RESUMO
Pyruvate kinase M2 (PKM2) plays a pivotal role in the growth, survival and metabolic reprogramming of cancer cells. Here, we presented for the first time that tanshinone â ¡A inhibited human esophagus cancer cell growth through miR-122-mediated PKM2 down-regulation pathway. Tanshinone â ¡A inhibited cell proliferation and induced cell cycle arrest in S phase in human Ec109 cells. As expected, tanshinone â ¡A down-regulated PKM2 mRNA and protein expression in Ec109 cells. Given these findings, we further investigated microRNAs regulation of PKM2 and confirmed miR-122 for targeting PKM2. Moreover, we found that tanshinone â ¡A-induced up-regulation of miR-122 expression inhibited PKM2 expression in Ec109 cells. Meanwhile, tanshinone â ¡A inhibited proliferation through miR122-medated PKM2 down-regulation. It was demonstrated that the anticancer activity of tanshinone â ¡A was targeted at metabolic regulation of miR-122/PKM2 in human esophagus cancer cells. Taken together, our results revealed tanshinone â ¡A targeting at PKM2-mediated metabolic reprogramming play an important role in inhibition of esophageal cancer cell growth.
Assuntos
Abietanos/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/biossíntese , Proteínas de Neoplasias/biossíntese , Piruvato Quinase/biossíntese , RNA Neoplásico/biossíntese , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Células HeLa , HumanosRESUMO
BACKGROUND: Concurrence of high glucose or diabetes in patients with dyslipidemia is presenting major challenges for clinicians. Although sporadically reported, a rational basis for the use of fibrates for the treatment of dyslipidemia with concurrent metabolic syndrome has not been established. METHODS: In this study, wild-type (WT) and Ppara-null (KO) mice were fed a serial gemfibrozil- and fenofibrate-containing diet under the same experimental conditions for 14 days. Glucose level in the blood, glycogen storage in the liver tissues, and the potential toxic responses were assayed. Genes involved in glucose metabolism were determined by quantitative polymerase chain reaction analysis. RESULTS: Both the blood glucose level and the glycogen content in the liver were down-regulated by gemfibrozil but not by fenofibrate in WT mice, in a dose-dependent manner. This decrement did not occur in KO mice for either fibrate agent. Secondary regulation on the transcription of pyruvate kinase, and gluconolactonase were observed following gemfibrozil treatment, which was differential between WT mice and KO mice. CONCLUSIONS: Gemfibrozil, not fenofibrate, down-regulates systemic glucose level and glycogen storage in the liver dependent on PPARα, suggesting its potential value for treatment of dyslipidemia with concurrent diabetes or high glucose levels.
Assuntos
Fenofibrato/farmacologia , Genfibrozila/farmacologia , Glucose/metabolismo , Hipolipemiantes/farmacologia , PPAR alfa/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Glicogênio/metabolismo , Hepatomegalia/genética , Hepatomegalia/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , PPAR alfa/genética , Piruvato Quinase/biossínteseRESUMO
Influence of γ-irradiation of barley seeds (Nur variety) at the doses of 8-50 Gy on catalase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and guaiacol peroxidase activities was studied in the seedlings on the 3, 5 and 7 days after germination. It has been shown that activities of the studied enzymes increase in the dose range that causes the growth stimulation in the seedlings (16-20 Gy).
