RESUMO
INTRODUCTION: Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified. METHODS: The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database. RESULTS: The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10-3 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10-2 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10-3 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10-2 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10-4 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10-2 )] and increased exponentially as the expression of LDHA and PFKP increased. CONCLUSIONS: LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making.
Assuntos
Fosfofrutoquinases , Neoplasias do Colo do Útero , Feminino , Humanos , Biomarcadores/metabolismo , Glicólise/genética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5/metabolismo , Fosfofrutoquinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias do Colo do Útero/genéticaRESUMO
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 SubstratoRESUMO
The African most prevalent tropical disease after malaria is schistosomiasis and this disease in the developing countries is a massive socio-economic and public health burden. The disease also caused over 200,000 deaths. The development and design of new and novel antischistosomal drugs is now very important, as there are no vaccines currently and there is only one drug at the moment for the treatment of schistosomiasis. In this article, 6-gingerol was docked against the Schistosoma mansoni phosphofructokinase and the docking result was compared to those obtained from the docking of its modified analogues against the same enzyme. The chemical structure of 6-gingerol was obtained from the PubChem database while the modified analogues were designed using the ChemAxon software. The molecular docking procedure was carried out with the aid of the AutoDock Vina software while polar interactions which were eventually used in predicting the amino acid residues at the Schistosoma mansoni phosphofructokinase active site were visualized using the Pymol software. The Schistosoma mansoni phosphofructokinase 3D crystallized structure was modeled using the Swiss Model server. The molecular docking result showed that the modifications made on 6-gingerol had a positive effect on the binding energy of the compound to the enzyme active site as an appreciable increase was observed. 6-Gingerol and its modified analogues also violated none of the Lipinski's rule with suggests that the experimental compounds are drug-like. The C2H5 analogue of 6 gingerol was selected as the ideal therapeutic agent based on the pharmacokinetics study and the exhibited binding energy(AU)
La enfermedad tropical con más prevalencia en África después de la malaria es la esquistosomiasis; en los países en vías de desarrollo constituye una carga socio-económica y de salud pública enorme. La enfermedad ha ocasionado más de 200.000 muertes anuales. El desarrollo y diseño de nuevas y novedosas drogas antiesquistosomales es muy importante, ya que actualmente no existe vacuna disponible y solo hay una sola droga licenciada para su tratamiento. En esta investigación, el compuesto 6-gingerol se acopló a la enzima fosfofructoquinasa de Schistosoma mansoni y se comparó con los resultados obtenidos a partir de las interacciones de sus análogos modificados a la misma enzima. La estructura química del 6-gingerol se obtuvo de la base de datos PubChem, mientras que los análogos modificados se diseñaron utilizando el software ChemAxon. El procedimiento de acoplamiento molecular se llevó a cabo con la ayuda del software AutoDockVina, mientras las interacciones polares eventualmente utilizadas para predecir los residuos de aminoácidos en el sitio activo de la enzima fosfofructoquinasa de Schistosoma mansoni se visualizaron empleando el software Pymol. La estructura cristalizada tridimensional de la enzima fosfofructoquinasa de Schistosoma mansoni se modeló utilizando el programa Swiss Model. Se demostró que las modificaciones realizadas en el 6-gingerol tuvieron un efecto positivo en la energía de unión del compuesto al sitio activo de la enzima, tras observarse un aumento apreciable de dicha energía. El compuesto 6-Gingerol y sus análogos modificados tampoco violaron ninguna de las reglas de Lipinski, lo que sugiere que estos compuestos experimentales tienen propiedades similares a los medicamentos. El análogo C2H5 de 6-gingerol se seleccionó como el agente terapéutico ideal, basados en el estudio de farmacocinética y la energía de enlace demostrada(AU)
Assuntos
Esquistossomose mansoni/tratamento farmacológico , Esquistossomose mansoni/epidemiologia , Farmacocinética , Fosfofrutoquinases/uso terapêutico , ÁfricaRESUMO
This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.
