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1.
Biomolecules ; 11(12)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34944532

RESUMO

It has been considered that proline dehydrogenase/proline oxidase (PRODH/POX) is involved in antineoplastic activity of metformin (MET). The aim of this study is identification of key metabolites of glycolysis, pentose phosphate pathway (PPP), tricarboxylic acids (TCA), urea cycles (UC) and some amino acids in MET-treated MCF-7 cells and PRODH/POX-knocked out MCF-7 (MCF-7crPOX) cells. MCF-7crPOX cells were generated by using CRISPR-Cas9. Targeted metabolomics was performed by LC-MS/MS/QqQ. Expression of pro-apoptotic proteins was evaluated by Western blot. In the absence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to similar inhibition of glycolysis (drastic increase in intracellular glucose and pyruvate) and increase in the utilization of phospho-enol-pyruvic acid, glucose-6-phosphate and some metabolites of TCA and UC, contributing to apoptosis. However, in the presence of glutamine, MET treatment or PRODH/POX-knock out of MCF-7 cells contributed to utilization of some studied metabolites (except glucose), facilitating pro-survival phenotype of MCF-7 cells in these conditions. It suggests that MET treatment or PRODH/POX-knock out induce similar metabolic effects (glucose starvation) and glycolysis is tightly linked to glutamine metabolism in MCF-7 breast cancer cells. The data provide insight into mechanism of anticancer activity of MET as an approach to further studies on experimental breast cancer therapy.


Assuntos
Neoplasias da Mama/metabolismo , Glutamina/metabolismo , Metabolômica/métodos , Metformina/farmacologia , Prolina Oxidase/genética , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Cromatografia Líquida , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Glicólise/efeitos dos fármacos , Humanos , Células MCF-7 , Via de Pentose Fosfato/efeitos dos fármacos , Espectrometria de Massas em Tandem , Ácidos Tricarboxílicos/metabolismo , Ureia/metabolismo
2.
Elife ; 102021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34709178

RESUMO

Cellular metabolism has key roles in T cells differentiation and function. CD4+ T helper-1 (Th1), Th2, and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis. Here, we showed that blocking 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shifts toward Th1, Th2, and Th17 phenotypes which led to the development of fetal inflammatory disorder in mice model. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD blocked Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.


Assuntos
Via de Pentose Fosfato , Fosfogluconato Desidrogenase/genética , Linfócitos T Reguladores/fisiologia , Animais , Camundongos , Fosfogluconato Desidrogenase/metabolismo
3.
J Biotechnol ; 342: 54-63, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34687809

RESUMO

Pseudomonas aeruginosa is an important chassis for production of polyhydroxyalkanoates (PHA) and rhamnolipids (RHL). Advances in the understanding of the biosynthesis metabolism of these biocompounds are crucial for increasing yield. 13C-Metabolic Flux Ratio Analysis (13C-MFA) is a technique to estimate in vivo metabolic fluxes ratios. PHA and RHL are essentially non-growth associated products of biotechnological interest and both contain hydroxyalkanoates (HAs), whose labeling patterns could be accessed by GC-MS. In this study, to reveal the relative contributions of the Entner-Doudoroff (ED) pathway and the non-oxidative Pentose Phosphate (PP) pathway to PHA and RHL production, 13C-MFA was performed in Pseudomonas aeruginosa LFM634 when supplied with labeled glucose. This bacterial strain lacks both functional EMP and the oxidative PP branch. Labeling patterns in HAs were measured. Experiments with [U-13C] glucose indicated a low flux though PP pathway. An optimal design of labeling experiment showed that [6-13C] glucose would be the best substrate to enable an estimation of the ED flux with high accuracy. Results of experiments performed with this isotope indicated that about two-thirds of glyceraldehyde 3-phosphate is recycled through a cyclic ED architecture, suggesting that P. aeruginosa utilizes that cycle to regulate the NADPH/Acetyl-CoA ratio for PHA and RHL biosynthesis.


