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1.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
2.
J Biol Chem ; 296: 100219, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839685

RESUMO

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFKs), specific glucokinases (ADP-GKs), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, none exhibits fructose-6-phosphate (F6P) at the active site. Using an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with F6P at its active site. Key residues for sugar binding and catalysis were identified by alanine scanning, D36 being a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding because its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semiclosed, and closed).


Assuntos
Proteínas Arqueais/química , Frutosefosfatos/química , Glucoquinase/química , Methanosarcinales/química , Fosfofrutoquinases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Frutosefosfatos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucoquinase/genética , Glucoquinase/metabolismo , Cinética , Ligantes , Methanosarcinales/enzimologia , Methanosarcinales/genética , Modelos Moleculares , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
3.
Biochim Biophys Acta Proteins Proteom ; 1869(6): 140642, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33647452

RESUMO

Anhydrobiotic organisms accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered proteins (IDPs) reported to improve cellular tolerance to water stress. Here we show that AfrLEA6, a Group 6 LEA protein only recently discovered in animals, protects lactate dehydrogenase (LDH), citrate synthase (CS) and phosphofructokinase (PFK) against damage during desiccation. In some cases, protection is enhanced by trehalose, a naturally-occurring protective solute. An open question is whether gain of secondary structure by LEA proteins during drying is a prerequisite for this stabilizing function. We used incremental drying (equilibration to a series of relative humidities, RH) to test the ability of AfrLEA2, a Group 3 LEA protein, to protect desiccation-sensitive PFK. AfrLEA2 was chosen due to its exceptional ability to protect PFK. In parallel, circular dichroism (CD) spectra were obtained for AfrLEA2 across the identical range of relative water contents. Protection of PFK by AfrLEA2, above that observed with trehalose and BSA, coincides with simultaneous gain of α-helix in AfrLEA2. At 100% RH, the CD spectrum for AfrLEA2 is typical of random coil, while at decreasing RH, the spectrum shows higher ellipticity at 191 nm and minima at 208 and 220 nm, diagnostic of α-helix. This study provides experimental evidence linking the gain of α-helix with stabilization of a target protein across a graded series of hydration states. Mechanistically, it is intriguing that certain other functions of these IDPs, like preventing aggregation of target proteins, can occur in fully hydrated cells and apparently do not require gain of α-helix.


Assuntos
Artemia/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Fosfofrutoquinases/metabolismo , Animais , Artemia/química , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Dicroísmo Circular , Dessecação , Fosfofrutoquinases/química , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Estabilidade Proteica
4.
Microb Cell Fact ; 20(1): 32, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531004

RESUMO

BACKGROUND: Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and "bifid shunt", to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). RESULT: Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. CONCLUSION: This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.


Assuntos
Sistemas CRISPR-Cas/genética , Escherichia coli/enzimologia , Glucosefosfato Desidrogenase/metabolismo , Glicólise , Ácido Mevalônico/metabolismo , Fosfofrutoquinases/metabolismo , Trifosfato de Adenosina/metabolismo , Isótopos de Carbono , Regulação para Baixo , Metabolismo Energético , Fermentação , Análise do Fluxo Metabólico , NADP/metabolismo , Via de Pentose Fosfato , Regiões Promotoras Genéticas/genética
5.
Nat Commun ; 12(1): 1052, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594070

RESUMO

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.


Assuntos
Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Trypanosoma/enzimologia , Tripanossomíase Africana/metabolismo , Tripanossomíase Africana/parasitologia , Doença Aguda , Regulação Alostérica/efeitos dos fármacos , Animais , Células Hep G2 , Humanos , Concentração Inibidora 50 , Estimativa de Kaplan-Meier , Camundongos , Parasitos/efeitos dos fármacos , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Relação Estrutura-Atividade , Trypanosoma/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico
6.
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 , Fosfofrutoquinases/metabolismo , 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 , Fosfofrutoquinases/antagonistas & inibidores , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
7.
Plant Cell Physiol ; 62(3): 401-410, 2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-33416847

