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1.
Theriogenology ; 141: 62-67, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518730

RESUMO

Primordial germ cells (PGCs) are precursors of sperms and oocytes and responsible for passing the genetic information from one generation to the next. Chicken PGCs segregate from somatic cells in early embryo and could be isolated and cultured in vitro, making it a useful tool to produce genetically modified animals. However, the number of PGCs isolated from embryo is limited and these cells are not efficient to proliferation in vitro. GSK-3 plays an important role in multiple intracellular signaling pathways and inhibition of GSK-3-mediated ß-catenin phosphorylation is known to reduce apoptosis and promote proliferation in T cells and embryo stem cells (ESC). In this study, we investigate the effect of GSK-3 inhibitor on the proliferation of PGCs in vitro and found significant increases of cell proliferation in the culture supplemented with CHIR. We further found that CHIR regulates PGC cell cycle by activating Wnt signaling and antagonizing the apoptosis of PGCs by inhibition of the expression of caspase-3 and Beclin-1. PGCs treated with CHIR expressed the germ cell-related markers and retain the capability to colonize the embryonic gonad after re-introduction to vasculature of HH stage-15 embryos. These results suggest that GSK-3 is involved in cell renewal and apoptosis in chicken PGCs.


Assuntos
Embrião de Galinha/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Germinativas/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Via de Sinalização Wnt/efeitos dos fármacos
2.
PLoS Genet ; 15(8): e1008136, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381575

RESUMO

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/genética , Hidroxiureia/toxicidade , Nucleotídeos/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética
3.
Mol Carcinog ; 58(11): 2077-2090, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31411358

RESUMO

The plasma membrane (PM) is considered as a major druggable site. More than 50% of the existing drugs target PM proteins. In the wake of emerging data indicating a key role of estrogens in prostate cancer (PCa) pathogenesis, the study was undertaken to explore whether the estrogen binding sites exist on the PM and if such sites are functionally relevant in PCa. Estradiol (E2) binding to the PM was detected in androgen-dependent (LNCaP), androgen-independent (PC3, DU145) PCa cell lines, nontumorigenic (RWPE1) prostate epithelial cell line, and rat prostate cells. Conventional estrogen receptors (nuclear estrogen receptors), known for their nuclear localization, were detected in the PM enriched extracts. This was indirectly confirmed by reduced localization of ERs on the PM of cells, silenced for the expression of their cognate genes. Further, unlike cell-permeable E2, stimulation with cell-impermeable estradiol (E2-BSA) did not induce proliferation in LNCaP cells. However, stimulation with E2-BSA led to alterations in the phosphorylation status of several kinases including GSK3 and AKT, along with the hyperphosphorylation of cytoskeletal proteins such as ß-actin and cytokeratin 8 in LNCaP. This was accompanied by epithelial-to-mesenchymal (EMT) features such as increased migration and invasion; higher vimentin expression, and a concomitant decrease in the E-cadherin expression. These effects were not observed in RWPE1 cells. Interestingly, cell-permeable E2 failed to induce EMT in PCa cells. This in vitro study is the first to suggest that the PM-initiated estrogen signaling contributes to higher invasiveness in PCa cells. Plasma membrane ERs may act as novel targets for PCa therapeutics.


Assuntos
Androgênios/metabolismo , Membrana Celular/genética , Estrogênios/metabolismo , Neoplasias da Próstata/genética , Animais , Caderinas/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Transição Epitelial-Mesenquimal/genética , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Humanos , Queratina-8/genética , Masculino , Camundongos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Ratos , Transdução de Sinais
4.
Biomed Res Int ; 2019: 9628065, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31467921

RESUMO

The reparative process following spinal cord injury (SCI) is extremely complicated. Cells in the microenvironment express multiple inhibitory factors that affect axonal regeneration over a prolonged period of time. The axon growth inhibitory factor glycogen synthase kinase-3 (GSK-3) is an important factor during these processes. TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione) is the most effective and specific non-ATP-competitive inhibitor of GSK-3. Here, we show that administering TDZD-8 after SCI was associated with significantly inhibited neuronal apoptosis, upregulated GAP-43 expression, increased density of cortical spinal tract fibers around areas of injury, and increased Basso, Beattie, and Bresnahan (BBB) scores in the lower limbs. These findings support the notion that GSK-3 inhibitors promote neuronal cell regeneration and lower limb functional recovery.


Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Regeneração Nervosa/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Tiadiazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Axônios/efeitos dos fármacos , Modelos Animais de Doenças , Proteína GAP-43/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Humanos , Atividade Motora/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Recuperação de Função Fisiológica/efeitos dos fármacos , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia
5.
J Toxicol Sci ; 44(7): 493-503, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31270305

RESUMO

Methamphetamine (METH) is a potent and highly addictive central nervous system stimulant. The association between METH exposure and Alzheimer's disease (AD) has gained more attention, but, the mechanisms behind METH-induced neuron-related adverse outcomes remain poorly understood. With the western blot assay, our results revealed that METH exposure significantly increased the expression of AD-associated pathological proteins, including the amyloid precursor protein (APP) and the phosphorylated tau protein (p-tau). Meanwhile, the insulin signaling was disturbed after the administration of METH, since the key insulin signaling proteins, such as p-AKT, p-GSK3α, p-GSK3ß and p-ERK, were reduced. Additionally, the linking between the pathological proteins and the insulin signaling mediated by METH in the present work was verified by the treatment with the insulin signaling enhancer rosiglitazone, which was shown to improve the insulin signaling and decrease APP and p-tau expression. Thus, targeting insulin signaling may provide novel insights into potential therapeutic intervention for METH-mediated AD-like neurodegeneration.


Assuntos
Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Estimulantes do Sistema Nervoso Central/efeitos adversos , Expressão Gênica , Insulina/fisiologia , Metanfetamina/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas tau/genética , Proteínas tau/metabolismo , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/genética , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia
6.
Food Funct ; 10(8): 4725-4738, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31304955

RESUMO

Antrodia camphorata is a well-known traditional Chinese mushroom used as a functional food and nutraceutical in Taiwan and China. The aim of this study was to explore the protective effects and mechanism(s) of the ethyl acetate crude extract of A. camphorata (EtOAc-AC) and its active constituent ergostatrien-7,9(11),22-trien-3ß-ol (EK100) in an acute ischemic stroke (AIS) murine model. Treating mice with induced AIS injury by using EtOAc-AC (0.3-0.6 g kg-1, p.o.) and EK100 (60 and 120 mg kg-1, p.o.) 2 h after AIS induction significantly increased the tracking distance and reduced brain infarction. Both EtOAc-AC and EK-100 reduced the expression levels of p65NF-κB and caspase 3 near the peri-infarct cortex and promoted the expression of neurogenesis-associated protein doublecortin (DCX) near the hippocampus, accompanied by glycogen synthase kinase 3 (GSK-3) inhibition and ß-catenin upregulation. Signaling pathway analysis revealed that the advantageous effects of EtOAc-AC and EK-100 involved triggering the activation of PI3K/Akt and inhibition of GSK-3. Our findings suggest that EtOAc-AC and its active constituent EK100 display anti-inflammatory and anti-apoptotic activities. Both EtOAc-AC and EK100 reduce ischemic brain injury by decreasing p65NF-κB and caspase 3 expression, and they promote neurogenesis (DCX) and neuroprotection (Bcl2) by activating the PI3k/Akt-associated GSK3 inhibition and ß-catenin activation.


Assuntos
Antrodia/química , Isquemia Encefálica/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Ergosterol/análogos & derivados , Neurogênese/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Apoptose/efeitos dos fármacos , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Caspase 3/genética , Caspase 3/metabolismo , Cateninas/genética , Cateninas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Ergosterol/administração & dosagem , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
7.
Blood ; 134(4): 363-373, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31101621

RESUMO

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and ß are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3ß compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3ß genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3ß binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3ß is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3ß correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.


Assuntos
Quinase 3 da Glicogênio Sintase/genética , Linfoma/etiologia , Terapia de Alvo Molecular , Animais , Biomarcadores Tumorais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Marcação de Genes/métodos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Indóis/farmacologia , Linfoma/diagnóstico , Linfoma/mortalidade , Linfoma/terapia , Maleimidas/farmacologia , Camundongos , Camundongos Transgênicos , Mitose/efeitos dos fármacos , Mitose/genética , Terapia de Alvo Molecular/efeitos adversos , Terapia de Alvo Molecular/métodos , Fuso Acromático/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
8.
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
9.
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
10.
PLoS One ; 14(4): e0214610, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969984

RESUMO

Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel/uso terapêutico , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Aberrações Cromossômicas/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Paclitaxel/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo
11.
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
12.
PLoS One ; 14(3): e0199484, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30856175

RESUMO

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate.