Assuntos
Poli-Hidroxialcanoatos , Glucose , Glicolipídeos , Via de Pentose Fosfato , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas aeruginosa/metabolismo
4.
Tohoku J Exp Med ; 255(1): 49-55, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34526430

RESUMO

Diamond-Blackfan anemia is a congenital bone marrow failure syndrome characterized by red blood cell (RBC) aplasia with varied malformations in infants. Elevated activity of adenosine deaminase (ADA) has been considered as a useful biomarker of Diamond-Blackfan anemia, and ADA assay has been shown to be more sensitive than genetic diagnosis. Approximately, 80% of the examined patients showed elevated ADA activity, whereas genetic tests of ribosome subunit genes identified mutations in approximately 60% of the patients. We previously reported that reduced glutathione (GSH) levels in RBCs may serve as a biomarker of Diamond-Blackfan anemia. In this study, to confirm the universality of our data, we extended the analysis to seven RBC enzymes and GSH of 14 patients with Diamond-Blackfan anemia and performed a cross-analysis study using enzyme activity assay and recently reported proteome data. Statistical analysis revealed that both data exhibited high similarity, upregulation in the hexokinase and pentose-phosphate pathway, and downregulation in glycolytic enzymes such as phosphofructokinase and pyruvate kinase, in the RBCs obtained from the subjects with Diamond-Blackfan anemia. The only discrepancy between enzyme activity and proteome data was observed in glucose-6-phosphate dehydrogenase (G6PD), as increased G6PD activity showed no relation with the significant elevation in protein levels. These results suggest that our enzymatic activity data of Diamond-Blackfan anemia are universal and that the enzymatic activation of G6PD via a hitherto-unveiled mechanism is another metabolic feature of RBCs of Diamond-Blackfan anemia.


Assuntos
Anemia de Diamond-Blackfan/sangue , Anemia de Diamond-Blackfan/enzimologia , Eritrócitos/enzimologia , Adolescente , Aminoidrolases/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Regulação para Baixo , Glucosefosfato Desidrogenase/sangue , Glutationa/sangue , Glicólise , Humanos , Lactente , Japão , Via de Pentose Fosfato , Regulação para Cima
5.
J Chromatogr A ; 1656: 462531, 2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34520889

RESUMO

Highly selective methods for the analysis of intermediate metabolites involved in glycolysis and phosphate pentose pathways are essential for metabolism and metabolic flux studies. However, the successful separation of phosphorylated compounds is difficult due to their high polarity, as well as their structural isomers. In this study, phosphorylated compounds in spiked serum samples were analyzed using capillary electrophoresis tandem mass spectrometry (CE-MS/MS) and gas chromatography (GC)-MS/MS. Following liquid-liquid extraction, ultrafiltration and derivatization steps were needed to perform CE-MS/MS and GC-MS/MS, respectively. The CE-MS/MS method allowed for the identification and quantification of all 15 biologically important phosphorylated compounds, whereas only 13 compounds were identified and quantified by GC-MS/MS. Both methods demonstrated wide linear ranges, good interday (<9.6%: CE-MS/MS; <14.7%: GC-MS/MS) and intraday (<13.0%: CE-MS/MS; <14.9%: GC-MS/MS) variability, and limits of detection (LODs) in the ranges of 0.25-2 and 0.05-0.5 µmol/L for CE-MS/MS and GC-MS/MS, respectively. In the phosphorylated compound stability test, the instability of glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) was observed during freeze-thaw and long-term storage due to reversible isomerization. The results of CE-MS/MS and GC-MS/MS analysis showed that the concentrations of phosphorylated compounds determined using the two methods matched closely, while that of glycerol 3-phosphate (G3P) showed some variability in cell extracts. Therefore, while both CE-MS/MS and GC-MS/MS are suitable for analyzing metabolites resulting from the glycolysis and pentose phosphate pathways, additional validation is needed for some compounds, depending on the background matrix.


Assuntos
Via de Pentose Fosfato , Espectrometria de Massas em Tandem , Eletroforese Capilar , Cromatografia Gasosa-Espectrometria de Massas , Glicólise
6.
Cells ; 10(9)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34572080

RESUMO

Embryonic stem cells (ESCs) are pluripotent cells with indefinite self-renewal ability and differentiation properties. To function properly and maintain genomic stability, ESCs need to be endowed with an efficient repair system as well as effective redox homeostasis. In this study, we investigated different aspects involved in ESCs' response to iron accumulation following stable knockdown of the ferritin heavy chain (FTH1) gene, which encodes for a major iron storage protein with ferroxidase activity. Experimental findings highlight unexpected and, to a certain extent, paradoxical results. If on one hand FTH1 silencing does not correlate with increased ROS production nor with changes in the redox status, strengthening the concept that hESCs are extremely resistant and, to a certain extent, even refractory to intracellular iron imbalance, on the other, the differentiation potential of hESCs seems to be affected and apoptosis is observed. Interestingly, we found that FTH1 silencing is accompanied by a significant activation of the nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signaling pathway and pentose phosphate pathway (PPP), which crosstalk in driving hESCs antioxidant cascade events. These findings shed new light on how hESCs perform under oxidative stress, dissecting the molecular mechanisms through which Nrf2, in combination with PPP, counteracts oxidative injury triggered by FTH1 knockdown.