Assuntos
Citrus sinensis/enzimologia , Fosfofrutoquinases/metabolismo , Fosfotransferases/metabolismo , Citrus sinensis/genética , Clonagem Molecular , Difosfatos/metabolismo , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Expressão Gênica , Cinética , Complexos Multiproteicos , Fosfofrutoquinases/genética , Fosforilação , Fosfotransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas RecombinantesRESUMO
Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis, being considered the pacemaker of this pathway. In mammals, this enzyme exists as three different isoforms, PFKM, PFKL and PFKP, presenting different regulatory and catalytic properties. The expression of these isoforms is tissue-specific and vary according to the cell differentiation and signalization. Although it is known that the expression of the different PFK isoforms directly affects cell function, the information regarding the regulation of PFK isoforms expression is scarce. In the present work, we evaluate the role of insulin signalization on the expression of three PFK isoforms on skeletal muscle, liver, and epididymal white adipose tissue (eWAT) of mice. For this, Swiss mice were treated with streptozotocin (STZ) to disrupt pancreatic ß-cells and, thus, insulin production. Control group were treated with citrate buffer (STZ vehicle). These groups were then treated with insulin or saline twice a day for ten consecutive days when animals were euthanized and tissues used for the evaluation of PFK isoforms expression by quantitative PCR (qPCR). Our results revealed that the lack of insulin significantly impacted the expression of PFKL, presenting mild effects on PFKM and no effects on PFKP. The decrease of PFKL and PFKM mRNA levels observed on the group treated with STZ was reversed by the treatment with insulin. In conclusion, insulin, the most known regulator of glucose consumption, specifically regulates the expression of PFKL and PFKM, which impact the regulation of glycolysis in the cell.
Assuntos
Insulina/farmacologia , Fígado/enzimologia , Músculo Esquelético/enzimologia , Fosfofrutoquinase-1/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/enzimologia , Animais , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacosRESUMO
The genome of Methanosarcinales organisms presents both ADP-dependent glucokinase and phosphofructokinase genes. However, Methanococcoides burtonii has a truncate glucokinase gene with a large deletion at the C-terminal, where the catalytic GXGD motif is located. Characterization of its phosphofructokinase annotated protein shows that is a bifunctional enzyme able to supply the absence of the glucokinase activity. Moreover, kinetic analyses of the phosphofructokinase annotated enzyme from, Methanohalobium evestigatum demonstrated that this enzyme is also bifunctional. The high conservation of the active site residues of all the enzymes from the order Methanosarcinales suggest that they should be bifunctional, as was previously reported for the ADP-dependent kinases from Methanococcales, highlighting the redundancy of the glucokinase activity in this archaeal group. The presence of active glycolytic enzymes would be important when glycogen storage of these organisms needs to be degraded to be used as energy source. Kinetic and structural information allows us to establish a substrate specificity signature that identifies specific GK or PFK, and bifunctional enzymes in this family.
Assuntos
Difosfato de Adenosina/química , Proteínas Arqueais/química , Glucoquinase/química , Methanosarcinales/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glucoquinase/genética , Glucoquinase/metabolismo , Cinética , Methanosarcinales/classificação , Methanosarcinales/genética , Modelos Moleculares , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , 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 , TermodinâmicaRESUMO
Although respiration is the principal cause of the loss of sucrose in postharvest sugarbeet (Beta vulgaris L.), the internal mechanisms that control root respiration rate are unknown. Available evidence, however, indicates that respiration rate is likely to be controlled by the availability of respiratory substrates, and glycolysis has a central role in generating these substrates. To determine glycolytic changes that occur in sugarbeet roots after harvest and to elucidate relationships between glycolysis and respiration, sugarbeet roots were stored for up to 60 days, during which activities of glycolytic enzymes and concentrations of glycolytic substrates, intermediates, cofactors, and products were determined. Respiration rate was also determined, and relationships between respiration rate and glycolytic enzymes and metabolites were evaluated. Glycolysis was highly variable during storage, with 10 of 14 glycolytic activities and 14 of 17 glycolytic metabolites significantly altered during storage. Changes in glycolytic enzyme activities and metabolites occurred throughout the 60 day storage period, but were greatest in the first 4 days after harvest. Positive relationships between changes in glycolytic enzyme activities and root respiration rate were abundant, with 10 of 14 enzyme activities elevated when root respiration was elevated and 9 glycolytic activities static during periods of unchanging respiration rate. Major roles for pyruvate kinase and phosphofructokinase in the regulation of postharvest sugarbeet root glycolysis were indicated based on changes in enzymatic activities and concentrations of their substrates and products. Additionally, a strong positive relationship between respiration rate and pyruvate kinase activity was found indicating that downstream TCA cycle enzymes were unlikely to regulate or restrict root respiration in a major way. Overall, these results establish that glycolysis is not static during sugarbeet root storage and that changes in glycolysis are closely related to changes in sugarbeet root respiration.
