GSK inhibitor for 24, 48 and 72 hours. Induction of apoptosis was assessed by DNA fragmentation (TUNEL) assay. EGFR and NF-kB expression was evaluated by immunofluorescence analyses. RESULTS: GSK-3 inhibitor IX induced cytotoxicity and apoptosis in dose-dependent manner in GBM cells. Our results indicated that GSK-3 inhibitor IX induces apoptosis, resulting in a significant decrease in the expression of NF-kB and EGF. CONCLUSION: Inhibition through GSK-3 has been found promising in creating therapeutic management of GBM cells. Proliferation, differentiation, cell cycle regulation, and apoptosis are mechanisms that must be interpreted as a whole. Components associated with EGFR, NF-kB, and apoptosis affect the mechanism solely and collectively. Our collective data suggest that GSK-3 inhibitor IX inhibited cellular proliferation and induced apoptotic events by modulating EGFR and NF-kB expression in GBM cells. GSK-3 inhibition holds promise for the development of new approaches for the therapeutic management of GBM cells.


Assuntos
Apoptose/fisiologia , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
7.
Nat Commun ; 10(1): 1582, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952843

RESUMO

A major target of insulin signaling is the FoxO family of Forkhead transcription factors, which translocate from the nucleus to the cytoplasm following insulin-stimulated phosphorylation. Here we show that the Forkhead transcription factors FoxK1 and FoxK2 are also downstream targets of insulin action, but that following insulin stimulation, they translocate from the cytoplasm to nucleus, reciprocal to the translocation of FoxO1. FoxK1/FoxK2 translocation to the nucleus is dependent on the Akt-mTOR pathway, while its localization to the cytoplasm in the basal state is dependent on GSK3. Knockdown of FoxK1 and FoxK2 in liver cells results in upregulation of genes related to apoptosis and down-regulation of genes involved in cell cycle and lipid metabolism. This is associated with decreased cell proliferation and altered mitochondrial fatty acid metabolism. Thus, FoxK1/K2 are reciprocally regulated to FoxO1 following insulin stimulation and play a critical role in the control of apoptosis, metabolism and mitochondrial function.


Assuntos
Fatores de Transcrição Forkhead/fisiologia , Insulina/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Fatores de Transcrição Forkhead/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Camundongos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
8.
Chem Biol Interact ; 305: 180-194, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30928401

RESUMO

Didymin is a naturally occurring orally active flavonoid glycoside (isosakuranetin 7-O-rutinoside) found in various citrus fruits, which has been previously reported to possess a wide variety of pharmacological activities including anticancer, antioxidant, antinociceptive, neuroprotective, hepatoprotective, inflammatory, and cardiovascular. However, there have not been any reports concerning its anti-diabetic potential until now. Therefore, we evaluated the anti-diabetic potential of didymin via inhibition of α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), rat lens aldose reductase (RLAR), human recombinant AR (HRAR), and advanced glycation end-product (AGE) formation inhibitory assays. Didymin strongly inhibited PTP1B, α-glucosidase, HRAR, RLAR, and AGE in the corresponding assays. Kinetic study revealed that didymin exhibited a mixed type inhibition against α-glucosidase and HRAR, while it competitively inhibited PTP1B and RLAR. Docking simulations of didymin demonstrated negative binding energies and close proximity to residues in the binding pocket of HRAR, RLAR, PTP1B and α-glucosidase, indicating that didymin have high affinity and tight binding capacity towards the active site of these enzymes. Furthermore, we also examined the molecular mechanisms underlying the anti-diabetic effects of didymin in insulin-resistant HepG2 cells which significantly increased glucose uptake and decreased the expression of PTP1B in insulin-resistant HepG2 cells. In addition, didymin activated insulin receptor substrate (IRS)-1 by increasing phosphorylation at tyrosine 895 and enhanced the phosphorylations of phosphoinositide 3-kinase (PI3K), Akt, and glycogen synthasekinase-3(GSK-3). Interestingly, didymin reduced the expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, two key enzymes involved in the gluconeogenesis and leading to a diminished glucose production. The results of the present study clearly demonstrated that didymin will be useful for developing multiple target-oriented therapeutic modalities for treatment of diabetes, and diabetes-associated complications.