RESUMO

Various proteins in plant chloroplasts are subject to thiol-based redox regulation, allowing light-responsive control of chloroplast functions. Most redox-regulated proteins are known to be reductively activated in the light in a thioredoxin (Trx)-dependent manner, but its regulatory network remains incompletely understood. Using a biochemical procedure, we here show that a specific form of phosphofructokinase (PFK) is a novel redox-regulated protein whose activity is suppressed upon reduction. PFK is a key enzyme in the glycolytic pathway. In Arabidopsis thaliana, PFK5 is targeted to chloroplasts and uniquely contains an insertion sequence harboring two Cys residues (Cys152 and Cys157) in the N-terminal region. Redox shift assays using a thiol-modifying reagent indicated that PFK5 is efficiently reduced by a specific type of Trx, namely, Trx-f. PFK5 enzyme activity was lowered with the Trx-f-dependent reduction. PFK5 redox regulation was bidirectional; PFK5 was also oxidized and activated by the recently identified Trx-like2/2-Cys peroxiredoxin pathway. Mass spectrometry-based peptide mapping analysis revealed that Cys152 and Cys157 are critical for the intramolecular disulfide bond formation in PFK5. The involvement of Cys152 and Cys157 in PFK5 redox regulation was further supported by a site-directed mutagenesis study. PFK5 catalyzes the reverse reaction of fructose 1,6-bisphosphatase (FBPase), which is reduced and activated specifically by Trx-f. Our data suggest that PFK5 redox regulation, together with that of FBPase, constitutes a checkpoint for switching light/dark metabolism in chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Oxirredução , Fosfofrutoquinases/metabolismo , Nucleotídeos de Adenina/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , Cisteína/metabolismo , Redes e Vias Metabólicas , Peroxirredoxinas/metabolismo , Fosfofrutoquinases/genética
8.
Neurochem Res ; 45(10): 2474-2486, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761296

RESUMO

Neuronostatin (NST) is an endogenous peptide hormone, it has the ability to improve oligomeric Aß (oAß)-induced cognitive impairments and increase blood glucose levels in mice. However, the relationship between NST and oAß regarding brain glucose metabolism has not yet been established. The present study defined the contributions of NST and oAß in the brain glucose metabolism in mice. It was found that i.c.v. co-administration of NST (3 nmol/mouse) and oAß (1 nmol/mouse) decreased the mRNA expressions of glucose-6-phosphate dehydrogenase and phosphofructokinase. The treatments were observed to reduce ATP production and the enzyme activities of glucose-6-phosphate dehydrogenase and hexokinase in both the cortex and hippocampus. Simultaneously, co-injection of NST and oAß inhibited the mRNA and protein expression of glucose transporters GLUT3 and GLUT1 in the cortex and hippocampus. NST promoted the oAß-induced decreased the cortical NeuN staining, while oAß increased the levels of NST in both the cortex and hippocampus. I.c.v. co-administration of NST and oAß led to increase the levels of GPR107 expression and the phosphorylation of PKA, Akt, PERK and eIF-2α in the cortex. These findings suggest that NST promoted oAß-induced dysfunctional glucose metabolism through the GPR107/PKA/Akt signaling pathway and PERK/eIF2α axis in the brain, which thus contributes to metabolic dysfunction and Alzheimer's disease (AD) pathophysiology.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Córtex Cerebral/metabolismo , Glucose/metabolismo , Hipocampo/metabolismo , Fragmentos de Peptídeos/farmacologia , Hormônios Peptídicos/farmacologia , Trifosfato de Adenosina/metabolismo , Peptídeos beta-Amiloides/química , Animais , Combinação de Medicamentos , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glicólise/efeitos dos fármacos , Hexoquinase/metabolismo , Masculino , Camundongos , Fragmentos de Peptídeos/química , Fosfofrutoquinases/metabolismo , Multimerização Proteica , Transdução de Sinais/fisiologia
9.
Physiol Rep ; 8(11): e14473, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32512646

RESUMO

The aim of this study was to investigate effects of short-term hypoxic training on lactate metabolism in the gluteus medius muscle of Thoroughbreds. Using crossover design (3 months washout), eight Thoroughbred horses were trained for 2 weeks in normoxia (FI O2  = 21%) and hypoxia (FI O2  = 18%) each. They ran at 95% maximal oxygen consumption (V̇O2max ) on a treadmill inclined at 6% for 2 min (3 days/week) measured under normoxia. Before and after each training period, all horses were subjected to an incremental exercise test (IET) under normoxia. Following the 2-week trainings, V̇O2max in IET increased significantly under both oxygen conditions. The exercise duration in IET increased significantly only after hypoxic training. The monocarboxylate transporter (MCT) 1 protein levels remained unchanged after training under both oxygen conditions, whereas MCT4 protein levels increased significantly after training in hypoxia but not after training in normoxia. Phosphofructokinase activity increased significantly only after hypoxic training, whereas cytochrome c oxidase activity increased significantly only after normoxic training. Our results suggest that hypoxic training efficiently enhances glycolytic capacity and levels of the lactate transporter protein MCT4, which facilitates lactate efflux from the skeletal muscle.