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hordeum/efeitos da radiação , Plântula/efeitos da radiação , Sementes/efeitos da radiação , Catalase/biossíntese , Raios gama , Germinação/efeitos da radiação , Glucosefosfato Desidrogenase/biossíntese , Hordeum/enzimologia , Hordeum/crescimento & desenvolvimento , Peroxidase/biossíntese , Piruvato Quinase/biossíntese , Plântula/enzimologia , Sementes/enzimologiaRESUMO
BACKGROUND: ChREBP (carbohydrate response element binding protein) is a glucose-responsive transcription factor that is known to be an important regulator of glycolytic and lipogenic genes in response to glucose. We hypothesized that activation of ChREBP could be relevant to anoxia survival by the anoxia-tolerant turtle, Trachemys scripta elegans. METHODS: Expression of ChREBP in response to 5 and 20h of anoxia was examined using RT-PCR and Western immunoblotting. In addition, subcellular localization and DNA-binding activity of ChREBP protein were assessed and transcript levels of liver pyruvate kinase (LPK), a downstream gene under ChREBP control were quantified using RT-PCR. RESULTS: ChREBP was anoxia-responsive in kidney and liver, with transcript levels increasing by 1.2-1.8 fold in response to anoxia and protein levels increasing by 1.8-1.9 fold. Enhanced nuclear presence under anoxia was also observed in both tissues by 2.2-2.8 fold. A 4.2 fold increase in DNA binding activity of ChREBP was also observed in liver in response to 5h of anoxia. In addition, transcript levels of LPK increased by 2.1 fold in response to 5h of anoxia in the liver. CONCLUSIONS: The results suggest that activation of ChREBP in response to anoxia might be a crucial factor for anoxia survival in turtle liver by contributing to elevated glycolytic flux in the initial phases of oxygen limitation. GENERAL SIGNIFICANCE: This study provides the first demonstration of activation of ChREBP in response to anoxia in a natural model of anoxia tolerance, further improving our understanding of the molecular nature of anoxia tolerance.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipóxia/metabolismo , Fígado/metabolismo , Piruvato Quinase/biossíntese , Animais , Hipóxia/patologia , Fatores de Tempo , TartarugasRESUMO
Kaposi's sarcoma (KS) is a vascular neoplasm caused by infection of endothelial or endothelial precursor cells with the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8). Research efforts have focused on defining the molecular events explaining how KSHV promotes pathological angiogenesis and KS tumor formation. mTOR/HIF-1 is a fundamental pathway driving these processes through the upregulation of angiogenic and inflammatory proteins, including VEGF, ANGPTL4, and ANGPT2. Interestingly, HIF-1 has also been implicated in the upregulation of metabolic genes associated with aerobic glycolysis and the growth of solid tumors. However, whether HIF-1 plays a role in regulating cell metabolism in KS remains unexplored. Here, we show that the HIF-1 metabolic effector, pyruvate kinase 2 (PKM2), is upregulated upon KSHV infection of endothelial cells and is necessary to maintain aerobic glycolysis in infected cells. We further demonstrate that PKM2 regulates KS angiogenic phenotype by acting as a coactivator of HIF-1 and increasing the levels of HIF-1 angiogenic factors, including VEGF. Indeed, inhibition of PKM2 expression blocked endothelial cell migration and differentiation and the angiogenic potential of KSHV-infected cells. We also investigated whether PKM2 regulates the angiogenic dysregulation induced by the KSHV-encoded G protein-coupled receptor (vGPCR), a viral oncogene that promotes Kaposi's sarcomagenesis through the upregulation of HIF angiogenic factors. Interestingly, we found that PKM2 controls vGPCR-induced VEGF paracrine secretion and vGPCR oncogenesis. Our findings provide a molecular mechanism for how HIF-1 dysregulation fuels both angiogenesis and tumor metabolism in KS and support further investigations on therapeutic approaches targeting HIF-1 and PKM2 for KS treatment.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicólise , Herpesvirus Humano 8/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Piruvato Quinase/biossíntese , Sarcoma de Kaposi/metabolismo , Regulação para Cima , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Patológica/patologia , Sarcoma de Kaposi/patologiaRESUMO
The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned in the bacterial expression vector pProEX HTc. The construct carried a hexahistidine tag in N-terminal position to facilitate purification of the recombinant protein. Production of high levels of soluble recombinant PKc1 in Escherichia coli was only possible when using a co-expression strategy with the chaperones GroES-GroEL. Purification of the protein by Ni(2 +) chelation chromatography yielded a single protein with an apparent molecular mass of 58kDa and a specific activity of 34unitsmg(-1) protein. The recombinant enzyme had an optimum pH between 6 and 7. It was relatively heat stable as it retained 80% of its activity after 2min at 75°C. Hyperbolic saturation kinetics were observed with ADP and UDP whereas sigmoidal saturation was observed during analysis of phosphoenolpyruvate binding. Among possible effectors tested, aspartate and glutamate had no effect on enzyme activity, whereas α-ketoglutarate and citrate were the most potent inhibitors. When tested on phosphoenolpyruvate saturation kinetics, these latter compounds increased S0.5. These findings suggest that S. tuberosum PKc1 is subject to a strong control by respiratory metabolism exerted via citrate and other tricarboxylic acid cycle intermediates.