Assuntos
Ferritinas/genética , Células-Tronco Embrionárias Humanas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Oxirredutases/genética , Elementos de Resposta Antioxidante , Apoptose , Diferenciação Celular , Células Cultivadas , Ferritinas/farmacologia , Inativação Gênica , Humanos , Oxirredução , Oxirredutases/metabolismo , Via de Pentose Fosfato , Transdução de Sinais
7.
J Agric Food Chem ; 69(33): 9625-9631, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34382797

RESUMO

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA, pfkB, and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.


Assuntos
Via de Pentose Fosfato , Xilitol , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Deleção de Genes , Glucose , Glicólise , NADP/metabolismo , Fosfatos , Xilose
8.
Elife ; 102021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34382934

RESUMO

Alterations to the androgen receptor (AR) signalling axis and cellular metabolism are hallmarks of prostate cancer. This study provides insight into both hallmarks by uncovering a novel link between AR and the pentose phosphate pathway (PPP). Specifically, we identify 6-phosphogluoconate dehydrogenase (6PGD) as an androgen-regulated gene that is upregulated in prostate cancer. AR increased the expression of 6PGD indirectly via activation of sterol regulatory element binding protein 1 (SREBP1). Accordingly, loss of 6PGD, AR or SREBP1 resulted in suppression of PPP activity as revealed by 1,2-13C2 glucose metabolic flux analysis. Knockdown of 6PGD also impaired growth and elicited death of prostate cancer cells, at least in part due to increased oxidative stress. We investigated the therapeutic potential of targeting 6PGD using two specific inhibitors, physcion and S3, and observed substantial anti-cancer activity in multiple models of prostate cancer, including aggressive, therapy-resistant models of castration-resistant disease as well as prospectively collected patient-derived tumour explants. Targeting of 6PGD was associated with two important tumour-suppressive mechanisms: first, increased activity of the AMP-activated protein kinase (AMPK), which repressed anabolic growth-promoting pathways regulated by acetyl-CoA carboxylase 1 (ACC1) and mammalian target of rapamycin complex 1 (mTORC1); and second, enhanced AR ubiquitylation, associated with a reduction in AR protein levels and activity. Supporting the biological relevance of positive feedback between AR and 6PGD, pharmacological co-targeting of both factors was more effective in suppressing the growth of prostate cancer cells than single-agent therapies. Collectively, this work provides new insight into the dysregulated metabolism of prostate cancer and provides impetus for further investigation of co-targeting AR and the PPP as a novel therapeutic strategy.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Linhagem Celular , Emodina/análogos & derivados , Retroalimentação , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Via de Pentose Fosfato , Neoplasias da Próstata/genética , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
9.
Biochem Pharmacol ; 192: 114736, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34411567

RESUMO

Reprogramming of energy metabolism is a hallmarkofcancer, and the pentose phosphate pathway (PPP) is a major glucose metabolic pathway important for meeting the cellular demands of biosynthesis and anti-oxidant defense. Our previous study showed that phosphoinositide 3-kinase enhancer-activating Akt (PIKE-A) plays an important role in glioblastoma cell survival and growth under cellular energy stress condition. However, the crucial functions of PIKE-A in cancer energy metabolism are poorly understood.In the present study, we show that PIKE-A promotes DNA biosynthesis, NADPH production and inhibits reactive oxygen species (ROS) production, leading to increasing proliferation and growth of glioblastoma cell and suppressing cellular senescence. Mechanistically, PIKE-A binds to STAT3 and stimulates its phosphorylation mediated by tyrosine kinase Fyn, which enhances transcription of the rate-limitting enzyme glucose-6-phosphate dehydrogenase (G6PD) in the PPP. Finally, targeting PIKE-A-G6PD axis sensitizes glioblastoma to temozolomide (TMZ)treatment. This study reveals that STAT3 is a novel binding partner of PIKE-A which recruits Fyn to phosphorylate STAT3, contributing to the expression of G6PD, leading to promoting tumor growth and suppressing cellular senescence. Thus, the PIKE-A/STAT3/G6PD axis strongly links the PPP to carcinogenesis and may become a promising cancer therapeutic target.