RESUMO
The aim of this work was to study the regulation of PFK 1 and G6PDH, two key enzymes involved in glucose metabolism in cumulus oocyte-complexes (COCs), and its relationship with the oocyte maturation process. It was observed that the activity of PFK 1 in the presence of ATP was inhibited whereas the addition of AMP increased the activity (P < 0.05). To verify the effect of the physiological modulators on the COC glycolytic pathway, the lactate production during IVM and the maturation rate were evaluated. In accordance with the enzymatic activity, the glycolytic activity evaluated by lactate production and the maturation rate diminished (P < 0.05) with the addition of ATP. While the AMP had a dose response effect on the lactate production, the maturation rate remained unaltered. It was observed that NADPH inhibited the activity of the G6PDH and the addition of NADP increased the activity of the enzyme (P < 0.05). To verify the effect of the physiological modulators on the COC pentose phosphate pathway (PPP), the proportion of colourless oocytes evaluated by brilliant cresyl blue (BCB) and the maturation rate were carried out. In presence of NADPH an inhibition (P < 0.05) on PPP and maturation rate was observed. On the other hand, NADP had no effect on PPP activity and maturation rate. The present study shows that the regulation of key enzymes of glucose metabolism in bovine COCs are regulated mainly by the energetic charge and the redox status. We also reported a tight relation between the activity of the PFK 1 and G6PDH enzymes, glycolytic and PPP activities and the oocyte maturation process.
Assuntos
Animais , Bovinos , Bovinos/metabolismo , Fosfofrutoquinase-1 , Glucosefosfato DesidrogenaseRESUMO
The aim of this work was to study the regulation of PFK 1 and G6PDH, two key enzymes involved in glucose metabolism in cumulus oocyte-complexes (COCs), and its relationship with the oocyte maturation process. It was observed that the activity of PFK 1 in the presence of ATP was inhibited whereas the addition of AMP increased the activity (P < 0.05). To verify the effect of the physiological modulators on the COC glycolytic pathway, the lactate production during IVM and the maturation rate were evaluated. In accordance with the enzymatic activity, the glycolytic activity evaluated by lactate production and the maturation rate diminished (P < 0.05) with the addition of ATP. While the AMP had a dose response effect on the lactate production, the maturation rate remained unaltered. It was observed that NADPH inhibited the activity of the G6PDH and the addition of NADP increased the activity of the enzyme (P < 0.05). To verify the effect of the physiological modulators on the COC pentose phosphate pathway (PPP), the proportion of colourless oocytes evaluated by brilliant cresyl blue (BCB) and the maturation rate were carried out. In presence of NADPH an inhibition (P < 0.05) on PPP and maturation rate was observed. On the other hand, NADP had no effect on PPP activity and maturation rate. The present study shows that the regulation of key enzymes of glucose metabolism in bovine COCs are regulated mainly by the energetic charge and the redox status. We also reported a tight relation between the activity of the PFK 1 and G6PDH enzymes, glycolytic and PPP activities and the oocyte maturation process.(AU)
Assuntos
Animais , Bovinos , Bovinos/metabolismo , Fosfofrutoquinase-1 , Glucosefosfato DesidrogenaseRESUMO
We have proposed an allosteric ATP inhibition mechanism of Pfk-2 determining the structure of different forms of the enzyme together with a kinetic enzyme analysis. Here we complement the mechanism by using hybrid oligomers of the homodimeric enzyme to get insights about the allosteric communication pathways between the same sites or different ones located in different subunits. Kinetic analysis of the hybrid enzymes indicate that homotropic interactions between allosteric sites for ATP or between substrate sites for fructose-6-P have a minor effect on the enzymatic inhibition induced by ATP. In fact, the sigmoid response for fructose-6-P observed at elevated ATP concentrations can be eliminated even though the enzymatic inhibition is still operative. Nevertheless, leverage coupling analysis supports heterotropic interactions between the allosteric ATP and fructose-6-P binding occurring between and within each subunit.