Assuntos
Citosol/química , Fosfoenolpiruvato/química , Proteínas de Plantas/isolamento & purificação , Piruvato Quinase/isolamento & purificação , Solanum tuberosum/química , Difosfato de Adenosina/química , Ácido Cítrico/química , Clonagem Molecular , Citosol/enzimologia , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Concentração de Íons de Hidrogênio , Ácidos Cetoglutáricos/química , Cinética , Peso Molecular , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Solanum tuberosum/enzimologia , Difosfato de Uridina/químicaRESUMO
Severe aplastic anemia (SAA) is a syndrome of severe bone marrow failure with high mortality. Our previous studies have demonstrated that both immature and activated DC1 increased in the bone marrow of SAA patients, and the balance of DC1 subsets shifted the stable form to active one, which might promote Th0 cells to polarize to Th1 cells and cause the over-function of T lymphocytes and hematopoiesis failure in SAA. So we assumed myeloid dendritic cells (mDCs) may be the key immune cells that cause destruction of hematopoietic cells in SAA, but the mechanism of activation of mDCs is unclear. Here, we investigated the proteome of mDCs in SAA patients to further explore the pathogenesis of SAA and the possible antigen that leads to immune activation in SAA. mDCs from 12 SAA patients, 12 remission patients and 12 controls were sorted by flow cytometry and examined by two-dimensional gel electrophoresis and mass spectrometry. Intensity changes of 41 spots were detected with statistical significance. Nine of the 41 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels were found in the SAA group. These changes reveal that abnormal expression of cofilin, glucose-6-phosphate dehydrogenase and pyruvate kinase enzyme M2 in mDCs from SAA patients may be the reason for mDC hyperfunction.
Assuntos
Anemia Aplástica/metabolismo , Células Dendríticas/metabolismo , Células Mieloides/metabolismo , Células Th1/metabolismo , Adolescente , Adulto , Medula Óssea/metabolismo , Células Cultivadas , Cofilina 1/biossíntese , Células Dendríticas/imunologia , Feminino , Expressão Gênica , Glucosefosfato Desidrogenase/biossíntese , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Proteoma/biossíntese , Proteoma/metabolismo , Piruvato Quinase/biossíntese , Células Th1/imunologia , Adulto JovemRESUMO
This study aimed at investigating the extracellular and cell wall-associated proteins (exoproteome) of Lactobacillus plantarum DC400 when cultivated on modified chemically defined medium (CDM) supplemented with the chemically synthesized pheromone plantaricin A (PlnA) or cocultured with L. plantarum DPPMA20 or Lactobacillus sanfranciscensis DPPMA174. Compared to monoculture, two-dimensional gel electrophoresis (2-DE) analysis showed that the exoproteome of L. plantarum DC400 was affected by PlnA and cocultivation with strains DPPMA20 and, especially, DPPMA174. The highest similarity of the 2-DE maps was found between DC400 cells cultivated in monoculture and in coculture with strain DPPMA20. Almost all extracellular proteins (22 spots) and cell wall-associated proteins (40 spots) which showed decreased or increased levels of synthesis during growth in CDM supplemented with PlnA and/or in coculture with strain DPPMA20 or DPPMA174 were identified. On the basis of the sequences in the Kyoto Encyclopedia of Genes and Genomes database, changes to the exoproteome concerned proteins involved in quorum sensing (QS), the transport system, stress response, carbohydrate metabolism and glycolysis, oxidation/reduction processes, the proteolytic system, amino acid metabolism, cell wall and catabolic processes, and cell shape, growth, and division. Cultivation with PlnA and cocultivation with strains DPPMA20 and, especially, DPMMA174 markedly increased the capacity of L. plantarum DC400 to form biofilms, to adhere to human Caco-2 cells, and to prevent the adhesion of potential intestinal pathogens. These phenotypic traits were in part related to oversynthesized moonlighting proteins (e.g., DnaK and GroEL, pyruvate kinase, enolase, and glyceraldehyde-3-phosphate dehydrogenase) in response to QS mechanisms and interaction with L. plantarum DPPMA20 and, especially, L. sanfranciscensis DPPMA174.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Bacteriocinas/farmacologia , Lactobacillus plantarum/fisiologia , Proteínas de Bactérias/biossíntese , Biofilmes/crescimento & desenvolvimento , Células CACO-2 , Linhagem Celular , Chaperonina 60/biossíntese , Chaperonina 60/metabolismo , Técnicas de Cocultura , Regulação Bacteriana da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Proteínas de Membrana/metabolismo , Fosfopiruvato Hidratase/biossíntese , Fosfopiruvato Hidratase/metabolismo , Proteoma/efeitos dos fármacos , Piruvato Quinase/biossíntese , Piruvato Quinase/metabolismo , Percepção de QuorumRESUMO
This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.