Assuntos
Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio/farmacologia , Triptofano/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Detergentes/farmacologia , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/fisiologia , Genes Fúngicos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos
13.
Dev Biol ; 449(2): 99-106, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30826399

RESUMO

The developmental biology of neural crest cells in humans remains unexplored due to technical and ethical challenges. The use of pluripotent stem cells to model human neural crest development has gained momentum. We recently introduced a rapid chemically defined approach to induce robust neural crest by WNT/ß-CATENIN activation. Here we investigate the temporal requirements of ectopic WNT activation needed to induce neural crest cells. By altering the temporal activation of canonical WNT/ß-CATENIN with a GSK3 inhibitor we find that a 2 Day pulse of WNT/ß-CATENIN activation via GSK3 inhibition is optimal to generate bona fide neural crest cells, as shown by their capacity to differentiate to neural crest specific fates including peripheral neurons, glia, melanoblasts and ectomesenchymal osteocytes, chondrocytes and adipocytes. Although a 2 Day pulse can impart neural crest character when GSK3 is inhibited days after seeding, optimal results are obtained when WNT is activated from the beginning, and we find that the window of competence to induce NCs from non-neural ectodermal/placodal precursors closes by day 3 of culture. The reduced requirement for exogenous WNT activation offers an approach that is cost-effective, and we show that this adherent 2-dimensional approach is efficient in a broad range of culture platforms ranging from 96-well vessels to 10 cm dishes.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias Humanas/metabolismo , Crista Neural/metabolismo , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ectoderma/citologia , Ectoderma/embriologia , Ectoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Crista Neural/citologia , Crista Neural/embriologia , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Células-Tronco Pluripotentes/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismo
14.
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 , Peptídeos e Proteínas de Sinalização Intracelular , Gotículas Lipídicas/metabolismo , Fígado/patologia , Lisina Acetiltransferase 5/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana , 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
15.
Nat Commun ; 10(1): 403, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679422

RESUMO

Albuminuria affects millions of people, and is an independent risk factor for kidney failure, cardiovascular morbidity and death. The key cell that prevents albuminuria is the terminally differentiated glomerular podocyte. Here we report the evolutionary importance of the enzyme Glycogen Synthase Kinase 3 (GSK3) for maintaining podocyte function in mice and the equivalent nephrocyte cell in Drosophila. Developmental deletion of both GSK3 isoforms (α and ß) in murine podocytes causes late neonatal death associated with massive albuminuria and renal failure. Similarly, silencing GSK3 in nephrocytes is developmentally lethal for this cell. Mature genetic or pharmacological podocyte/nephrocyte GSK3 inhibition is also detrimental; producing albuminuric kidney disease in mice and nephrocyte depletion in Drosophila. Mechanistically, GSK3 loss causes differentiated podocytes to re-enter the cell cycle and undergo mitotic catastrophe, modulated via the Hippo pathway but independent of Wnt-ß-catenin. This work clearly identifies GSK3 as a critical regulator of podocyte and hence kidney function.


Assuntos
Albuminúria/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Nefropatias/metabolismo , Rim/fisiologia , Podócitos/metabolismo , Albuminúria/sangue , Albuminúria/patologia , Albuminúria/urina , Animais , Ciclo Celular , Linhagem Celular , Modelos Animais de Doenças , Drosophila , Deleção de Genes , Inativação Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Estimativa de Kaplan-Meier , Rim/patologia , Nefropatias/sangue , Nefropatias/patologia , Nefropatias/urina , Masculino , Camundongos , Podócitos/enzimologia , Podócitos/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Ratos Wistar , Insuficiência Renal , Verteporfina/farmacologia , beta Catenina/metabolismo
16.
Behav Neurosci ; 133(1): 135-143, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30688489

RESUMO

Akt protein family (Akt1, Akt2 and Akt3) of serine/threonine kinases, also known as protein kinase B, are enzymes implicated in many physiological and pathological processes in the central nervous system. A striking feature of these enzymes is their ability to interact with several molecular targets such as the glycogen synthase kinase 3 (GSK-3). Among Akt isoforms, the Akt3 is significantly more expressed in the brain and the present investigation was designed to determine whether the Akt3/GSK-3 pathway plays a role in the learning of a complex motor skill. Using the accelerating rotarod task, known to reproduce different motor learning phases, we demonstrated in mouse models that genetic deletion of GSK-3α or GSK-3ß had no effect on rotarod performances. However, Akt3 deletion robustly compromised rotarod learning when compared with wild-type animals. Biochemical analysis in the striatum revealed modifications in the levels of both phosphorylated GSK-3 and tau in Akt3-deficient mice, which are reminiscent of enhanced GSK-3 activity. In this line, we observed that both biochemical and motor learning impairments were prevented in Akt3-deficent mice by chronic treatments with lithium, a well-known GSK-3 inhibitor. Altogether, our findings raised the interesting possibility that interconnection between Akt3 and GSK-3 kinases is required in the learning of new complex motor tasks. (PsycINFO Database Record (c) 2019 APA, all rights reserved).