Assuntos
Flavonoides/farmacologia , Glucose/metabolismo , Glicosídeos/farmacologia , Hipoglicemiantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Citrus/química , Citrus/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Resistência à Insulina , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo
9.
PLoS Genet ; 15(3): e1008012, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865627

RESUMO

orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. Here, we report that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. We show that repolarization of the oocyte also requires orb and that orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Animais Geneticamente Modificados , Polaridade Celular/genética , Polaridade Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genes de Insetos , Quinase 3 da Glicogênio Sintase/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Oócitos/citologia , Oogênese/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo
10.
Mol Carcinog ; 58(7): 1181-1193, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30834573

RESUMO

Junctional adhesion molecule A (JAM-A) is a transmembrane protein that contributes to different biological process, including the epithelial to mesenchymal transition (EMT). Through an EMT profiler array, we explored the molecular players associated with human thyroid cancer progression and identified JAM-A as one of the genes mostly deregulated. The quantitative real-time polymerase chain reaction and immunohistochemistry analyses showed that downregulation of JAM-A occurred in anaplastic thyroid carcinoma (ATC) compared with normal thyroid (NT) and papillary thyroid carcinoma (PTC) tissues and correlated with extrathyroid infiltration, tumor size, and ATC histotype. In ATC cell lines, JAM-A restoration suppressed malignant hallmarks of transformation including cell proliferation, motility, and transendothelial migration. Accordingly, knockdown of JAM-A enhanced thyroid cancer cell proliferation and motility in PTC cells. Through the proteome profiler human phospho-kinase array, we demonstrated that higher expression of JAM-A was associated with a significant increased level of phosphorylation of p53 and GSK3 α/ß proteins. In conclusion, our findings highlight a novel role of JAM-A in thyroid cancer progression and suggest that JAM-A restoration could have potential clinical relevance in thyroid cancer treatment.


Assuntos
Moléculas de Adesão Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Receptores de Superfície Celular/metabolismo , Câncer Papilífero da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/patologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Neoplasias da Glândula Tireoide/patologia
11.
Cell Commun Signal ; 17(1): 21, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30832675

RESUMO

BACKGROUND: Platelet-activating factor (PAF) is a potent lipid mediator whose involvement in the onset and progression of atherosclerosis is mediated by, among others, the modulation of cytokine expression patterns. The presence of multiple potential protein-tyrosine phosphatase (PTP) 1B substrates in PAF receptor signaling pathways brought us to investigate its involvement in PAF-induced cytokine expression in monocyte-derived dendritic cells (Mo-DCs) and to study the pathways involved in this modulation. METHODS: We used in-vitro-matured human dendritic cells and the HEK-293 cell line in our studies. PTP1B inhibition was though siRNAs and a selective inhibitor. Cytokine expression was studied with RT-PCR, luciferase assays and ELISA. Phosphorylation status of kinases and transcription factors was studied with western blotting. RESULTS: Here, we report that PTP1B was involved in the modulation of cytokine expression in PAF-stimulated Mo-DCs. A study of the down-regulation of PAF-induced IL-8 expression, by PTP1B, showed modulation of PAF-induced transactivation of the IL-8 promoter which was dependent on the presence of the C/EBPß -binding site. Results also suggested that PTP1B decreased PAF-induced IL-8 production by a glycogen synthase kinase (GSK)-3-dependent pathway via activation of the Src family kinases (SFK). These kinases activated an unidentified pathway at early stimulation times and the PI3K/Akt signaling pathway in a later phase. This change in GSK-3 activity decreased the C/EBPß phosphorylation levels of the threonine 235, a residue whose phosphorylation is known to increase C/EBPß transactivation potential, and consequently modified IL-8 expression. CONCLUSION: The negative regulation of GSK-3 activity by PTP1B and the consequent decrease in phosphorylation of the C/EBPß transactivation domain could be an important negative feedback loop by which cells control their cytokine production after PAF stimulation.


Assuntos
Interleucina-8/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Interleucina-8/genética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo
12.
Genes Cells ; 24(5): 354-365, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30838725