Assuntos
Transportadores de Ácidos Monocarboxílicos/metabolismo , Fosfofrutoquinases/metabolismo , Condicionamento Físico Animal/métodos , Condicionamento Físico Animal/fisiologia , Animais , Estudos Cross-Over , Feminino , Cavalos , Hipóxia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Consumo de Oxigênio
10.
PLoS One ; 15(6): e0233745, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32542029

RESUMO

The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (ß-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/µg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.


Assuntos
Alérgenos/química , Análise de Alimentos/métodos , Peptídeos/química , Proteólise , Alérgenos/metabolismo , Animais , Cromatografia Líquida/métodos , Inocuidade dos Alimentos , Hemoglobinas/química , Hemoglobinas/metabolismo , Lactalbumina/química , Lactalbumina/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Peptídeos/metabolismo , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Suínos , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismo
11.
PLoS One ; 15(3): e0229669, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163433

RESUMO

Exogenously hypercholesterolemic (ExHC) rats develop diet-induced hypercholesterolemia (DIHC) when fed with dietary cholesterol. Previously, we reported that, under the high-sucrose-diet-feeding condition, a loss-of-function mutation in Smek2 results in low activity of fatty acid synthase (FAS) followed by the shortage of hepatic triacylglycerol content in ExHC rats and the onset of DIHC. However, the relationship between the Smek2 mutation and FAS dysfunction is still unclear. Here, we focused on carbohydrate metabolism, which provides substrates for FAS, and analyzed carbohydrate and lipid metabolisms in ExHC rats to clarify how the deficit of Smek2 causes DIHC. Male ExHC and SD rats were fed high-sucrose or high-starch diets containing 1% cholesterol for 2 weeks. Serum cholesterol levels of the ExHC rats were higher, regardless of the dietary carbohydrate. Hepatic triacylglycerol levels were higher in only the SD rats fed the high-sucrose diet. Moreover, the ExHC rats exhibited a diabetes-like status and accumulation of hepatic glycogen and low hepatic mRNA levels of liver-type phosphofructokinase (Pfkl), which encodes a rate-limiting enzyme for glycolysis. These results suggest that the glucose utilization, particularly glycolysis, is impaired in the liver of ExHC rats. To evaluate how the diet with extremely low glucose affect to DIHC, ExHC.BN-Dihc2BN, a congenic strain that does not develop DIHC, and ExHC rats were fed a high-fructose diet containing 1% cholesterol for 2 weeks. The serum cholesterol and hepatic triacylglycerol levels were similar in the strains. Results of water-soluble metabolite analysis with primary hepatocytes, an increase in fructose-6-phosphate and decreases in succinate, malate and aspartate in ExHC rats, support impaired glycolysis in the ExHC rats. Thus, the Smek2 mutation causes abnormal hepatic glucose utilization via downregulation of Pfkl expression. This abnormal glucose metabolism disrupts hepatic fatty acid synthesis and causes DIHC in the ExHC rats.


Assuntos
Glucose/metabolismo , Hipercolesterolemia/etiologia , Hipercolesterolemia/metabolismo , Fígado/metabolismo , Animais , Animais Congênicos , Colesterol na Dieta/administração & dosagem , Colesterol na Dieta/efeitos adversos , Modelos Animais de Doenças , Regulação para Baixo , Ácido Graxo Sintase Tipo I/metabolismo , Glicólise/genética , Hipercolesterolemia/genética , Mutação com Perda de Função , Masculino , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley
12.
Nature ; 578(7796): 621-626, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32051585

RESUMO

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility1-3. Although all of these processes consume energy4,5, it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.


Assuntos
Microambiente Celular , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glucose/metabolismo , Glicólise , Dureza , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Brônquios/citologia , Bovinos , Diferenciação Celular , Linhagem Celular , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ribonucleoproteínas/metabolismo , Fibras de Estresse/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
13.
Food Chem ; 314: 126203, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31978718

RESUMO

The activity, expression and S-nitrosylation of glycogen phosphorylase (GP), phosphofructokinase (PFK) and pyruvate kinase (PK) was compared between pale, soft and exudative (PSE) and red, firm and non-exudative (RFN) pork. The nitric oxide synthase (NOS) activity of RFN pork was higher than PSE pork (P < 0.05). Glycogen and lactic acid content were significantly different between PSE and RFN samples at 1 h postmortem (P < 0.05). Compared to PSE pork, RFN pork had lower activities and higher S-nitrosylation levels of GP, PFK and PK (P < 0.05). Moreover, GP expression in RFN pork was lower (P < 0.05) while no significant differences of PFK and PK expression were observed between these two groups. These data suggest that protein S-nitrosylation can presumably regulate glycolysis by modulating glycolytic enzymes activities and then regulate the development of PSE pork.


