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1.
Exp Parasitol ; 208: 107790, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31697939

RESUMO

Neospora caninum is a protozoan that has tropism for the central nervous system. The aim of this study was to determine whether experimental infection of gerbils would interfere with activity of enzymes associated with energy metabolism. We randomized 20 gerbils into two groups (ten animals per group): the control group (healthy animals; uninfected) and the infected group (experimentally infected with dose 7.8 × 102 tachyzoites of N. caninum per gerbil). On day six and twelve post-infection (PI), brain and spleen tissues were collected for biochemical and histopathological analyses. No histopathological lesions were observed in the brains of infected animals; however, inflammatory infiltrates were found in the spleen. Significantly greater levels of reactive oxygen species (ROS) were observed in the brain and spleen of infected gerbils than in the control group at 12 days PI. Cytosolic creatine kinase (CK-CYT), mitochondrial creatine kinase (CK-MIT), and pyruvate kinase (PK) activities were lower in the brains of infected gerbils than in those of the control group on day 12 PI. There was significantly less CK-CYT activity in the spleens of infected gerbils on day 6 and 12 PI. Finally, there was significantly less sodium-potassium ion pump (Na+/K+ ATPase) activity in the brains and spleens of infected gerbils on day 12 PI. These data suggest that experimental infection with N. caninum interfered with energy metabolism associated with ATP homeostasis in the brain and spleen, directly or indirectly, apparently mediated by ROS overproduction, contributing to inhibition of Na+/K+ ATPase activity.


Assuntos
Encéfalo/enzimologia , Coccidiose/enzimologia , Metabolismo Energético , Neospora , Baço/enzimologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Coccidiose/metabolismo , Creatina Quinase/metabolismo , Citosol/enzimologia , Gerbillinae , Masculino , Mitocôndrias/enzimologia , Piruvato Quinase/metabolismo , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Baço/química , Baço/patologia
2.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 9-17, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31867609

RESUMO

Increased glycolysis is involved in the proliferation and migration of vascular smooth muscle cells (VSMCs). Pyruvate kinase isoform M2 (PKM2), a key rate-limiting enzyme in glycolysis, accelerates the proliferation and migration of tumor cells. Although the intracellular mechanisms associated with oxidized low-density lipoprotein (oxLDL)-stimulated VSMC proliferation and migration have been extensively explored, it is still unclear whether oxLDL promotes the proliferation and migration of VSMCs by enhancing PKM2-dependent glycolysis. In the present study, we detected PKM2 expression and pyruvate kinase activity in oxLDL-treated VSMCs and explored the regulation of PKM2 in oxLDL-treated VSMCs and apoE-/- mice. The results showed that PKM2 expression in VSMCs was higher in the intima than in the media in plaques from atherosclerotic rabbits. Moreover, PKM2 level in VSMCs was increased during atherosclerosis progression in apoE-/- mice. Both PKM2 expression and pyruvate kinase activity were found to be upregulated by oxLDL stimulation in VSMCs. Shikonin (SKN), a specific inhibitor of PKM2, was found to inhibit the oxLDL-induced proliferation and migration in VSMCs, in addition to delaying the atherosclerosis progression in apoE-/- mice. More importantly, oxLDL increased glucose uptake, ATP and lactate production, and the extracellular acidification rate in VSMCs, which could be reversed by SKN. Meanwhile, oxygen consumption rate was unchanged after oxLDL stimulation, suggesting that glycolysis is the main contributor to the energy supply in oxLDL-treated VSMCs. Our results suggest that oxLDL induces VSMC proliferation and migration by upregulating PKM2-dependent glycolysis, thereby contributing to the atherosclerosis progression. Thus, targeting PKM2-dependent glycolysis might provide a novel therapeutic approach for the treatment of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Proliferação de Células , Glicólise/genética , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Piruvato Quinase/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout para ApoE , Naftoquinonas/farmacologia , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Coelhos , Ratos , Ratos Sprague-Dawley , Hormônios Tireóideos/genética
3.
Plant Sci ; 290: 110285, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31779905

RESUMO

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.