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/metabolismo , Frutosefosfatos/metabolismo , Fosfofrutoquinase-2/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Simulação por Computador , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Frutosefosfatos/química , Cinética , Modelos Moleculares , Estrutura Molecular , Mutação , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/química , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade por SubstratoRESUMO
Marine organisms are exposed to hypoxia in natural ecosystems and during farming. In these circumstances marine shrimp survive and synthesize ATP by anaerobic metabolism. Phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in carbohydrate metabolism. Here we report the cDNA of FBP from the shrimp Litopenaeus vannamei hepatopancreas and expression of PFK and FBP under normoxia and hypoxia. Hypoxia induces PFK and FBP expression in hepatopancreas but not in gills and muscle. Induction in hepatopancreas of the glycolytic and gluconeogenic key enzymes, PFK and FBP, suggests that PFK could be a key factor for increasing anaerobic rate, while FBP is probably involved in the activation of gluconeogenesis or the pentose-phosphates pathway during hypoxia in the highly active metabolism of hepatopancreas.
Assuntos
Hipóxia Celular/fisiologia , Frutose-Bifosfatase/genética , Regulação Enzimológica da Expressão Gênica , Penaeidae/enzimologia , Penaeidae/genética , Fosfofrutoquinases/genética , Sequência de Aminoácidos , Animais , Hepatopâncreas/enzimologia , Hepatopâncreas/fisiopatologia , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
BACKGROUND: Fructose administration rapidly induces oxidative stress that triggers compensatory hepatic metabolic changes. We evaluated the effect of an antioxidant, R/S-α-lipoic acid on fructose-induced oxidative stress and carbohydrate metabolism changes. METHODS: Wistar rats were fed a standard commercial diet, the same diet plus 10% fructose in drinking water, or injected with R/S-α-lipoic acid (35mg/kg, i.p.) (control+L and fructose+L). Three weeks thereafter, blood samples were drawn to measure glucose, triglycerides, insulin, and the homeostasis model assessment-insulin resistance (HOMA-IR) and Matsuda indices. In the liver, we measured gene expression, protein content and activity of several enzymes, and metabolite concentration. RESULTS: Comparable body weight changes and calorie intake were recorded in all groups after the treatments. Fructose fed rats had hyperinsulinemia, hypertriglyceridemia, higher HOMA-IR and lower Matsuda indices compared to control animals. Fructose fed rats showed increased fructokinase gene expression, protein content and activity, glucokinase and glucose-6-phosphatase gene expression and activity, glycogen storage, glucose-6-phosphate dehydrogenase mRNA and enzyme activity, NAD(P)H oxidase subunits (gp91(phox) and p22(phox)) gene expression and protein concentration and phosphofructokinase-2 protein content than control rats. All these changes were prevented by R/S-α-lipoic acid co-administration. CONCLUSIONS: Fructose induces hepatic metabolic changes that presumably begin with increased fructose phosphorylation by fructokinase, followed by adaptive changes that attempt to switch the substrate flow from mitochondrial metabolism to energy storage. These changes can be effectively prevented by R/S-α-lipoic acid co-administration. GENERAL SIGNIFICANCE: Control of oxidative stress could be a useful strategy to prevent the transition from impaired glucose tolerance to type 2 diabetes.
Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Frutose/farmacologia , Fígado/metabolismo , Estresse Oxidativo , Ácido Tióctico/farmacologia , Animais , Glucoquinase/análise , Glucoquinase/genética , Masculino , NADPH Oxidases/análise , NADPH Oxidases/genética , Ratos , Ratos WistarRESUMO
The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.