Assuntos
Glicólise , Músculo Esquelético/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Carne de Porco/análise , Animais , Cor , Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Músculo Esquelético/enzimologia , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , Suínos
14.
FEBS J ; 287(13): 2847-2861, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31838765

RESUMO

Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK - to a much larger extent than Trypanosoma PFK - is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5 F6P value by 10-fold, compared to only a 1.3- and 1.4-fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme's 'reaching arms' and 'embracing arms' that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T-to-R equilibria for the two families, with the low-melting Leishmania PFK favouring the flexible inactive T-state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP-dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.


Assuntos
Monofosfato de Adenosina/metabolismo , Glicólise , Guanosina Trifosfato/metabolismo , Leishmania/enzimologia , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Trypanosoma brucei brucei/enzimologia , Monofosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Domínio Catalítico , Cristalografia por Raios X , Gluconeogênese , Guanosina Trifosfato/química , Humanos , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade da Espécie , Especificidade por Substrato
15.
J Ethnopharmacol ; 249: 112383, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733308

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The absence of scientific data on the age long folkloric use of Digitaria exilis grains by sufferers of diabetes prompted the present investigation. This study was aimed at evaluating the antidiabetic activity of aqueous extract of Digitaria exilis grains in streptozotocin (STZ)-induced diabetic rats. MATERIAL AND METHODS: Forty two male rats (166.43 ±â€¯3.32 g) were completely randomized into six groups (A-F) of 7 animals each. Animals in group A (control) were administered 0.5 ml of distilled water while those in groups B, C, D, E and F which were induced with diabetes mellitus (by intraperitoneal administration of 60 mg/kg body weight of STZ) were also administered distilled water, 50 mg/kg body weight of metformin (a reference antidiabetic drug), 50, 100 and 200 mg/kg body weight of aqueous extract of D. exilis grains respectively, twice daily for 14 days. Blood glucose levels and some relevant biomolecules were determined 14 days post-administration. RESULTS: Alkaloids, flavonoids, saponins, tannins, anthraquinones, terpenoids, cardiac glycosides, phlobatannins, phenolics and cardenolides were detected in the extract with alkaloids (30.20 mg/ml) occurring the most and phlobatannins (0.22 mg/ml) the least. Streptozotocin significantly (p < 0.05) increased the levels of blood glucose, serum albumin, urea, creatinine and cholesterol, activities of glucose-6-phosphatase and fructose-1,6-bisphosphatase in the liver and intake of feed and water. Body weight, weight of pancreas, pancreatic insulin, liver glycogen content, red blood cell and white blood cell and their related indices, liver hexokinase and phosphofructokinase activities were significantly reduced by STZ. In contrast, the extract significantly reversed all those STZ-treatment induced changes with the 200 mg/kg body weight of the extract producing profound values that compared favourably with the distilled water treated non-diabetic animals and metformin treated diabetic animals. CONCLUSION: Overall, this study revealed that Digitaria exilis grains possess antidiabetic activity via increased insulin secretion, as plasma concentrations of insulin were not determined, enhanced activities of hexokinase and phosphofuctokinase and repletion of hepatic glycogen content.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Digitaria/química , Hipoglicemiantes/farmacologia , Extratos Vegetais/farmacologia , Animais , Relação Dose-Resposta a Droga , Hexoquinase/metabolismo , Hipoglicemiantes/administração & dosagem , Insulina/metabolismo , Masculino , Metformina/farmacologia , Fosfofrutoquinases/metabolismo , Extratos Vegetais/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Wistar , Estreptozocina
16.
BMC Res Notes ; 12(1): 682, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640766

RESUMO

OBJECTIVE: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). RESULTS: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.


Assuntos
Metabolismo dos Carboidratos , Ensaios Enzimáticos/métodos , Glucose/metabolismo , Glicólise , Animais , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Estudos de Viabilidade , Frutose-Bifosfato Aldolase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Células HEK293 , Células Hep G2 , Hexoquinase/metabolismo , Humanos , Camundongos , Fenótipo , Fosfofrutoquinases/metabolismo , Fosfoglucomutase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Projetos Piloto
17.
J Agric Food Chem ; 67(38): 10637-10645, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31513389

