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1.
Nat Commun ; 12(1): 1718, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741961

RESUMO

Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.


Assuntos
Proliferação de Células/fisiologia , Citrulinação/fisiologia , Glicólise/fisiologia , Neoplasias/metabolismo , Proteína-Arginina Desiminase do Tipo 1/metabolismo , Proteína-Arginina Desiminase do Tipo 3/metabolismo , Piruvato Quinase/metabolismo , Regulação Alostérica , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Melanoma , Proteínas de Membrana , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Neoplasias/genética , Proteína-Arginina Desiminase do Tipo 1/genética , Proteína-Arginina Desiminase do Tipo 3/genética , Hormônios Tireóideos , Regulação para Cima
2.
Artigo em Chinês | MEDLINE | ID: mdl-33691363

RESUMO

Objective: To study the cytotoxicity and malignant transformation ability of chrysotile on MeT-5A cells. Methods: In June 2016, lactate dehydrogenase (LDH) method was used to detect the cytotoxicity of chrysotile to MeT-5A cells. MeT-5A cells were treated with 5 µg/cm(2) chrysotile intermittently for 24 h a time, once a week and a total of 28 times. After the cells showed anchorage independent growth, the cell features of malignant transformation were identified by colony forming frequency in soft agar, and the soft agar colony formation rates were calculated. The activities of key speed limiting enzymes of glycolysis metabolism including hexokinase (HK) , phosphofructokinase (PFK) and pyruvate kinase (PK) were determined by UV colorimetry. Results: Chrysotile was cytotoxic to MeT-5A cells in a concentration-dependent decline. Compared with the control group, the relative survival rates of MeT-5A cells were significantly decreased after exposed to chrysotile at 10, 20, 40 and 80 µg/cm(2) (P<0.05) . After 28 times of exposure, the growth rate of the cells in chrysotile transformed MeT-5A cells was accelerated, the arrangement was disordered, the contact inhibition was lost, and the double layer growth appeared, which could grow on soft agar. The colony forming rate of the chrysotile transformed MeT-5A cells was 18.33‰±2.49‰. Compared with the control group (0) , the difference was statistically significant (P<0.01) . The activities of glycolysis related kinase including PK [ (19.51±1.52) U/L], PFK[ (0.12±0.02) U/10(4) cell] and HK[ (0.26±0.01) U/10(4) cell] were increased in the chrysotile transformed MeT-5A cells compared with control group [ (25.00±1.04) U/L、(0.15±0.01) U/10(4) cell and (0.33±0.01) U/10(4) cell] (P<0.01) . Conclusion: Chrysotile can induce malignant transformation of MeT-5A cells and increase the activities of glycolysis related kinases including PK, PFK and HK.


Assuntos
Asbestos Serpentinas , Fosfofrutoquinase-1 , Asbestos Serpentinas/toxicidade , Glicólise , Hexoquinase/metabolismo , Fosfofrutoquinase-1/metabolismo , Piruvato Quinase/metabolismo
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540748

RESUMO

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Proteínas de Bactérias/antagonistas & inibidores , Catálise , Indução Enzimática , Retroalimentação Fisiológica , Frutosedifosfatos/biossíntese , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Gluconeogênese , Glicólise , Hexosefosfatos/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , L-Lactato Desidrogenase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Oxigênio/farmacologia , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
4.
Carbohydr Polym ; 255: 117532, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436261

RESUMO

Aldolase A (ALDOA) facilitated aerobic glycolysis in cancer cells is a potential target in the treatment of hepatocellular carcinoma (HCC). However, only few effective inhibitors of ALDOA have been reported until now. In this research, we found a polysaccharide called HDPS-4II from Holotrichia diomphalia Bates, which can specifically bind to ALDOA with a dissociation constant of 2.86 µM. HDPS-4II with a molecular weight of 19 kDa was a linear triple-helix glucan composed of ɑ-d-1,4-Glcp and ɑ-d-1,6-Glcp in a ratio of 1.0:10.0. HDPS-4II significantly inhibited aldolase enzyme activity, glycolysis, and further inhibited the expression of phosphorylated AMPKα in HCC cells. Through analyzing ALDOA-overexpressing and -knockdown cells, it was confirmed that ALDOA mediated the viability and glycolysis inhibition of HDPS-4II. Moreover, HDPS-4II administration markedly inhibited tumor growth in mice xenografted with HCCs. These findings suggest that HDPS-4II, as an ALDOA antagonist, is a promising remedy in the treatment and prevention of HCC.