RESUMO

The biological relation between ubiquitin ligases and their substrates has been largely unclear. We previously developed a method-differential proteomics-based identification of ubiquitylation substrates (DiPIUS)-for the comprehensive identification of substrates for a given ubiquitin ligase. We have now applied DiPIUS to the F-box protein Fbxw7 in three cell lines (mHepa, Neuro2A and C2C12) and thereby identified Krüppel-like factor 7 (KLF7) as a candidate substrate of the SCFFbxw7 ubiquitin ligase complex. KLF7 was shown to interact with Fbxw7 and to undergo Fbxw7-mediated polyubiquitylation. The stability of KLF7 was increased by depletion of Fbxw7, mutation of a putative Cdc4 phosphodegron (CPD) of KLF7 or exposure to inhibitors of glycogen synthase kinase-3 (GSK-3). Over-expression of Fbxw7 in Neuro2A cells down-regulated expression of the p21Cip1 gene, which is a transcriptional target of KLF7 in neuronal differentiation and maintenance. Despite the presence of an almost identical CPD sequence in KLF6, the closest paralog of KLF7, mutation of this sequence affected neither the interaction of KLF6 with Fbxw7 nor its half-life. Our results suggest that KLF7, but not KLF6, is a bona fide substrate of SCFFbxw7 , and that control of KLF7 abundance by SCFFbxw7 might contribute to the regulation of neuronal differentiation and maintenance.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteólise , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Fosforilação , Ubiquitinação
13.
Eur J Med Chem ; 171: 221-234, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30925338

RESUMO

Glycogen synthase kinase 3α (GSK-3α) plays a constitutive role in various physiological processes and has been proved to be a therapeutic target for acute myeloid leukemia (AML). In this paper, by means of computer-aided drug design, we discovered a novel chemical series of GSK-3α inhibitors with an IC50 value of 0.033-2.804 µM. The preliminary structure-activity relationship was concluded and, notably, the most potent and isoform-selective compound G28_14 was identified with IC50 values of 33 nM and 218 nM against GSK-3α and -3ß, respectively, exhibiting a nearly ten-fold isoform-selectivity. Further cell viability assays and colony formation assays revealed that G28_14 suppressed cell survival by impairing cell proliferation by up to 90% in two AML cell lines. Moreover, surface marker expression analysis demonstrated that G28_14 induced terminal differentiation with a high level of CD11b, CD11c, and CD14. Western immunoblotting showed that G28_14 isoform-selectively inhibited the phosphorylation of GSK-3α in-cell without activating Wnt/ß-catenin signaling. In addition, to elucidate its structure-activity relationship, the binding mode of this chemical series was proposed using molecular docking and molecular dynamics simulations. Taken together, this chemical series is worth developing as differentiation therapies for the treatment of AML.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
14.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871024

RESUMO

To better understand the inflammation-associated mechanisms modulating and terminating tumor necrosis factor (TNF-)induced signal transduction and the development of TNF tolerance, we analyzed both the proteome and the phosphoproteome in TNF long term-incubated (i.e., 48 h) primary human monocytes using liquid chromatography-mass spectrometry. Our analyses revealed the presence of a defined set of proteins characterized by reproducible changes in expression and phosphorylation patterns in long term TNF-treated samples. In total, 148 proteins and 569 phosphopeptides were significantly regulated (103 proteins increased, 45 proteins decreased; 377 peptides with increased and 192 peptides with decreased phosphorylation). A variety of these proteins are associated with the non-canonical nuclear factor κB (NF-κB) pathway (nuclear factor κB (NFKB) 2, v-rel reticuloendotheliosis viral oncogene homolog (REL) B, indolamin-2,3-dioxygenase (IDO), kynureninase (KYNU)) or involved in the negative regulation of the canonical NF-κB system. Within the phosphopeptides, binding motifs for specific kinases were identified. Glycogen synthase kinase (GSK) 3 proved to be a promising candidate, since it targets NF-κB inhibiting factors, such as CCAAT/enhancer binding protein (C/EBP) ß. Our experiments demonstrate that both proteome and phosphoproteome analysis can be effectively applied to study protein/phosphorylation patterns of primary monocytes. These results provide new regulatory candidates and evidence for a complex network of specific but synergistically acting/cooperating mechanisms enabling the affected cells to resist sustained TNF exposure and resulting in the resolution of inflammation.