Assuntos
Butanóis/metabolismo , Clostridium acetobutylicum/metabolismo , Expressão Gênica , Fosfofrutoquinase-1/biossíntese , Piruvato Quinase/biossíntese , Acetona/metabolismo , Trifosfato de Adenosina/metabolismo , Biotecnologia/métodos , Clostridium acetobutylicum/genética , Etanol/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , NAD/metabolismo , Fosfofrutoquinase-1/genética , Piruvato Quinase/genéticaRESUMO
OBJECTIVE: To investigate the enhancement effect of the combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin in the treatment of lung cancer. METHODS: Twenty five BABL/nu/nu mice bearing A549 lung cancer were divided into 5 groups (NS control, psh-Control, psh-PKM2 treated group, Endostar treated group, psh-PKM2+Endostar treated group) and treated with shRNA interfering plasmid targeting PKM2 and recombinant Endostatin respectively or in combination. The expression of PKM2 in A549 detected with immunofluorescent assay. The interference effect of psh-PKM2 was determined by Western blot. The tumor volume, microvessel density (MVD), apoptosis index (AI) and side effects were observed. RESULTS: The combination treatment of RNA interfering plasmid targeting PKM2 with recombinant Endostatin inhibited tumor growth obviously (P < 0.05); The combination group revealed a decreased MVD and an increased AI (P < 0.05). CONCLUSION: The combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin might enhance anti-tumor effect by increasing the apoptosis of the cancer cell.
Assuntos
Apoptose/fisiologia , Endostatinas/uso terapêutico , Neoplasias Pulmonares/terapia , Piruvato Quinase/genética , RNA Interferente Pequeno/genética , Animais , Linhagem Celular Tumoral , Endostatinas/biossíntese , Endostatinas/genética , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Plasmídeos/genética , Piruvato Quinase/biossíntese , Proteínas Recombinantes/uso terapêuticoRESUMO
The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) induces a battery of cytoprotective genes after oxidative stress. Nrf2 aids in liver regeneration by altering insulin signaling; however, whether Nrf2 participates in hepatic glucose homeostasis is unknown. Compared with wild-type mice, mice lacking Nrf2 (Nrf2-null) have lower basal serum insulin and prolonged hyperglycemia in response to an intraperitoneal glucose challenge. In the present study, blood glucose, serum insulin, urine flow rate, and hepatic expression of glucose-related genes were quantified in male diabetic wild-type and Nrf2-null mice. Type 1 diabetes was induced with a single intraperitoneal dose (200 mg/kg) of streptozotocin (STZ). Histopathology and serum insulin levels confirmed depleted pancreatic beta-cells in STZ-treated mice of both genotypes. Five days after STZ, Nrf2-null mice had higher blood glucose levels than wild-type mice. Nine days after STZ, polyuria occurred in both genotypes with more urine output from Nrf2-null mice (11-fold) than wild-type mice (7-fold). Moreover, STZ-treated Nrf2-null mice had higher levels of serum beta-hydroxybutyrate, triglycerides, and fatty acids 10 days after STZ compared with wild-type mice. STZ reduced hepatic glycogen in both genotypes, with less observed in Nrf2-null mice. Increased urine output and blood glucose in STZ-treated Nrf2-null mice corresponded with enhanced gluconeogenesis (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase)- and reduced glycolysis (pyruvate kinase)-related mRNA expression in their livers. Furthermore, the Nrf2 activator oltipraz lowered blood glucose in wild-type but not Nrf2-null mice administered STZ. Collectively, these data indicate that the absence of Nrf2 worsens hyperglycemia in type I diabetic mice and Nrf2 may represent a therapeutic target for reducing circulating glucose levels.