Assuntos
Antineoplásicos/farmacologia , Besouros/química , Frutose-Bifosfato Aldolase/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucanos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Frutose-Bifosfato Aldolase/metabolismo , Glucanos/química , Glucanos/isolamento & purificação , Glicólise/efeitos dos fármacos , Glicólise/genética , Células Hep G2 , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Larva/química , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 12(1): 594, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500413

RESUMO

Telomeres are organized into a heterochromatin structure and maintenance of silent heterochromatin is required for chromosome stability. How telomere heterochromatin is dynamically regulated in response to stimuli remains unknown. Pyruvate kinase Pyk1 forms a complex named SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex) to regulate gene expression by phosphorylating histone H3T11 (H3pT11). Here, we identify a function of SESAME in regulating telomere heterochromatin structure. SESAME phosphorylates H3T11 at telomeres, which maintains SIR (silent information regulator) complex occupancy at telomeres and protects Sir2 from degradation by autophagy. Moreover, SESAME-catalyzed H3pT11 directly represses autophagy-related gene expression to further prevent autophagy-mediated Sir2 degradation. By promoting H3pT11, serine increases Sir2 protein levels and enhances telomere silencing. Loss of H3pT11 leads to reduced Sir2 and compromised telomere silencing during chronological aging. Together, our study provides insights into dynamic regulation of silent heterochromatin by histone modifications and autophagy in response to cell metabolism and aging.


Assuntos
Instabilidade Cromossômica , Histonas/metabolismo , Complexos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/genética , Telômero/metabolismo , Autofagia , Regulação Fúngica da Expressão Gênica , Heterocromatina/metabolismo , Fosforilação , Proteólise , Piruvato Quinase/metabolismo , Saccharomyces cerevisiae/enzimologia , Serina/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo
7.
Nat Commun ; 11(1): 4509, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908151

RESUMO

Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/enzimologia , Glicólise/fisiologia , Mitocôndrias/enzimologia , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Metabolismo Energético/fisiologia , Mutação , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/fisiologia , Piruvato Quinase/genética , Piruvato Quinase/metabolismo
8.
Am J Pathol ; 190(11): 2267-2281, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32805235

RESUMO

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The in vitro and in vivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs in vitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis in vivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.


Assuntos
Células Estreladas do Fígado/enzimologia , Cirrose Hepática/enzimologia , Multimerização Proteica , Piruvato Quinase/metabolismo , Acetilação , Animais , Ciclina D1/metabolismo , Feminino , Células Estreladas do Fígado/patologia , Histonas/metabolismo , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos , Compostos Orgânicos/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo
9.
Ecotoxicol Environ Saf ; 205: 111146, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32827965

RESUMO

Cadmium (Cd) exposure in environment is associated with development of esophageal cancer. However, the mechanisms of Cd-induced carcinogenesis are still not been fully cleared, and the present study aimed to explore the possible etiological mechanism of Cd-induced esophageal cancer. Human esophageal epithelial cell lines (HET-1A and KYSE450) were treated with CdCl2 at 0.05 mg/l for 12, 24 h, and the then the apoptosis were detected using flow cytometry with annexin-V-FITC/PI staining. Results showed that apoptosis of treatment groups was significantly inhibited, and decreased reactive oxygen species (ROS) production played a key role in the inhibitory effects by upregulating Bcl-2 and downregulating Caspase-3/9. The relief of oxidative stress during Cd exposure was actively promoted by the increased nicotinamide adenine dinucleotide phosphoric acid and glutathione levels. To investigate the causes of enhanced intracellular antioxidant capacity, the activity of pyruvate kinase (PK), a key enzyme of glycolysis, was detected. Our results showed that PK activity was inhibited, suggesting that glycolysis process was blocked which promoted more intermediate metabolites of glycolysis to be used for reduced nicotinamide adenine dinucleotide phosphoric acid (NADPH) or other antioxidants synthesis. PK activity was closely correlated with phosphorylation of pyruvate kinase M2 (PKM2), and a highly negative correlation (correlation coefficients: -0.835, p < 0.05) between them was found. Western blotting showed the overphosphorylation of PKM2 in Cd-exposed cells, resulting from increased expression of cyclin-dependent kinases 6 (CDK6). These results suggested a possible mechanism of carcinogenic: Cd-induced upregulation of CDK6 in esophageal cell lines caused PKM2 overphosphorylation inhibiting PK activity, thereby shunting glucose-derived carbon into the pentose phosphate pathway and promoting the production of NADPH and reduced glutathione (GSH) to neutralize ROS, which finally results in the inhibited apoptosis.