Assuntos
Diospyros/fisiologia , Frutas/fisiologia , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Diospyros/enzimologia , Diospyros/genética , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Paladar , Fatores de Transcrição/metabolismo
4.
Adv Gerontol ; 32(4): 572-580, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31800186

RESUMO

The present work introduces data on studying the activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) and the state of the cardiovascular system in elderly and senile people who applied to polyclinic "Health zone" in Baku. 60 people on an enzimopatiya and 87 people on a condition of cardiovascular system were examined. The examined persons were found the decreased myocardial blood flow, ischemic heart disease (IHD), against increased PK and LDH. Statistically significant differences in the activity of enzymes depending on gender, age were established, and changes in the bioelectric activity of the heart during an ECG were detected.


Assuntos
Sistema Cardiovascular , L-Lactato Desidrogenase , Miocárdio , Piruvato Quinase , Idoso , Idoso de 80 Anos ou mais , Azerbaijão , Sistema Cardiovascular/enzimologia , Sistema Cardiovascular/patologia , Feminino , Coração/fisiopatologia , Humanos , L-Lactato Desidrogenase/metabolismo , Masculino , Isquemia Miocárdica/enzimologia , Miocárdio/enzimologia , Piruvato Quinase/metabolismo
5.
Emerg Microbes Infect ; 8(1): 1604-1618, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31711375

RESUMO

The incidence of Vibrio alginolyticus infections has increased in recent years due to the influence of climate change and rising sea temperature. Vibrio virulence regulatory RNA 1 (Vvrr1) is a newly found noncoding RNA (ncRNA) predicted to be closely related to the adhesion ability of V. alginolyticus based on the previous RNA-seq. In this study, the target genes of Vvrr1 were fully screened and verified by constructing Vvrr1-overexpressing strains and using the proteome sequencing technology. Pyruvate kinase I (pykF) gene was predicted to be a chief target gene of Vvrr1 involved in virulence regulation. The adhesion ability, biofilm formation and virulence were significantly reduced in the Vvrr1-overexpressing and the pykF-silenced strain compared with the wild strains. Similar to the overexpression of Vvrr1, the silencing of pykF also reduced the expression level of virulence genes, such as ndk, eno, sdhB, glpF, and cysH. Meanwhile, by constructing the "pykF-GFP" fusion expression plasmid and using the GFP reporter gene analysis in Escherichia coli, the fluorescence intensity of the strain containing Vvrr1 whole ncRNA sequence vector was found to be significantly weakened. These indicated that Vvrr1 participated in the virulence regulation mechanism of V. alginolyticus by interacting with the virulence gene pykF.


Assuntos
Doenças dos Peixes/microbiologia , RNA Bacteriano/genética , RNA não Traduzido/genética , Vibrioses/veterinária , Vibrio alginolyticus/genética , Vibrio alginolyticus/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peixes , Regulação Bacteriana da Expressão Gênica , Proteômica , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Vibrioses/microbiologia , Vibrio alginolyticus/metabolismo , Virulência
6.
Mol Cells ; 42(9): 628-636, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31564074

RESUMO

Altered genetic features in cancer cells lead to a high rate of aerobic glycolysis and metabolic reprogramming that is essential for increased cancer cell viability and rapid proliferation. Pyruvate kinase muscle (PKM) is a rate-limiting enzyme in the final step of glycolysis. Herein, we report that PKM is a potential therapeutic target in triple-negative breast cancer (TNBC) cells. We found that PKM1 or PKM2 is highly expressed in TNBC tissues or cells. Knockdown of PKM significantly suppressed cell proliferation and migration, and strongly reduced S phase and induced G2 phase cell cycle arrest by reducing phosphorylation of the CDC2 protein in TNBC cells. Additionally, knockdown of PKM significantly suppressed NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity by reducing the phosphorylation of p65 at serine 536, and also decreased the expression of NF-kB target genes. Taken together, PKM is a potential target that may have therapeutic implications for TNBC cells.


Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Técnicas de Silenciamento de Genes , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Piruvato Quinase/metabolismo , Hormônios Tireóideos/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos
7.
Ecotoxicol Environ Saf ; 183: 109576, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31509928

RESUMO

Formaldehyde (FA), a ubiquitous indoor environmental pollutant, has been classified as a carcinogen. There are many studies showed that low levels of FA could promote cell proliferation, however, little is known about the signal pathways. To determine the potential molecular mechanisms, human chronic myeloid leukemia cells (K562 cells) and human bronchial epithelial cells (16HBE cells) were exposed to different concentrations of FA. The data showed that FA at 0-125 µM or 0-60 µM promoted the proliferation of K562 cells or 16HBE cells respectively, indicating that FA did have the Hormesis effect. FA at 75 µM (K562 cells) and 40 µM (16HBE cells) significantly promoted cell proliferation, increased intracellular reactive oxygen species (ROS) levels, and decreased glutathione (GSH) content. At the same time, FA treatment induced a marked increase in the key molecules of cell division like CyclinD-cdk4 and E2F1. In addition, pyruvate kinase isozyme M2 (PKM2), glucose, glucose transporter 1 (GLUT1), lactic acid and lactate dehydrogenase A (LDHA) content in the Warburg effect were increased. Administering Vitamin E (VE), significantly disrupted cell division and disturbed the Warburg effect, effectively indicating the decrease of cell activity. Conclusively, these findings suggested that low concentrations of FA could promote cell proliferation by accelerating cell division process or enhancing the Warburg effect to embody the Hormesis effect.


Assuntos
Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Formaldeído/toxicidade , Hormese/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Glucose/metabolismo , Humanos , Células K562 , Piruvato Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Colloids Surf B Biointerfaces ; 182: 110405, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31377611

RESUMO

Tumor cells are sensitive to the disturbance of mitochondrial functions. Attenuation of dysfunctional mitochondria by natural compounds is an emerging strategy for the recovery of abnormal energy metabolism of cancer. To develop a nano-sized curcumin (CUR) in attenuating the energy metabolism of cancer cells, herein, a coral-shaped nano-transporter DNA-FeS2-DA nanoparticle was synthesized using double-stranded DNA rich in 'GAG' and 'GC' series as a template and poly-dopamine as an adhesive. CUR was successfully loaded to DNA-FeS2-DA with a molar ratio of ssDNA: CUR of 1:16, forming CUR@DNA-FeS2-DA. This nano-curcumin can readily enter mitochondrion in MCF-7 cancer cells. The CUR@DNA-FeS2-DA nanocomposite displays desirable photothermal effect and stability, while its CUR can be released gradually in the weak acid environment. The expression of both pyruvate kinase M2 and fatty acid synthase in the MCF-7 cancer cells were noticeably inhibited by CUR@DNA-FeS2-DA. Given the controlled release and mitochondria-targeting properties, this CUR@DNA-FeS2-DA nanocomposite is a promising new drug entity for intervening the energy metabolism of cancer cells.


Assuntos
Curcumina/farmacologia , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Nanopartículas/química , Piruvato Quinase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Curcumina/farmacocinética , DNA/química , Dopamina/química , Liberação Controlada de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Ferro/química , Células MCF-7 , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Piruvato Quinase/metabolismo , Sulfetos/química
9.
Exp Parasitol ; 204: 107726, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299264