Assuntos
Diabetes Mellitus Tipo 1/genética , Intolerância à Glucose/genética , Hiperglicemia/genética , Fator 2 Relacionado a NF-E2/genética , Animais , Glicemia/análise , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Intolerância à Glucose/patologia , Intolerância à Glucose/fisiopatologia , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/genética , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Insulina/análise , Fígado/metabolismo , Glicogênio Hepático/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD(P)H Desidrogenase (Quinona)/biossíntese , NAD(P)H Desidrogenase (Quinona)/genética , Pâncreas/metabolismo , Pâncreas/patologia , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Pirazinas/farmacologia , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , RNA Mensageiro/biossíntese , Tionas , Tiofenos , UrodinâmicaRESUMO
A multifaceted approach is presented as a general strategy to identify new drug targets in a breast cancer stem cell-containing side population. The approach we have utilized combines side population cell sorting and stable isotope labeling by amino acids in cell culture with mass spectrometry to compare and identify proteins with differential expression profiles between side population cells, know to be enriched in cancer stem cells, and nonside population cells, which are depleted in cancer stem cells, for two breast cancer cell lines, MCF7 and MDA-MB231. Almost 900 proteins were quantified, and several important proteins in cell cycle control and differentiation were found to be upregulated in the cancer stem cell-containing side population. Most interestingly, a splice isoform of pyruvate kinase M2 as well as peroxiredoxin 6 were found to be downregulated. The differential levels of three of these proteins, thymosin beta4 (TB4), proliferation-associated protein 2G4, and SIAH-interacting protein, were validated using Western blot. Furthermore, functional validation provided clear evidence that elevated TB4 expression contributes to drug resistance in the stem cell population. Small interfering RNA silencing of TB4 led to a loss of chemoresistance in two separate breast cancer populations. These proteins likely contribute to resistance in the cancer stem cell-containing side population, and their altered expression in a tumor causes clinical resistance to chemotherapy. The ability to perform quantitative mass spectrometry has enabled the identification of a series of proteins that could serve as future therapeutic targets.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Regulação Neoplásica da Expressão Gênica , Espectrometria de Massas/métodos , Células-Tronco Neoplásicas/citologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Feminino , Humanos , Camundongos , Camundongos SCID , Proteínas Nucleares , Peroxirredoxina VI/biossíntese , Piruvato Quinase/biossíntese , Proteínas de Ligação a RNA , Timosina/biossínteseRESUMO
The soluble cytoplasmic protein pyruvate kinase (PK) has been expressed at the cell surface in a membrane-anchored form (APK). The hybrid protein contains the NH2-terminal signal/anchor domain of a class II integral membrane protein (hemagglutinin/neuraminidase, of the paramyxovirus SV5) fused to the PK NH2 terminus. APK contains a cryptic site that is used for N-linked glycosylation but elimination of this site by site-specific mutagenesis does not prevent cell surface localization. Truncated forms of the APK molecule, with up to 80% of the PK region of APK removed, can also be expressed at the cell surface. These data suggest that neither the complete PK molecule nor its glycosylation are necessary for intracellular transport of PK to the cell surface, and it is possible that specific signals may not be needed in the ectodomain of this hybrid protein to specify cell surface localization.