Assuntos
Cádmio/toxicidade , Quinase 6 Dependente de Ciclina/metabolismo , Apoptose/efeitos dos fármacos , Cádmio/metabolismo , Caspase 3 , Neoplasias Esofágicas , Glicólise/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2 , Piruvato Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
10.
Ecotoxicol Environ Saf ; 201: 110861, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32544748

RESUMO

Marine biota have been co-challenged with ocean warming and mercury (Hg) pollution over many generations because of human activities; however, the molecular mechanisms to explain their combined effects are not well understood. In this study, a marine planktonic copepod Pseudodiaptomus annandalei was acutely exposed to different temperature (22 and 25 °C) and Hg (0 and 118 µg/L) treatments in a 24-h cross-factored experiment. Hg accumulation and its subcellular fractions were determined in the copepods after exposure. The expression of the genes of superoxide dismutase (SOD), glutathione peroxidase (GPx), metallothionein1 (mt1), heat shock protein 70 (hsp70), hsp90, hexokinase (hk), and pyruvate kinase (pk) was also analyzed. Both the Hg treatment alone and the combined exposure of warmer temperature plus Hg pollution remarkably facilitated Hg bioaccumulation in the exposed copepods. Compared with the Hg treatment alone, the combined exposure increased total Hg accumulation and also the amount of Hg stored in the metal-sensitive fractions (MSF), suggesting elevated Hg toxicity in P. annandalei under a warmer environment, given that the MSF is directly related to metal toxicity. The warmer temperature significantly up-regulated the mRNA levels of mt1, hsp70, hsp90, and hk, indicating the copepods suffered from thermal stress. With exposure to Hg, the mRNA level of SOD increased strikingly but the transcript levels of hsp90, hk, and pk decreased significantly, indicating that Hg induced toxic events (e.g., oxidative damage and energy depletion). Particularly, in contrast to the Hg treatment alone, the combined exposure significantly down-regulated the mRNA levels of SOD and GPx but up-regulated the mRNA levels of mt1, hsp70, hsp90, hk, and pk. Collectively, the results of this study indicate that ocean warming will potentially boost Hg toxicity in the marine copepod P. annandalei, which is information that will increase the accuracy of the projections of marine ecosystem responses to the joint effects of climate change stressors and metal pollution on the future ocean.


Assuntos
Copépodes/efeitos dos fármacos , Temperatura Alta , Mercúrio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Copépodes/genética , Copépodes/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Mercúrio/farmacocinética , Metalotioneína/genética , Metalotioneína/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , RNA Mensageiro/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para Cima , Poluentes Químicos da Água/farmacocinética
11.
Nat Commun ; 11(1): 3162, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572027

RESUMO

Interacting with proteins is a crucial way for long noncoding RNAs (lncRNAs) to exert their biological responses. Here we report a high throughput strategy to characterize lncRNA interacting proteins in vivo by combining tobramycin affinity purification and mass spectrometric analysis (TOBAP-MS). Using this method, we identify 140 candidate binding proteins for lncRNA highly upregulated in liver cancer (HULC). Intriguingly, HULC directly binds to two glycolytic enzymes, lactate dehydrogenase A (LDHA) and pyruvate kinase M2 (PKM2). Mechanistic study suggests that HULC functions as an adaptor molecule that enhances the binding of LDHA and PKM2 to fibroblast growth factor receptor type 1 (FGFR1), leading to elevated phosphorylation of these two enzymes and consequently promoting glycolysis. This study provides a convenient method to study lncRNA interactome in vivo and reveals a unique mechanism by which HULC promotes Warburg effect by orchestrating the enzymatic activities of glycolytic enzymes.