RESUMO

The aims of this study were to evaluate if the use of copper oxide wire particles, isolated or in association with closantel, in lambs infected with Haemonchus contortus enhances the anthelmintic efficacy of closantel, as well as to evaluate the effects of treatment in hepatic energy metabolism, inflammatory markers and hematological and biochemical tests. The lambs were randomly divided into five groups (6 animals each), as follows: uninfected animals (Control); animals infected with H. contortus (HC); infected and treated with closantel (HC + CL); infected and treated with copper oxide wire particles (HC + Cu); and infected and treated with closantel plus copper oxide wire particles (HC + CL + Cu). The animals of infected groups were infected orally with H. contortus (5,000 L3 -larvae) and on day 14 post infection (p.i) the treatments were initiated. The egg per gram of feces (EPG), butyrylcholinesterase (BuChE), myeloperoxidase (MPO), adenylate kinase (AK) and pyruvate kinase (PK) activities and hematological and biochemical tests were evaluated. Treatments with copper oxide (isolated and associated) were able to reduce the EPG count on days 28, 35, 42 and 49 p.i when compared to HC group, while closantel was able to reduce EPG only from day 35 p.i. Moreover, treatment with closantel (isolated or associated) was able to prevent the inhibition of hepatic AK and PK activities caused by H. contortus infection, which may contribute to efficient intracellular energetic communication in order to maintain the balance between cellular ATP consumption and production. Butyrylcholinesterase and MPO activities were higher in infected lambs compared to uninfected, while treated groups showed lower enzymatic activity compared to the group HC. The use of all therapeutic protocols was able to reduce the EPG count. Based on these evidences, the use of copper oxide plus closantel may be considered an alternative to treat lambs infected by H. contortus.


Assuntos
Anti-Helmínticos/administração & dosagem , Cobre/administração & dosagem , Hemoncose/veterinária , Inflamação/veterinária , Salicilanilidas/administração & dosagem , Doenças dos Ovinos/tratamento farmacológico , Abomaso/metabolismo , Adenilato Quinase/metabolismo , Administração Oral , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Análise Química do Sangue/veterinária , Butirilcolinesterase/sangue , Cápsulas , Metabolismo Energético/efeitos dos fármacos , Contagem de Eritrócitos/veterinária , Fezes/parasitologia , Hemoncose/complicações , Hemoncose/tratamento farmacológico , Hemoncose/metabolismo , Hematócrito/veterinária , Hemoglobinas/análise , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Contagem de Ovos de Parasitas/veterinária , Peroxidase/sangue , Piruvato Quinase/metabolismo , Distribuição Aleatória , Salicilanilidas/farmacologia , Salicilanilidas/uso terapêutico , Ovinos , Doenças dos Ovinos/metabolismo , Doenças dos Ovinos/prevenção & controle
10.
Poult Sci ; 98(10): 4346-4358, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31287882

RESUMO

Cytochrome P-450 2C45 (CYP2C45) is the most highly expressed cytochrome P-450 isoform in chicken liver, and may play an important role in avian liver biology. However, information regarding the function of CYP2C45 in fatty liver is generally limited. The aim of this study was to investigate the role of CYP2C45 during the development of goose fatty liver. Our result indicated that the transcription of CYP2C45, together with PK and ALOX5, was increased in goose liver upon overfeeding for 19 D (P < 0.05). In goose primary hepatocytes, CYP2C45 RNA expression was also upgraded by the treatment with various chemicals like insulin, the fatty acids, and PPAR agonists (P < 0.05). We also found that both CYP2C45 overexpression and troglitazone treatment could increase the expression of pyruvate kinase (PK) and arachidonate 5-lipoxygenase (ALOX5), and furthermore, showed that the up-regulation of PK and ALOX5 induced by troglitazone could be suppressed by small interfering RNAs targeting CYP2C45 (P < 0.05). These findings suggest that fatty acids treatment and the overfeeding can induce the up-regulation of CYP2C45 expression possibly via PPARγ and that the induction of PK and ALOX5 in goose fatty liver is at least partially attributed to fatty acid-induced expression of CYP2C45. Thus, our data provides an insight into the mechanism by which glycolysis and arachidonic acid metabolism are modulated in goose fatty liver.