Assuntos
Proliferação de Células/fisiologia , Proteínas de Ligação ao GTP/biossíntese , Proteínas Ativadoras de GTPase/biossíntese , Glioblastoma/metabolismo , Glucosefosfato Desidrogenase/biossíntese , Via de Pentose Fosfato/fisiologia , Fator de Transcrição STAT3/biossíntese , Animais , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes/métodos , Glioblastoma/patologia , Glucosefosfato Desidrogenase/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Nus , Fosforilação/fisiologia
10.
Cancer Sci ; 112(10): 4112-4126, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363719

RESUMO

Evi1 is a transcription factor essential for the development as well as progression of acute myeloid leukemia (AML) and high Evi1 AML is associated with extremely poor clinical outcome. Since targeting metabolic vulnerability is the emerging therapeutic strategy of cancer, we herein investigated a novel therapeutic target of Evi1 by analyzing transcriptomic, epigenetic, and metabolomic profiling of mouse high Evi1 leukemia cells. We revealed that Evi1 overexpression and Evi1-driven leukemic transformation upregulate transcription of gluconeogenesis enzyme Fbp1 and other pentose phosphate enzymes with interaction between Evi1 and the enhancer region of these genes. Metabolome analysis using Evi1-overexpressing leukemia cells uncovered pentose phosphate pathway upregulation by Evi1 overexpression. Suppression of Fbp1 as well as pentose phosphate pathway enzymes by shRNA-mediated knockdown selectively decreased Evi1-driven leukemogenesis in vitro. Moreover, pharmacological or shRNA-mediated Fbp1 inhibition in secondarily transplanted Evi1-overexpressing leukemia mouse significantly decreased leukemia cell burden. Collectively, targeting FBP1 is a promising therapeutic strategy of high Evi1 AML.


Assuntos
Frutose-Bifosfatase/metabolismo , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Via de Pentose Fosfato , Animais , Modelos Animais de Doenças , Progressão da Doença , Elementos Facilitadores Genéticos , Epigênese Genética , Frutose-Bifosfatase/antagonistas & inibidores , Frutose-Bifosfatase/genética , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/patologia , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Via de Pentose Fosfato/genética , RNA Interferente Pequeno , Ensaio Tumoral de Célula-Tronco , Regulação para Cima
11.
PLoS Pathog ; 17(8): e1009326, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34339477

RESUMO

Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and thus represent potential targets for antibacterial strategies. Here we focused on the role of the pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of Francisella novicida. The involvement of the PPP in the intracellular life cycle of Francisella was first demonstrated by studying PPP inactivating mutants. Indeed, we observed that inactivation of the tktA, rpiA or rpe genes severely impaired intramacrophage multiplication during the first 24 hours. However, time-lapse video microscopy demonstrated that rpiA and rpe mutants were able to resume late intracellular multiplication. To better understand the links between PPP and other metabolic networks in the bacterium, we also performed an extensive proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections to glycolysis, the tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Altogether our study highlights how PPP plays a key role in the pathogenesis and growth of Francisella in its intracellular niche.


Assuntos
Proteínas de Bactérias/metabolismo , Drosophila melanogaster/metabolismo , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Metaboloma , Via de Pentose Fosfato , Proteoma , Animais , Proteínas de Bactérias/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/microbiologia , Francisella/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicólise , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
12.
Biomolecules ; 11(8)2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34439897

RESUMO

Several studies reported that metformin, the most widely used drug for type 2 diabetes, might affect cancer aggressiveness. The biguanide seems to directly impair cancer energy asset, with the consequent phosphorylation of AMP-activated protein kinase (AMPK) inhibiting cell proliferation and tumor growth. This action is most often attributed to a well-documented blockage of oxidative phosphorylation (OXPHOS) caused by a direct interference of metformin on Complex I function. Nevertheless, several other pleiotropic actions seem to contribute to the anticancer potential of this biguanide. In particular, in vitro and in vivo experimental studies recently documented that metformin selectively inhibits the uptake of 2-[18F]-Fluoro-2-Deoxy-D-Glucose (FDG), via an impaired catalytic function of the enzyme hexose-6P-dehydrogenase (H6PD). H6PD triggers a still largely uncharacterized pentose-phosphate pathway (PPP) within the endoplasmic reticulum (ER) that has been found to play a pivotal role in feeding the NADPH reductive power for both cellular proliferation and antioxidant responses. Regardless of its exploitability in the clinical setting, this metformin action might configure the ER metabolism as a potential target for innovative therapeutic strategies in patients with solid cancers and potentially modifies the current interpretative model of FDG uptake, attributing PET/CT capability to predict cancer aggressiveness to the activation of H6PD catalytic function.