Assuntos
Membrana Celular/enzimologia , Citoplasma/enzimologia , Piruvato Quinase/metabolismo , Animais , Imunofluorescência , Regulação da Expressão Gênica , Glicosilação , Proteína HN , Plasmídeos , Testes de Precipitina , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Zonula occludens proteins (ZOPs) are essential scaffold proteins involved in the organization of epithelial and endothelial intercellular junctions. Based on their molecular domain architecture, they are members of the large family of membrane-associated guanylate kinase-like (MAGUK) proteins. As all other MAGUKs, ZOPs contain a core of several PDZ, an src homology-3, and a guanylate kinase-like domain, indicating that these proteins may serve both structural and signaling functions. In addition, ZOPs exhibit some unique motifs not shared by other MAGUKs, i.e., several nuclear localization (NLS) and nuclear export signals (NES), allowing these proteins to shuttle between the cytoplasm and the nucleus. However, the stimuli leading to the nuclear accumulation of ZOPs and the resulting physiological consequences remain poorly defined. We have previously reported the direct binding of nuclear ZO-2 to scaffold attachment factor B, a heterogeneous nuclear ribonucleoprotein involved in chromatin organization and the transcriptional control of eukaryotic genes. We now report that the nuclear accumulation of ZO-2 leads to an increase in the expression of the M2 type of pyruvate kinase (M2-PK) in epithelial and endothelial cells. Further, the proliferative activity was increased, while the intercellular junctional stability of Madin-Darby canine kidney cells was reduced. Our data provide evidence to suggest that ZO-2 exerts a junction-unrelated function that further supports the notion of a general "dual" role of junctional MAGUKs, being an indispensable structural component at cell-cell junctions and a nuclear factor influencing gene expression and cell behavior.
Assuntos
Células Endoteliais/fisiologia , Células Epiteliais/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células , Cães , Regulação da Expressão Gênica/fisiologia , Junções Intercelulares/metabolismo , Proteínas Nucleares/genética , Piruvato Quinase/biossíntese , Transfecção , Proteína da Zônula de Oclusão-2RESUMO
Dietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element-binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Glicólise/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP , Animais , Células Cultivadas , Gorduras Insaturadas na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Ácido Eicosapentaenoico/farmacologia , Ácido Graxo Sintases/biossíntese , Ácido Graxo Sintases/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Glicólise/fisiologia , Hepatócitos/enzimologia , Lipogênese/fisiologia , Masculino , Camundongos , Complexos Multienzimáticos/metabolismo , Proteínas Nucleares/metabolismo , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Nerve growth factor (NGF) is a potent mediator, which regulates characteristics of mast cells, but its biological function is not well characterized. This study aimed to screen proteins associated with the maturation of human mast cells-1 (HMC-1) or mouse bone marrow-derived mast cells (BMMCs) cultured with NGF, and to examine the functions of proteins involved. METHODS: NGF (10 ng/ml) was added to cell culture medium every other day for 10 days for HMC-1 or twice a week for 5 weeks for BMMCs. Granule formation was determined by electron microscopy or May-Grunwald-Giemsa staining, TNF-alpha by ELISA, expressions of various proteins by two-dimensional gel electrophoresis (2-DE), siRNA transfection by Lipofectamine 2000, and the expressions of pyruvate kinase and annexin I by immunoblotting. RESULTS: After NGF treatment, granule formation and total amounts of granular mediator, TNF-alpha increased in both mast cells. This TNF-alpha was released by calcium ionophore or by antigen/antibody reaction. Expressions of pyruvate kinase and annexin I obtained by 2-DE were confirmed by immunoblotting and siRNA-transfected HMC-1 cells. Expressions of proteins, granule formation and TNF-alpha content were blocked by both the TrkA inhibitor, K252a, and the ERK inhibitor, PD98059, but not by the PI3 kinase inhibitors, LY294002 and wortmannin. CONCLUSION: These data suggest that pyruvate kinase and annexin I expressed by NGF contribute to granule formation containing TNF-alpha as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway.
Assuntos
Anexina A1/biossíntese , Grânulos Citoplasmáticos/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Piruvato Quinase/biossíntese , Androstadienos/farmacologia , Animais , Anexina A1/imunologia , Carbazóis/farmacologia , Linhagem Celular Tumoral , Cromonas/farmacologia , Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/ultraestrutura , Eletroforese em Gel Bidimensional , Inibidores Enzimáticos/farmacologia , Feminino , Flavonoides/farmacologia , Humanos , Immunoblotting , Alcaloides Indólicos/farmacologia , Mastócitos/citologia , Mastócitos/enzimologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Morfolinas/farmacologia , Fator de Crescimento Neural/imunologia , Fosforilação , Piruvato Quinase/imunologia , RNA Interferente Pequeno/imunologia , Transfecção , Fator de Necrose Tumoral alfa/imunologia , WortmaninaRESUMO
The African clawed frog, Xenopus laevis, can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis. Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower Km for phosphoenolpyruvate (PEP) (38%), a lower Ka for fructose-1,6-bisphosphate (F1,6P2) (32%), and a greater activation of PK via F1,6P2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.