Assuntos
Monócitos/metabolismo , Proteoma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Inflamação/metabolismo , NF-kappa B/metabolismo , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Células THP-1
15.
Malar J ; 18(1): 89, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898128

RESUMO

BACKGROUND: Malaria is one of the most prevalent tropical infectious diseases. Since recently cases of artemisinin resistance were reported, novel anti-malarial drugs are required which differ from artemisinins in structure and biological target. The plasmodial glycogen synthase kinase-3 (PfGSK-3) was suggested as a new anti-malarial drug target. 4-Phenylthieno[2,3-b]pyridines were previously identified as selective PfGSK-3 inhibitors with antiplasmodial activity. The present study aims at identifying a molecular position on this scaffold for the attachment of side chains in order to improve solubility and antiplasmodial activity. Furthermore, the role of axial chirality in the compound class for antiplasmodial activity and PfGSK-3 inhibition was investigated. METHODS: 4-Phenylthieno[2,3-b]pyridines with substituents in 4-position of the phenyl ring were docked into the ATP binding site of PfGSK-3. The compounds were synthesized employing a Thorpe reaction as final step. The enantiomers of one congener were separated by chiral HPLC. All derivatives were tested for inhibition of asexual erythrocytic stages of transgenic NF54-luc Plasmodium falciparum. Selected compounds with promising antiplasmodial activity were further evaluated for inhibition of HEK293 cells as well as inhibition of isolated PfGSK-3 and HsGSK-3. The kinetic aqueous solubility was assessed by laser nephelometry. RESULTS: The para position at the 4-phenyl ring of the title compounds was identified as a suitable point for the attachment of side chains. While alkoxy substituents in this position led to decreased antiplasmodial activity, alkylamino groups retained antiparasitic potency. The most promising of these congeners (4h) was investigated in detail. This compound is a selective PfGSK-3 inhibitor (versus the human GSK-3 orthologue), and exhibits improved antiplasmodial activity in vitro as well as better solubility in aqueous media than its unsubstituted parent structure. The derivative 4b was separated into the atropisomers, and it was shown that the (+)-enantiomer acts as eutomer. CONCLUSIONS: The attachment of alkylamino side chains leads to the improvement of antiplasmodial activity and aqueous solubility of selective PfGSK-inhibitors belonging to the class of 4-phenylthieno[2,3-b]pyridines. These molecules show axial chirality, a feature of high impact for biological activity. The findings can be exploited for the development of improved selective PfGSK-3 inhibitors.


Assuntos
Antimaláricos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Piridinas/farmacologia , Células HEK293 , Humanos , Relação Estrutura-Atividade
16.
Mol Cell ; 73(4): 788-802.e7, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30704899

RESUMO

mTORC1 and GSK3 play critical roles in early stages of (macro)autophagy, but how they regulate late steps of autophagy remains poorly understood. Here we show that mTORC1 and GSK3-TIP60 signaling converge to modulate autophagosome maturation through Pacer, an autophagy regulator that was identified in our recent study. Hepatocyte-specific Pacer knockout in mice results in impaired autophagy flux, glycogen and lipid accumulation, and liver fibrosis. Under nutrient-rich conditions, mTORC1 phosphorylates Pacer at serine157 to disrupt the association of Pacer with Stx17 and the HOPS complex and thus abolishes Pacer-mediated autophagosome maturation. Importantly, dephosphorylation of Pacer under nutrient-deprived conditions promotes TIP60-mediated Pacer acetylation, which facilitates HOPS complex recruitment and is required for autophagosome maturation and lipid droplet clearance. This work not only identifies Pacer as a regulator in hepatic autophagy and liver homeostasis in vivo but also reveals a signal integration mechanism involved in late stages of autophagy and lipid metabolism.


Assuntos
Autofagossomos/enzimologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Quinase 3 da Glicogênio Sintase/metabolismo , Metabolismo dos Lipídeos , Fígado/enzimologia , Lisina Acetiltransferase 5/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Quinase 3 da Glicogênio Sintase/genética , Células HEK293 , Humanos , Gotículas Lipídicas/metabolismo , Fígado/patologia , Lisina Acetiltransferase 5/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Fosfato/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Transdução de Sinais , Transativadores/genética
17.
PLoS One ; 14(2): e0211239, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30721232