Assuntos
Glicólise , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/metabolismo , Piruvato Quinase/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Proteoma/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Ativação Transcricional
12.
J Cardiovasc Pharmacol Ther ; 25(4): 364-376, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32323562

RESUMO

BACKGROUND: The development of thoracic aortic aneurysm and dissection (TAAD) is mediated by inflammasome activation, which exacerbates the secretion of pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs), and reactive oxygen species (ROS). The glycolytic enzyme pyruvate kinase M2 (PKM2) has shown a protective role against various disorders with an inflammatory basis, such as sepsis, tumorigenesis, and diabetic nephropathy. However, its potential role in TAAD has not been investigated so far. APPROACH AND RESULTS: We analyzed aortic tissues from TAAD patients and the ß-aminopropionitrile fumarate (BAPN)-induced mouse model of TAAD and observed elevated levels of PKM2 in the aortic lesions of both. Treatment with the PKM2 activator TEPP-46 markedly attenuated the progression of TAAD in the mouse model as demonstrated by decreased morbidity and luminal diameter of the aorta. In addition, the thoracic aortas of the BAPN-induced mice showed reduced monocytes and macrophages infiltration and lower levels of IL-1ß, MMPs, and ROS when treated with TEPP-46. Furthermore, TEPP-46 treatment also suppressed the activation of the NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome by downregulating p-STAT3 and HIF1-α. CONCLUSION: Pyruvate kinase M2 plays a protective role in TAAD development, and its activation is a promising therapeutic strategy against the progression of TAAD.


Assuntos
Aneurisma Dissecante/prevenção & controle , Aorta Torácica/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Ativadores de Enzimas/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piruvato Quinase/farmacologia , Remodelação Vascular/efeitos dos fármacos , Aneurisma Dissecante/enzimologia , Aneurisma Dissecante/patologia , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/enzimologia , Aneurisma da Aorta Torácica/patologia , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
13.
Chemosphere ; 253: 126631, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32302917

RESUMO

We have previously demonstrated in a companion work that acclimation to 28 °C potentiated waterborne copper (Cu) toxic effects in Poecilia vivipara through oxidative stress-related processes. In the present study, we hypothesized that these results were related to kinetic metabolic adjustments in enzymes from aerobic and anaerobic pathways. To test this, P. vivipara was acclimated to two temperatures (22 °C or 28 °C) for three weeks and then exposed to Cu (control, 9 or 20 µg/L) for 96 h. The activity of enzymes from glycolysis (pyruvate kinase [PK] and lactate dehydrogenase [LDH]), Krebs cycle (citrate synthase [CS]) and the electron transport chain system (ETS) were assessed in gills, liver and muscle. Interactive effects were only seen for hepatic LDH activity, as both metal exposure and heat stress, combined or not, inhibited this enzyme, showing a suppression in anaerobic pathways. Conversely, a Cu main effect was present in the liver, expressed as an elevation in ETS activity, showing an enhancement in hepatic aerobic metabolism likely related with the very energy-demanding process of metal detoxification. Moreover, this study shows that P. vivipara has a remarkable ability to compensate heat stress in terms of energy metabolism, as we could not observe acclimation temperature effects for most of the cases. Nonetheless, a tissue-dependent effect of elevated temperature was observed, as we could observe an inhibition in muscular CS activity. Finally, it is concluded that kinetic adjustments in terms of the energy metabolism are not related with the temperature-dependent elevation of Cu toxicity in P. vivipara as we previously hypothesized.


Assuntos
Cobre/toxicidade , Metabolismo Energético/fisiologia , Poecilia/fisiologia , Temperatura , Poluentes Químicos da Água/toxicidade , Aclimatação/efeitos dos fármacos , Animais , Ciclo do Ácido Cítrico , Metabolismo Energético/efeitos dos fármacos , Fundulidae/metabolismo , Brânquias/metabolismo , Glicólise , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Metais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Poecilia/metabolismo , Piruvato Quinase/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(15): 8494-8502, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32229570

RESUMO

Human tuberculosis is caused by members of the Mycobacterium tuberculosis complex (MTBC) that vary in virulence and transmissibility. While genome-wide association studies have uncovered several mutations conferring drug resistance, much less is known about the factors underlying other bacterial phenotypes. Variation in the outcome of tuberculosis infection and diseases has been attributed primarily to patient and environmental factors, but recent evidence indicates an additional role for the genetic diversity among MTBC clinical strains. Here, we used metabolomics to unravel the effect of genetic variation on the strain-specific metabolic adaptive capacity and vulnerability. To define the functionality of single-nucleotide polymorphisms (SNPs) systematically, we developed a constraint-based approach that integrates metabolomic and genomic data. Our model-based predictions correctly classify SNP effects in pyruvate kinase and suggest a genetic basis for strain-specific inherent baseline susceptibility to the antibiotic para-aminosalicylic acid. Our method is broadly applicable across microbial life, opening possibilities for the development of more selective treatment strategies.