Assuntos
Desidratação/metabolismo , Estivação , Piruvato Quinase/biossíntese , Transcrição Gênica , Proteínas de Xenopus/biossíntese , Animais , Cinética , Especificidade de Órgãos , Xenopus laevisRESUMO
Recently, malic acid has gained attention due to its potential application in food, pharmaceutical, and medical industries. In this study, the synthetic scaffold complex strategy was employed between the two key enzymes pyruvate kinase (PykF) and malic enzyme (SfcA); SH3 ligand was attached to PykF, and the SH3 domain was attached to the C-terminus of ScfA. Synthetic scaffold systems can organize enzymes spatially and temporally to increase the local concentration of intermediates. In a flask culture, the recombinant strain harboring scaffold complex produced a maximum concentration of 5.72 g/L malic acid from 10 g/L glucose. The malic acid production was significantly increased 2.1-fold from the initial culture period. Finally, malic acid production was elevated to 30.2 g in a 5 L bioreactor from recombinant strain XL-1 blue.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Malato Desidrogenase , Malatos/metabolismo , Piruvato Quinase , Proteínas Recombinantes de Fusão , Domínios de Homologia de src , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Malato Desidrogenase/biossíntese , Malato Desidrogenase/genética , Piruvato Quinase/biossíntese , Piruvato Quinase/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
OBJECTIVE: To characterize the biologic featrues of hepatic oval cells and their protein expression profiles during induced differentiation in vitro. METHODS: Rat hepatic oval cells were treated with epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro, followed by morphological and molecular marker assessment by electromicroscopy, immunocytochemistry, RT-PCR and protein expression chip technology. RESULTS: Ten weeks after induction, the levels of GST-P mRNA and M2-PK mRNA were significantly reduced, whereas those of ALB and CK18 were elevated. Significant variations of expression was seen in 8 protein species during the course of the induced differentiation. CONCLUSION: Combined EGF and HGF treatment in vitro induces cell differentiation of hepatic oval cells, a process in which 8 protein species may play some regulatory roles.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Glutationa Transferase/biossíntese , Fator de Crescimento de Hepatócito/farmacologia , Hepatócitos/citologia , Piruvato Quinase/biossíntese , Albuminas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Glutationa Transferase/genética , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Imuno-Histoquímica , Queratina-18/metabolismo , Análise Serial de Proteínas , Piruvato Quinase/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Pyruvate kinase M2 (PKM2) is the key enzyme in the Warburg effect and plays a central role in cancer cell metabolic reprogramming. Recently, quite a few studies have investigated the correlation between PKM2 expression and prognosis in multiple cancer patients, but results were inconsistent. We therefore performed a meta-analysis to explore the prognostic value of PKM2 expression in patients with solid cancer. Here twenty-seven individual studies from 25 publications with a total of 4796 cases were included to explore the association between PKM2 and overall survival (OS) or disease-free survival (DFS)/ progression-free survival (PFS)/ recurrent-free survival (RFS) in subjects with solid cancer. Pooled analysis showed that high levels of PKM2 was significantly associated with a poorer overall survival (HR = 1.73; 95%CI = 1.48-2.03) and DFS/ PFS/ RFS (HR = 1.90; 95%CI = 1.39-2.59) irrespective of cancer types. Different analysis models (univariate or multivariate models), sample-sizes (≤100 or >100), and methods for data collection (direct extraction or indirect extraction) had no impact on the negative prognostic effect of PKM2 over-expression. Nevertheless, stratified by cancer type, high-expression of PKM2 was associated with an unfavorable OS in breast cancer, esophageal squamous carcinoma, hepatocellular carcinoma and gallbladder cancer; whereas was not correlated with a worse OS in pancreatic cancer and gastric cancer. In conclusion, over-expression of PKM2 is associated with poor prognosis in most solid cancers and it might be a potentially useful biomarker for predicting cancer prognosis in future clinical applications.