Assuntos
Araquidonato Lipoxigenases/genética , Proteínas Aviárias/genética , Ácidos Graxos/metabolismo , Fígado Gorduroso/veterinária , Gansos , Doenças das Aves Domésticas/genética , Piruvato Quinase/genética , Animais , Araquidonato Lipoxigenases/metabolismo , Proteínas Aviárias/metabolismo , Sequência de Bases , Sistema Enzimático do Citocromo P-450/genética , Fígado Gorduroso/genética , Masculino , PPAR gama/genética , Piruvato Quinase/metabolismo , Transdução de Sinais/genética
11.
EMBO J ; 38(17): e100938, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31328803

RESUMO

Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.


Assuntos
Substituição de Aminoácidos , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Piruvato Quinase/metabolismo , Animais , Células Cultivadas , Células Endoteliais , Homeostase , Humanos , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/genética , Oxirredução , Via de Pentose Fosfato , Ligação Proteica
12.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 461-469, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204694

RESUMO

Human liver pyruvate kinase (hLPYK) converts phosphoenolpyruvate to pyruvate in the final step of glycolysis. hLPYK is allosterically activated by fructose-1,6-bisphosphate (Fru-1,6-BP). The allosteric site, as defined by previous structural studies, is located in domain C between the phosphate-binding loop (residues 444-449) and the allosteric loop (residues 527-533). In this study, the X-ray crystal structures of four hLPYK variants were solved to make structural correlations with existing functional data. The variants are D499N, W527H, Δ529/S531G (called GGG here) and S531E. The results revealed a conformational toggle between the open and closed positions of the allosteric loop. In the absence of Fru-1,6-BP the open position is stabilized, in part, by a cation-π bond between Trp527 and Arg538' (from an adjacent monomer). In the S531E variant glutamate binds in place of the 6'-phosphate of Fru-1,6-BP in the allosteric site, leading to partial allosteric activation. Finally, the structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant.


Assuntos
Cátions/química , Frutosedifosfatos/metabolismo , Fígado/enzimologia , Mutação , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Sítio Alostérico , Sítios de Ligação , Cristalografia por Raios X , Frutosedifosfatos/química , Humanos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas , Piruvato Quinase/genética
13.
PLoS Biol ; 17(6): e2004413, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181072

RESUMO

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) ("intrabodies") and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Membranas Mitocondriais/fisiologia , Piruvato Quinase/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , GTP Fosfo-Hidrolases/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Isoformas de Proteínas/metabolismo , Piruvato Quinase/fisiologia , Transdução de Sinais , Anticorpos de Cadeia Única
14.
Food Chem ; 293: 537-544, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151645

RESUMO

To verify the effect of protein phosphorylation on glycolysis and elucidate the regulatory mechanism from the perspective of enzyme activity, ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor and the activities of glycogen phosphorylase, pyruvate kinase and phosphofructokinase were determined. The protein phosphorylation level was significantly different after incubation of muscle with kinase or phosphatase inhibitors. The pH value and lactate content revealed that a high phosphorylation level was the reason for the fast glycolysis. The glycogen phosphorylase, pyruvate kinase and phosphofructokinase activities were significantly higher in the phosphatase inhibitor group than in the other two groups (p < 0.05). Therefore, protein phosphorylation is involved in activating these three enzymes. In summary, protein phosphorylation plays a role in post-mortem glycolysis through the regulation of enzyme activity in ovine muscle.