Assuntos
Antituberculosos/farmacologia , Genômica/métodos , Interações Hospedeiro-Patógeno , Metaboloma , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleotídeo Único , Tuberculose/genética , Ácido Aminossalicílico/farmacologia , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Fenótipo , Filogenia , Piruvato Quinase/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Virulência
15.
Int J Mol Sci ; 21(5)2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32121259

RESUMO

Heat stress can particularly affect the kidney because of its high rate of adenosine triphosphate consumption. Competition between apoptosis and autophagy-mediated survival always exists in damaged tissue. And Hsp90 can enhance cellular protection to resist heat stress. However, the relationship between Hsp90 and the above competition and its underlying mechanism in the kidney are unclear. The present study found that heat stress induced obvious histopathological and oxidative injury, which was connected with cellular apoptosis and autophagy in the kidney and was associated with the levels of Hsp90 expression or function. The data showed that during heat stress, Hsp90 activated the PKM2-Akt signaling pathway to exert antiapoptotic effects and induce Hsp70 expression regulated by HSF-1, stimulated autophagy-mediated survival through the HIF-1α-BNIP3/BNIP3L pathway, and finally protected the kidney from heat-stress injury. Moreover, the nuclear translocation of PKM2, (p-) Akt, HSF-1, and HIF-1α was enhanced by heat stress, but only intranuclear p-Akt and HSF-1 were specifically influenced by Hsp90, contributing to regulate the cellular ability of resisting heat-stress damage. Our study provided new insights regarding the molecular mechanism of Hsp90 in the kidney in response to heat-stress injury, possibly contributing to finding new targets for the pharmacological regulation of human or animal acute kidney injury from heat stress in future research.


Assuntos
Apoptose , Autofagia , Proteínas de Choque Térmico HSP90/metabolismo , Resposta ao Choque Térmico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Quinase/metabolismo , Animais , Antioxidantes/metabolismo , Sobrevivência Celular , Proteínas de Choque Térmico HSP70/metabolismo , Rim/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Oxirredução , Fosforilação , Transdução de Sinais
16.
Biochem Biophys Res Commun ; 525(3): 726-732, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32143824

RESUMO

Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5' tRNA fragments derived from tyrosine pre-tRNA (5' Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5' Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5' Tyr-tRF. Our findings partially uncovered a mechanistic link between 5' Tyr-tRF and neuronal cell death that is regulated by PKM2.


Assuntos
Neurônios/enzimologia , Neurônios/patologia , Piruvato Quinase/metabolismo , Precursores de RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Tirosina/metabolismo , Animais , Morte Celular , Diferenciação Celular , Linhagem Celular , Embrião não Mamífero/metabolismo , Humanos , Peixe-Zebra/embriologia
17.
Life Sci ; 250: 117561, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198052

RESUMO

AIMS: Pyruvate kinase M2 (PKM2), a unique isoform of the pyruvate kinases, not only acts as a crucial metabolic enzyme when it locates in the cytoplasm, but also plays important roles in tumor formation and growth when it accumulates in the nuclei. Our aim was to investigate the potential role of PKM2 in liver regeneration in mice insulted with carbon tetrachloride (CCl4). MATERIAL AND METHODS: The liver regeneration model was established by intraperitoneal injection of CCl4 for 48 h in male BALB/c mice. The expression of PKM2, phospho-STAT3, STAT3, proliferating cell nuclear antigen (PCNA) and Cyclin D1 were evaluated by western blot. The distribution of PKM2 was verified by immunofluorescence staining. The degree of injured region was assessed by hematoxylin and eosin (HE) staining. The proliferation of liver cells was tested by Immunohistochemistry. KEY FINDINGS: The nuclear accumulation of PKM2 increased in the liver treated with CCl4, but treatment with ML-265 significantly suppressed CCl4-induced nuclear accumulation of PKM2. In addition, treatment with ML-265 suppressed the level of cyclin D1 and proliferating cell nuclear antigen (PCNA), reduced the count of Ki67-positive hepatocytes, and expanded the damaged region in histological examination. Meanwhile, treatment with ML-265 suppressed the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 by stattic made the same effects as ML-265. SIGNIFICANCE: These data uncovered the role of nuclear PKM2 in liver regeneration and the pro-proliferation effects of nuclear PKM2 may be through targeting its downstream transcription factor STAT3.