Assuntos
Glucose/metabolismo , Metformina/metabolismo , Neoplasias/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Pesquisa Biomédica , Desidrogenases de Carboidrato/metabolismo , Proliferação de Células , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Fluordesoxiglucose F18 , Humanos , Hipoglicemiantes/metabolismo , NADP/metabolismo , Fosforilação Oxidativa , Via de Pentose Fosfato , Fosforilação , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Reprodutibilidade dos Testes
13.
Environ Sci Pollut Res Int ; 28(44): 62919-62926, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34218382

RESUMO

Copper (Cu) is one of the toxic elements that cause environmental pollution. As a result of excessive accumulation of copper in the organism, it causes damage in various organs and tissues and hemolysis in erythrocytes. Astaxanthin (ATX) is a pigment belonging to the xanthophyll family, which is an oxygenated derivative of carotenoids. Thanks to its powerful antioxidant properties, ATX has an extraordinary potential to protect the organism against various diseases, especially cancer. The main objective of this study was to investigate the toxic effect of copper ions on the glucose 6-phosphate dehydrogenase (G6PD), 6-phospho-gluconate dehydrogenase (6PGD), glutathione reductase (GR), glutathione S-transferase (GST), and thioredoxin reductase (TrxR) enzymes and the role of astaxanthin in reducing this effect. In in vivo study, Wistar Albino male rats (n=28) were randomly divided into 4 groups: the control group, copper (Cu2+) group, astaxanthin (ATX) group, and copper + astaxanthin (Cu2++ATX) group. The results show that G6PD enzyme activity in Cu2+ group was strongly inhibited (p ˂ 0.05), while in other groups, there were no significant effects compared to the control group (p ⩾ 0.05). 6PGD enzyme activity was significantly reduced in Cu2+ group compared to that in the control group (p ˂ 0.05), and GR enzyme activity was lower in Cu2+ group compared to that in the control group (p ˂ 0.05). Similarly, when GST enzyme activity was evaluated, a strong decrease was observed in the Cu2+ group compared to that in the control group (p ˂ 0.05), while the enzyme activity in the Cu2++ATX group approached the control group (p ⩾ 0.05). When TrxR enzyme activity level was examined, a statistically significant decrease was observed in the Cu2+ and Cu2++ATX groups (p ˂ 0.05), and the enzyme activity in the ATX group was found to be close to that in the control group. When in vitro results were evaluated, it was observed that copper ions inhibited G6PD enzyme purified from rat erythrocyte tissues with IC50=1.90 µM value and Ki = 0.97 µM ± 0.082 value and the inhibition was non-competitive. From the results, it can be concluded that Cu2+ ions have an inhibitory effect on rat erythrocyte pentose phosphate pathway and antioxidant system enzymes both in vivo and in vitro, and astaxanthin reduces this effect.


Assuntos
Antioxidantes , Via de Pentose Fosfato , Animais , Cobre , Íons , Ratos , Ratos Wistar , Xantofilas
14.
Appl Microbiol Biotechnol ; 105(13): 5471-5489, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34250571