Assuntos
Glicogênio Fosforilase/metabolismo , Músculos/enzimologia , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , Animais , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piruvato Quinase/antagonistas & inibidores , Ovinos
15.
Bull Exp Biol Med ; 167(2): 263-266, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31243677

RESUMO

We studied the expression of genes encoding enzymes of carbohydrate and lipid metabolism ketohexokinase (Khk), glucokinase (Gck), pyruvate kinase (Pklr), acetyl-Co-carboxylase (Acaca), fatty acid synthase (Fasn), stearoyl-CoA desaturase (Scd), and their transcription regulators ChREBP (Mlxipl), SREBP-1c (Srebf1), and PPARα (Ppara) in rat liver. Control group rats received a semisynthetic ration over 20 weeks. Experimental group 1 received a semisynthetic ration and 20% fructose solution instead of drinking water. Experimental group 2 rats received a semisynthetic ration with quercetin (0.1% fodder weight) and 20% fructose solution. Consumption of 20% fructose solution (experimental group 1) led to an increase in Scd expression in comparison with the control and did not affect the expression of other genes. Addition of quercetin to the ration (experimental group 2) led to a decrease in the expression of Khk, Gck, Fasn, Scd, Mlxipl, and Ppara genes in comparison with experimental group 1. The results suggest that quercetin reduced the expression of genes of carbohydrate and lipid metabolism enzymes in the liver of rats receiving high-fructose ration.


Assuntos
Enzimas/genética , Frutose/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Quercetina/farmacologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Dieta , Enzimas/metabolismo , Frutoquinases/genética , Frutoquinases/metabolismo , Glucoquinase/genética , Glucoquinase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Masculino , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ratos , Ratos Wistar , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
16.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 794-801, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31202897

RESUMO

SIRT1 (Silent mating type information regulation 2 homolog 1) play a neuroprotective effect through deacetylation target proteins in various neuronal diseases. However, the precise mechanisms remain elusive. In this study, we aim to identify those novel interacting partners of SIRT1 in rat brain tissue. By using a pre-clear GST-Pull down assay followed by the LC-MS/MS analysis, we've identified potential SIRT1's interacting partners, which function annotation by GO and KEGG analysis indicating some metabolic pathways are among the most enriched. Then we confirmed two candidates Enolase-1 (and NSE (Neuron-Specific Enolase) in brain) and PKM (Pyruvate Kinase Muscle) are associated with SIRT1 in brain tissue lysis by co-immunoprecipitation. Furthermore, increase or decrease the SIRT1 enzyme activity by its agonist SRT1720 or antagonist EX527 could significantly affect the acetylation level of endogenous NSE and PKM, SIRT1 overexpression or knock out expreiments also showed the same results as use SIRT1's agonist or antagonist. Moreover, the acetylation changes on NSE or PKM could finally lead to affection on their catalytic activity. Taken together, our findings suggest that the function of SIRT1 binding proteins is enriched in metabolic pathways. NSE and PKM are new SIRT1 binding molecules. SIRT1 may regulate acetylation level of NSE and PKM through deacetylation and further regulate their catalytic activity. Our study provides new evidence for the involvement of SIRT1 in the mechanisms of metabolic regulation in central nervous system.


Assuntos
Encéfalo/enzimologia , Fosfopiruvato Hidratase/metabolismo , Piruvato Quinase/metabolismo , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Animais , Carbazóis/farmacologia , Catálise/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Fosfopiruvato Hidratase/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Piruvato Quinase/genética , Ratos , Ratos Sprague-Dawley , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética
17.
PLoS One ; 14(5): e0217131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31120964

RESUMO

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.


Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Piruvato Quinase/antagonistas & inibidores , RNA Interferente Pequeno/genética , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Feminino , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Transporte Proteico , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Frações Subcelulares , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
World J Gastroenterol ; 25(16): 1936-1949, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31086462

RESUMO

BACKGROUND: Study shows that signal transducer and activator of transcription 3 (STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2 (PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats. AIM: To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats. METHODS: A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with N-methyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen (PCNA), STAT3, and PKM2 were examined by Western blot and immunofluorescence. RESULTS: We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liver precancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression, PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells. Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2. CONCLUSION: The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.


Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Hepáticas/patologia , Lesões Pré-Cancerosas/patologia , Piruvato Quinase/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Óxidos S-Cíclicos/farmacologia , Modelos Animais de Doenças , Glicólise/efeitos dos fármacos , Hepatócitos , Humanos , Peróxido de Hidrogênio/toxicidade , Fígado/citologia , Fígado/patologia , Masculino , Metilnitronitrosoguanidina/toxicidade , Fosforilação/efeitos dos fármacos , Lesões Pré-Cancerosas/induzido quimicamente , Ratos , Ratos Wistar , Fator de Transcrição STAT3/antagonistas & inibidores , Células-Tronco , Regulação para Cima
19.
Cell Physiol Biochem ; 52(6): 1535-1552, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31135122

RESUMO

BACKGROUND/AIMS: Pyruvate kinase M2 (PKM2) is essential for aerobic glycolysis. Although high PKM2 expression is observed in various cancer tissues, its functional role in cancer metabolism is unclear. Here, we investigated the role of PKM2 in regulating autophagy and its associated pathways in prostate cancer cells. METHODS: Immunohistochemistry was performed to compare the expression level of PKM2 in prostate cancer patients and normal human, whereas expression of PKM2 in several cell lines was also examined by using western blot. PKM2 expression was silenced using various small interfering RNAs (siRNAs). Cell viability was examined using IncuCyte ZOOM™ live cell imaging system. Western blotting and immunofluorescence were performed to investigate the PKM2 knockdown on other cellular signaling molecules. Acridine orange and Monodansylcadaverine staining was performed to check effect of PKM2 knockdown on autophagy induction. High performance thin layer chromatography was carried out to quantify the level of different cellular metabolites (pyruvate and lactate). Colony formation assay was performed to determine the ability of a cells to form large colonies. RESULTS: PKM2 was highly expressed in prostate cancer patients as compared to normal human. PKM2 siRNA-transfected prostate cancer cells showed significantly reduced viability. Acridine orange, Monodansylcadaverine staining and western blotting analysis showed that PKM2 downregulation markedly increased autophagic cell death. Results of western blotting analysis showed that PKM2 knockdown affected protein kinase B/mechanistic target of rapamycin 1 pathway, which consequently downregulated the expression of glycolytic enzymes lactate dehydrogenase A and glucose transporter 1. Knockdown of PKM2 also reduced the colony formation ability of human prostate cancer cell DU145. CONCLUSION: To the best of our knowledge, this is the first study to show that PKM2 inhibition alters prostate cancer cell metabolism and induces autophagy, thus providing new perspectives for developing PKM2-targeting anticancer therapies for treating prostate cancer.


Assuntos
Autofagia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Quinase/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Transportador de Glucose Tipo 1/metabolismo , Humanos , Isoenzimas/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Neoplasias da Próstata/metabolismo , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
20.
Pathol Res Pract ; 215(6): 152409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31000383

RESUMO

Pyruvate kinase M2 (PKM2) serves as a key enzyme that promotes aerobic glycolysis. This study investigated the function of PKM2 in tumor growth and maintenance in gastric cancer (GC). Histological staining was applied to detect PKM2 expression in GC tissues. PCR and western blotting were used to measure PKM2 expression in GC cells. PKM2 was knocked down to examine the biological behavior of tumors, glycometabolism, and apoptosis. PKM2 was upregulated in GC tissues (65%, 34/52) compared with that in adjacent normal tissues (27%, 10/37). Moreover, PKM2 knockdown inhibited proliferation of BGC823 GC cells, and elevated PKM2 levels were associated with poor survival of GC patients. Furthermore, knockdown of PKM2 altered the biological behavior of BGC823 cells through induction of apoptosis. In conclusion, the results of this study indicated that inhibition of PKM2 could represent a novel strategy for gastric cancer treatment.


Assuntos
Glicólise/fisiologia , Piruvato Quinase/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Camundongos , Prognóstico , Neoplasias Gástricas/mortalidade
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