RESUMO

Previous studies have shown that selenite, a representative of inorganic form selenium, exerts its anticancer effect by inducing apoptosis in androgen-dependent LNCaP prostate cancer cells, but few studies have determined the nature of cell death induced by selenite in metastatic androgen-refractory PC-3 cells. Our study showed that necrosis-like cell death rather than apoptosis, pyroptosis, or autophagic cell death was caused by selenite in PC-3 cells. Mechanistically, this type of cell death was caused by ATP depletion (26.28 ± 3.39 nmol/mg of control versus 9.12 ± 2.44 nmol/mg of 10 µM selenite treatment) that resulted from phosphofructokinase activity reduction (100.17 ± 0.17% of control versus 21.74 ± 6.65% of 10 µM selenite treatment). Our study also showed that ROS production is necessary for the decrease in cellular ATP levels and in phosphofructokinase activity. To our knowledge, this is the first study showing that selenite can induce necrosis-like cell death in PC-3 cells. Our findings support selenite as an effective compound for the therapy of apoptosis-resistant prostate cancer.


Assuntos
Morte Celular/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Neoplasias da Próstata/fisiopatologia , Ácido Selenioso/farmacologia , Trifosfato de Adenosina/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Fosfofrutoquinases/metabolismo , Neoplasias da Próstata/metabolismo
18.
Biochemistry ; 58(52): 5294-5304, 2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-31478644

RESUMO

Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phosphate (Fru-6-P), is allosterically regulated by the K-type inhibitor phosphoenolpyruvate (PEP). The allosteric coupling between the substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P-bound and PEP-bound complexes on one side and the apo form and ternary complex on the other. Methyl-transverse relaxation-optimized spectroscopy (Me-TROSY) nuclear magnetic resonance was employed to gain structural information about BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly labeled with 15N and 2H and specifically labeled with δ-[13CH3]-isoleucine utilizing an isotopically labeled α-keto acid isoleucine precursor. Me-TROSY experiments were conducted on all four ligation states, and all 30 isoleucines, which are well dispersed throughout each subunit of the enzyme, are well-resolved in chemical shift correlation maps of 13C and 1H. Assignments for 17 isoleucines were determined through three-dimensional HMQC-NOESY experiments with [U-15N,2H];Ileδ1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2H,15N,13C]-BsPFK. The assignments allowed for the mapping of resonances representing isoleucine residues to a previously determined X-ray crystallography structure. This analysis, performed for all four states of ligation, has allowed specific regions of the enzyme influenced by the binding of allosteric ligands and those involved in the propagation of the allosteric effect to be identified and distinguished from one another.


Assuntos
Geobacillus stearothermophilus/enzimologia , Fosfofrutoquinases/química , Fosfofrutoquinases/metabolismo , Regulação Alostérica , Cinética , Espectroscopia de Ressonância Magnética , Multimerização Proteica , Estrutura Quaternária de Proteína
19.
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
20.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067671

RESUMO

Effects of fructose 1,6-bisphosphate (F-1,6-P2) towards N-methyl-d-aspartate NMDA excitotoxicity were evaluated in rat organotypic hippocampal brain slice cultures (OHSC) challenged for 3 h with 30 µM NMDA, followed by incubations (24, 48, and 72 h) without (controls) and with F-1,6-P2 (0.5, 1 or 1.5 mM). At each time, cell necrosis was determined by measuring LDH in the medium. Energy metabolism was evaluated by measuring ATP, GTP, ADP, AMP, and ATP catabolites (nucleosides and oxypurines) in deproteinized OHSC extracts. Gene expressions of phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase were also measured. F-1,6-P2 dose-dependently decreased NMDA excitotoxicity, abolishing cell necrosis at the highest concentration tested (1.5 mM). Additionally, F-1,6-P2 attenuated cell energy imbalance caused by NMDA, ameliorating the mitochondrial phosphorylating capacity (increase in ATP/ADP ratio) Metabolism normalization occurred when using 1.5 mM F-1,6-P2. Remarkable increase in expressions of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase (up to 25 times over the values of controls) was also observed. Since this phenomenon was recorded even in OHSC treated with F-1,6-P2 with no prior challenge with NMDA, it is highly conceivable that F-1,6-P2 can enter into intact cerebral cells producing significant benefits on energy metabolism. These effects are possibly mediated by changes occurring at the gene level, thus opening new perspectives for F-1,6-P2 application as a useful adjuvant to rescue mitochondrial metabolism of cerebral cells under stressing conditions.


Assuntos
Frutose-Bifosfatase/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/farmacologia , Animais , Metabolismo Energético , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Necrose , Fosfofrutoquinases/metabolismo , Nucleosídeos de Purina/metabolismo , Ratos , Ratos Wistar
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