Assuntos
Corpo Estriado/metabolismo , Glicogênio Sintase Quinase 3 beta/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Aprendizagem/fisiologia , Destreza Motora , Animais , Feminino , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta/genética , Masculino , Camundongos Knockout , Teste de Desempenho do Rota-Rod , Transdução de Sinais
17.
Biochem Cell Biol ; 97(2): 201-213, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30352171

RESUMO

B-cell lymphoma/leukemia 11B (Bcl11b) is a transcription factor critical for thymocyte development. We have previously characterized the kinetic post-translational modifications (PTMs) of Bcl11b in double-positive (DP) thymocytes during stimulation of the T cell receptor-activated MAP kinase pathway. However, the PTMs of Bcl11b in thymocytes from other developmental stages in the thymus, primarily double-negative (DN) cells, have not been previously identified. We found that kinetic modifications of Bcl11b in DN cells are somewhat different than the patterns observed in DP cells. Distinct from DP thymocytes, phosphorylation and sumoylation of Bcl11b in DN cells were not oppositely regulated in response to activation of MAP kinase, even though hyper-phosphorylation of Bcl11b coincided with near complete desumoylation. Additionally, prolonged stimulation of the MAP kinase pathway in DN cells, unlike DP thymocytes, did not alter Bcl11b levels of sumoylation or ubiquitinylation, or stability. On the other hand, activation of Wnt-Gsk3-dependent signaling in DN cells resulted in composite dephosphorylation and sumoylation of Bcl11b. Moreover, stimulation of MAP kinase and (or) Wnt signaling pathways differentially affects gene expression of some Bcl11b target and maturation-associated genes. Defining the signaling pathways and regulation of sequence-specific transcription factors by PTMs at various stages of thymopoiesis may improve our understanding of leukemogenesis.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Repressoras/metabolismo , Timócitos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , Quinase 3 da Glicogênio Sintase/genética , Ativação Linfocitária , Camundongos , Proteínas Repressoras/genética , Timócitos/citologia , Proteínas Supressoras de Tumor/genética
18.
Neurochem Int ; 122: 31-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30392874

RESUMO

Currently, no treatments exist that are able to directly treat against Alzheimer's disease (AD), and we are facing an inevitable increase in the near future of the amount of patients who will suffer from AD. Most animal models of AD are limited by not being able to recapitulate the entire pathology of AD. Recently an AD model in zebrafish was established by using the protein phosphatase 2A inhibitor, okadaic acid (OKA). Administering OKA to zebrafish was able to recapitulate most of the neuropathology associated with AD. Therefore, providing a drug discovery model for AD that is also time and cost efficient. This study was designed to investigate the effects of GSK3ß inhibition by 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) on this newly developed AD model. Fish were divided into 4 groups and each group received a different treatment. The fish were divided into a control group, a group treated with 1 µM TDZD-8 only, a group treated with 1 µM TDZD-8 + 100 nM OKA, and a group treated with 100 nM OKA only. Administering the GSK3ß inhibitor to zebrafish concomitantly with OKA proved to be protective. TDZD-8 treatment reduced the mortality rate, the ratio of active: inactive GSK3ß, pTau (Ser199), and restored PP2A activity. This further corroborates the use of GSKß inhibitors in the treatment against AD and bolsters the use of the OKA-induced AD-like zebrafish model for drug discovery.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Encéfalo/irrigação sanguínea , Glicogênio Sintase Quinase 3 beta/farmacologia , Tiadiazóis/farmacologia , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Okadáico/farmacologia , Peixe-Zebra , Proteínas tau/metabolismo
19.
J Cell Biol ; 218(3): 961-976, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30578283

RESUMO

Target of rapamycin complex-2 (TORC2), a conserved protein kinase complex, is an indispensable regulator of plasma membrane homeostasis. In budding yeast (Saccharomyces cerevisiae), the essential downstream effector of TORC2 is protein kinase Ypk1 and its paralog Ypk2. Muk1, a Rab5-specific guanine nucleotide exchange factor (GEF), was identified in our prior global screen for candidate Ypk1 targets. We confirm here that Muk1 is a substrate of Ypk1 and demonstrate that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Strikingly, yeast lacking its two Rab5 GEFs (Muk1 and Vps9) or its three Rab5 paralogs (Vps21/Ypt51, Ypt52, and Ypt53) or overexpressing Msb3, a Rab5-directed GTPase-activating protein, all exhibit pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Vps21 coimmunoprecipitates with TORC2, and immuno-enriched TORC2 is less active in vitro in the absence of Rab5 GTPases. Thus, TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling.


Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética
20.
PLoS One ; 13(12): e0208094, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30557366

RESUMO

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma.


Assuntos
Adenocarcinoma/tratamento farmacológico , Apoptose/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/patologia , Aminofenóis/farmacologia , Aminofenóis/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Cloreto de Lítio/farmacologia , Cloreto de Lítio/uso terapêutico , Maleimidas/farmacologia , Maleimidas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Neoplasias Gástricas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
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