Assuntos
Núcleo Celular/metabolismo , Regeneração Hepática , Piruvato Quinase/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Tetracloreto de Carbono , Proliferação de Células , Citoplasma/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Hepatopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo
18.
Nat Commun ; 11(1): 941, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071304

RESUMO

Oxidative stress is a major pathogenic mechanism in Parkinson's disease (PD). As an important cellular antioxidant, glutathione (GSH) balances the production and incorporation of free radicals to protect neurons from oxidative damage. GSH level is decreased in the brains of PD patients. Hence, clarifying the molecular mechanism of GSH deficiency may help deepen our knowledge of PD pathogenesis. Here we report that the astrocytic dopamine D2 receptor (DRD2) regulates GSH synthesis via PKM2-mediated Nrf2 transactivation. In addition we find that pyridoxine can dimerize PKM2 to promote GSH biosynthesis. Further experiments show that pyridoxine supplementation increases the resistance of nigral dopaminergic neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in wild-type mice as well as in astrocytic Drd2 conditional knockout mice. We conclude that dimerizing PKM2 may be a potential target for PD treatment.


Assuntos
Glutationa/biossíntese , Intoxicação por MPTP/patologia , Fator 2 Relacionado a NF-E2/genética , Fármacos Neuroprotetores/administração & dosagem , Piruvato Quinase/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Astrócitos , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Dopamina/metabolismo , Neurônios Dopaminérgicos , Intoxicação por MPTP/diagnóstico , Intoxicação por MPTP/tratamento farmacológico , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Cultura Primária de Células , Multimerização Proteica/efeitos dos fármacos , Piridoxina/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Receptores de Dopamina D2/genética , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia , Ativação Transcricional
19.
Sci Rep ; 10(1): 2990, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076076

RESUMO

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661 W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.


Assuntos
Apoptose/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Piruvato Quinase/metabolismo , Doenças Retinianas/tratamento farmacológico , Animais , Cegueira/etiologia , Cegueira/prevenção & controle , Linhagem Celular , Modelos Animais de Doenças , Ativadores de Enzimas/uso terapêutico , Glicólise/efeitos dos fármacos , Humanos , Injeções Intravítreas , Masculino , Camundongos , Camundongos Knockout , Células Fotorreceptoras/patologia , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Piruvato Quinase/genética , Coelhos , Ratos , Doenças Retinianas/complicações , Doenças Retinianas/patologia
20.
PLoS One ; 15(2): e0228894, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32078667

RESUMO

Lymphangioleiomyomatosis (LAM) is a devastating lung disease caused by inactivating gene mutations in either TSC1 or TSC2 that result in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). As LAM occurs predominantly in women during their reproductive age and is exacerbated by pregnancy, the female hormonal environment, and in particular estrogen, is implicated in LAM pathogenesis and progression. However, detailed underlying molecular mechanisms are not well understood. In this study, utilizing human pulmonary LAM specimens and cell culture models of TSC2-deficient LAM patient-derived and rat uterine leiomyoma-derived cells, we tested the hypothesis that estrogen promotes the growth of mTORC1-hyperactive cells through pyruvate kinase M2 (PKM2). Estrogen increased the phosphorylation of PKM2 at Ser37 and induced the nuclear translocation of phospho-PKM2. The estrogen receptor antagonist Faslodex reversed these effects. Restoration of TSC2 inhibited the phosphorylation of PKM2 in an mTORC1 inhibitor-insensitive manner. Finally, accumulation of phosphorylated PKM2 was evident in pulmonary nodule from LAM patients. Together, our data suggest that female predominance of LAM might be at least in part attributed to estrogen stimulation of PKM2-mediated cellular metabolic alterations. Targeting metabolic regulators of PKM2 might have therapeutic benefits for women with LAM and other female-specific neoplasms.


Assuntos
Estrogênios/metabolismo , Piruvato Quinase/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Animais , Linhagem Celular Tumoral , Estrogênios/fisiologia , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/fisiopatologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosforilação , Piruvato Quinase/fisiologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/genética
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