RESUMO

Serotonin-1B receptors (5-HT1BRs) modulate perseverative behaviors and prepulse inhibition (PPI) in humans and mice. These inhibitory G-protein-coupled receptors signal through a canonical G-protein-coupled pathway that is modulated by GSK-3ß, and a noncanonical pathway mediated by the adaptor protein ß-arrestin2 (Arrb2). Given the development of biased ligands that differentially affect canonical versus noncanonical signaling, we examined which signaling pathway mediates 5-HT1BR agonist-induced locomotor perseveration and PPI deficits, behavioral phenotypes observed in both obsessive-compulsive disorder (OCD) and autism spectrum disorder (ASD). To assess the role of canonical 5-HT1BR signaling, mice received acute pretreatment with a GSK-3 inhibitor (SB216763 or AR-A014418) and acute treatment with the 5-HT1A/1B receptor agonist RU24969 prior to assessing perseverative locomotor behavior in the open field, and PPI. To determine the role of noncanonical 5-HT1BR signaling, Arrb2 wild-type (WT), heterozygous (HT), and knockout (KO) mice received acute RU24969 treatment prior to behavioral testing. GSK-3 inhibition increased locomotor perseveration overall, and also failed to influence the RU24969-induced perseverative locomotor pattern in the open field. Yet, GSK-3 inhibition modestly reduced RU24969-induced PPI deficits. On the other hand, Arrb2 HT and KO mice showed reduced locomotion and no changes in perseveration overall, in addition to modest reductions in RU24969-induced locomotion and PPI deficits. In conclusion, our data do not support use of either GSK-3 inhibitors or ß-arrestin2 inhibition in treatment of perseverative behaviors.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Locomoção , Inibição Pré-Pulso , Receptor 5-HT1B de Serotonina/metabolismo , Transdução de Sinais , beta-Arrestina 2/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Feminino , Genótipo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Indóis/farmacologia , Locomoção/efeitos dos fármacos , Maleimidas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Inibição Pré-Pulso/efeitos dos fármacos , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , beta-Arrestina 2/genética
18.
Eur J Med Chem ; 168: 58-77, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30798053

RESUMO

Both cholinesterases (AChE and BChE) and kinases, such as GSK-3α/ß, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3α/ß (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5 nM for human acetylcholinesterase (hAChE) and 7 nM for GSK-3α/ß. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp.


Assuntos
Acetilcolinesterase/metabolismo , Antineoplásicos/farmacologia , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Triazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
19.
Dev Cell ; 48(2): 135-150, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30695696

RESUMO

Pluripotent embryonic stem cells (ESCs) are considered to have open and accessible chromatin relative to differentiated cells. However, as many studies supporting these conclusions relied on ESCs grown in serum, it has been suggested that some of these features are the result of culture conditions, particularly as more recent work using GSK3/MEK inhibitors ("2i") to mimic "ground-state" conditions of the pre-implantation blastocyst observed some altered epigenetic features. Here, we systematically review chromatin and epigenetic features in 2i- and serum-grown conditions to come to a clearer picture of what are genuine characteristics of pluripotency and what open chromatin features predict pluripotency.


Assuntos
Diferenciação Celular/fisiologia , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Animais , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia
20.
Planta ; 249(5): 1391-1403, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30673841

RESUMO

MAIN CONCLUSION: BR signaling pathways facilitate xylem differentiation and wood formation by fine tuning SlBZR1/SlBZR2-mediated gene expression networks involved in plant secondary growth. Brassinosteroid (BR) signaling and BR crosstalk with diverse signaling cues are involved in the pleiotropic regulation of plant growth and development. Recent studies reported the critical roles of BR biosynthesis and signaling in vascular bundle development and plant secondary growth; however, the molecular bases of these roles are unclear. Here, we performed comparative physiological and anatomical analyses of shoot morphological growth in a cultivated wild-type tomato (Solanum lycopersicum cv. BGA) and a BR biosynthetic mutant [Micro Tom (MT)]. We observed that the canonical BR signaling pathway was essential for xylem differentiation and sequential wood formation by facilitating plant secondary growth. The gradual retardation of xylem development phenotypes during shoot vegetative growth in the BR-deficient MT tomato mutant recovered completely in response to exogenous BR treatment or genetic complementation of the BR biosynthetic DWARF (D) gene. By contrast, overexpression of the tomato Glycogen synthase kinase 3 (SlGSK3) or CRISPR-Cas9 (CR)-mediated knockout of the tomato Brassinosteroid-insensitive 1 (SlBRI1) impaired BR signaling and resulted in severely defective xylem differentiation and secondary growth. Genetic modulation of the transcriptional activity of the tomato Brassinazole-resistant 1/2 (SlBZR1/SlBZR2) confirmed the positive roles of BR signaling pathways for xylem differentiation and secondary growth. Our data indicate that BR signaling pathways directly promote xylem differentiation and wood formation by canonical BR-activated SlBZR1/SlBZR2.


Assuntos
Brassinosteroides/metabolismo , Xilema/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica de Plantas , Quinase 3 da Glicogênio Sintase/metabolismo , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
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