RESUMO

6-phosphogluconate dehydrogenase (6PGDH) participates in pentose phosphate pathway of glucose metabolism by catalyzing oxidative decarboxylation of 6-phsophogluconate (6PG) and its absence has been lethal for several eukaryotes. Despite being a validated drug target in many organisms like Plasmodium, the enzyme has not been explored in leishmanial parasites. In the present study, 6PGDH of Leishmania donovani (Ld6PGDH) is cloned and purified followed by its characterization using biochemical and structural approaches. Ld6PGDH lacks the glycine-serine-rich sequence at its C-terminal that is present in other eukaryotes including humans. Leishmanial 6PGDH possesses more affinity for substrate (6PG) and cofactor (NADP) in comparison to that of human. The enzymatic activity is inhibited by gentamicin and cefuroxime through competitive mode with functioning more potently towards leishmanial 6PGDH than its human counterpart. CD analysis has shown higher α-helical content in the secondary structure of Ld6PGDH, while fluorescence studies revealed that tryptophan residues are not completely accessible to solvent environment. The three-dimensional structure was generated through homology modelling and docked with substrate and cofactor. The docking studies demonstrated two separate binding pockets for 6PG and NADP with higher affinity for the cofactor binding, and Asn105 is interacting with substrate as well as the cofactor. Additionally, MD simulation has shown complexes of Ld6PGDH with 6PG and NADP to be more stable than its apo form. Altogether, the present study might provide the foundation to investigate this enzyme as potential target against leishmaniasis. KEY POINTS: • Ld6PGDH enzymatic activity is competitively inhibited by gentamicin and cefuroxime. • It displays more helical contents and all structural characteristics of 6PGDH family. • Interaction studies demonstrate higher affinity of cofactor than substrate for Ld6PGDH.


Assuntos
Leishmania donovani , Fosfogluconato Desidrogenase , Humanos , Cinética , Leishmania donovani/metabolismo , Via de Pentose Fosfato , Fosfogluconato Desidrogenase/genética , Estrutura Secundária de Proteína
15.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207355

RESUMO

Astrocytes (also, astroglia) consume huge amounts of glucose and produce lactate regardless of sufficient oxygen availability, indicating a high capacity for aerobic glycolysis. Glycolysis in astrocytes is activated in accordance with neuronal excitation and leads to increases in the release of lactate from astrocytes. Although the fate of this lactate remains somewhat controversial, it is believed to fuel neurons as an energy substrate. Besides providing lactate, astrocytic glycolysis plays an important role in neuroprotection. Among the minor pathways of glucose metabolism, glucose flux to the pentose-phosphate pathway (PPP), a major shunt pathway of glycolysis, is attracting research interest. In fact, PPP activity in astrocytes is five to seven times higher than that in neurons. The astrocytic PPP plays a key role in protecting neurons against oxidative stress by providing neurons with a reduced form of glutathione, which is necessary to eliminate reactive oxygen species. Therefore, enhancing astrocytic glycolysis might promote neuronal protection during acute ischemic stroke. Contrariwise, the dysfunction of astrocytic glycolysis and the PPP have been implicated in the pathogenesis of various neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, since mitochondrial dysfunction and oxidative stress trigger and accelerate disease progression.


Assuntos
Astrócitos/metabolismo , Glicólise , Doenças Neurodegenerativas/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/genética , Via de Pentose Fosfato , Acidente Vascular Cerebral/genética
16.
Cells ; 10(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207480

RESUMO

The pentose phosphate pathway (PPP) is divided into an oxidative branch that makes pentose phosphates and a non-oxidative branch that consumes pentose phosphates, though the non-oxidative branch is considered reversible. A modified version of the non-oxidative branch is a critical component of the Calvin-Benson cycle that converts CO2 into sugar. The reaction sequence in the Calvin-Benson cycle is from triose phosphates to pentose phosphates, the opposite of the typical direction of the non-oxidative PPP. The photosynthetic direction is favored by replacing the transaldolase step of the normal non-oxidative PPP with a second aldolase reaction plus sedoheptulose-1,7-bisphosphatase. This can be considered an anabolic version of the non-oxidative PPP and is found in a few situations other than photosynthesis. In addition to the strong association of the non-oxidative PPP with photosynthesis metabolism, there is recent evidence that the oxidative PPP reactions are also important in photosynthesizing cells. These reactions can form a shunt around the non-oxidative PPP section of the Calvin-Benson cycle, consuming three ATP per glucose 6-phosphate consumed. A constitutive operation of this shunt occurs in the cytosol and gives rise to an unusual labeling pattern of photosynthetic metabolites while an inducible shunt in the stroma may occur in response to stress.


Assuntos
Via de Pentose Fosfato/fisiologia , Fotossíntese/fisiologia , Trifosfato de Adenosina/metabolismo , Citosol/metabolismo , Glucose-6-Fosfatase/metabolismo
17.
Int J Mol Sci ; 22(14)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34299056

RESUMO

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Concanavalina A/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Linfócitos/metabolismo , Mitógenos/farmacologia , Fosfatidilinositol 3-Quinases/química , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Apoptose , Proteínas Reguladoras de Apoptose/genética , Autofagia , Glicólise , Humanos , Linfócitos/efeitos dos fármacos , Via de Pentose Fosfato , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais
18.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299338

RESUMO

Obesity manifests itself with low-grade chronic inflammation that shapes immune responses during infection. Albeit obese individuals are at risk of higher mortality due to comorbidities, they are better protected from systemic inflammation. Recently, we showed that in the vasculature of obese mice kept on high-fat diet (HFD), neutrophils produce less neutrophil extracellular traps (NETs) than in lean controls (normal diet, ND). NETs are used by neutrophils to counteract severe infection, but they also cause collateral damage. Hardly anything is known about metabolic requirements for their formation, especially in the context of obesity and/or sepsis. Thus, we aimed to study the immunometabolism of NET formation by application of ex vivo neutrophil analyses (Seahorse analyzer, selective inhibitors, confocal imaging) and intravital microscopy. The obtained data show that glycolysis and/or pentose phosphate pathway are involved in NETs release by ND neutrophils in both physiological and inflammatory conditions. In contrast, such cells of septic HFD mice utilize these routes only to spontaneously cast NETs, while after secondary ex vivo activation they exhibit so called "exhausted phenotype", which manifests itself in diminished NET release despite high glycolytic potential and flexibility to oxidize fatty acids. Moreover, impact of ATP synthase inhibition on NET formation is revealed. Overall, the study shows that the neutrophil potential to cast NETs depends on both the metabolic and inflammatory state of the individual.


Assuntos
Armadilhas Extracelulares/metabolismo , Obesidade/metabolismo , Animais , Dieta Hiperlipídica , Armadilhas Extracelulares/imunologia , Glicólise , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/metabolismo , Obesidade/imunologia , Obesidade/patologia , Via de Pentose Fosfato , Sepse/metabolismo
19.
Redox Biol ; 46: 102065, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34293554

RESUMO

Although glucose, through pentose phosphate pathway (PPP), is the main source to generate NADPH, solid tumors are often deprived of glucose, hence alternative metabolic pathways to maintain NADPH homeostasis in cancer cells are required. Here, we report that lactate and glutamine support NADPH production via isocitrate dehydrogenase 1 (IDH1) and malic enzyme 1 (ME1), respectively, under glucose-deprived conditions. Isotopic tracing demonstrates that lactate participates in the formation of isocitrate. Malate derived from glutamine in mitochondria shuttles to cytosol to produce NADPH. In cells cultured in the absence of glucose, knockout of IDH1 and ME1 decreases NADPH/NADP+ and GSH/GSSG, increases ROS level and facilitates cell necrosis. In 4T1 murine breast tumors, knockout of ME1 retards tumor growth in vivo, with combined ME1/IDH1 knockout more strongly suppressing tumor growth. Our findings reveal two alternative NADPH-producing pathways that cancer cells use to resist glucose starvation, reflecting the metabolic plasticity and flexibility of cancer cells adapting to nutrition stress.


Assuntos
Glucose , Neoplasias , Animais , Glutamina , Ácido Láctico , Camundongos , NADP/metabolismo , Neoplasias/genética , Via de Pentose Fosfato
20.
Biomolecules ; 11(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065948

RESUMO

The pentose phosphate pathway (PPP) is a route that can work in parallel to glycolysis in glucose degradation in most living cells. It has a unidirectional oxidative part with glucose-6-phosphate dehydrogenase as a key enzyme generating NADPH, and a non-oxidative part involving the reversible transketolase and transaldolase reactions, which interchange PPP metabolites with glycolysis. While the oxidative branch is vital to cope with oxidative stress, the non-oxidative branch provides precursors for the synthesis of nucleic, fatty and aromatic amino acids. For glucose catabolism in the baker's yeast Saccharomyces cerevisiae, where its components were first discovered and extensively studied, the PPP plays only a minor role. In contrast, PPP and glycolysis contribute almost equally to glucose degradation in other yeasts. We here summarize the data available for the PPP enzymes focusing on S. cerevisiae and Kluyveromyces lactis, and describe the phenotypes of gene deletions and the benefits of their overproduction and modification. Reference to other yeasts and to the importance of the PPP in their biotechnological and medical applications is briefly being included. We propose future studies on the PPP in K. lactis to be of special interest for basic science and as a host for the expression of human disease genes.


Assuntos
Glucose/metabolismo , Kluyveromyces/metabolismo , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Animais , Glicólise , Humanos , Oxirredução , Estresse Oxidativo , Via de Pentose Fosfato
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