RESUMO
PURPOSE: This study examined the impact of a 5-wk sprint interval training (SIT) intervention on time to task failure (TTF) during severe-intensity constant work rate (CWR) exercise, as well as in glycolytic enzymatic content and activity, and glycogen content. METHODS: Fourteen active males were randomized into either a SIT group ( n = 8) composed of 15 SIT sessions over 5 wk, or a control group ( n = 6). At pretraining period, participants performed i) ramp incremental test to measure the cardiorespiratory function; ii) CWR cycling TTF at 150% of the power output (PO) at the respiratory compensation point (RCP-PO) with muscle biopsies at rest and immediately following task failure. After 5 wk, the same evaluations were repeated (i.e., exercise intensities matched to current training status), and an additional cycling CWR matched to pretraining 150% RCP-PO was performed only for TTF evaluation. The content and enzymatic activity of glycogen phosphorylase (GPhos), hexokinase (HK), phosphofructokinase (PFK), and lactate dehydrogenase (LDH), as well as the glycogen content, were analyzed. Content of monocarboxylate transporter isoform 4 (MCT4) and muscle buffering capacity were also measured. RESULTS: Despite improvements in total work performed at CWR posttraining, no differences were observed for TTF. The GPhos, HK, PFK, and LDH content and activity, and glycogen content also improved after training only in the SIT group. Furthermore, the MCT4 concentrations and muscle buffering capacity were also improved only for the SIT group. However, no difference in glycogen depletion was observed between groups and time. CONCLUSIONS: Five weeks of SIT improved the glycolytic pathway parameters and total work performed; however, glycogen depletion was not altered during CWR severe-intensity exercise, and TTF remained similar.
Assuntos
Glicogênio , Glicólise , Hexoquinase , Treinamento Intervalado de Alta Intensidade , Músculo Esquelético , Fosfofrutoquinases , Humanos , Masculino , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Treinamento Intervalado de Alta Intensidade/métodos , Glicólise/fisiologia , Fosfofrutoquinases/metabolismo , Hexoquinase/metabolismo , Adulto Jovem , L-Lactato Desidrogenase/metabolismo , Glicogênio Fosforilase/metabolismo , AdultoRESUMO
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
Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient-rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch. Here, we report that adenosine monophosphate (AMP) activates both enzymatic activities of the bifunctional adenosine diphosphate (ADP)-dependent phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK/GK). To understand this phenomenon, we performed a comprehensive kinetic characterisation, including determination of the kinetics, substrate inhibition and AMP activation mechanism of this enzyme. We determined that MmPFK/GK has an ordered-sequential mechanism, in which MgADP is the first substrate to bind and AMP is the last product released. The enzyme also displays substrate inhibition by both sugar substrates; we determined that this inhibition occurs through the formation of catalytically nonproductive enzyme complexes caused by sugar binding. For both activities, the AMP activation mechanism occurs primarily through incremental changes in the affinity for the sugar substrate, with this effect being higher in the GK than in the PFK activity. Interestingly, due to the increase in the sugar substrate affinity caused by AMP, an enhancement in the sugar substrate inhibition effect was also observed for both activities, which can be explained by an increase in sugar binding leading to the formation of dead-end complexes. These results shed light on the regulatory mechanisms of methanogenic archaeal sugar metabolism, a phenomenon that has been largely unexplored.
Assuntos
Mathanococcus , Fosfofrutoquinases , Difosfato de Adenosina , Monofosfato de Adenosina , Mathanococcus/genética , AçúcaresRESUMO
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
The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/1010 spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/1010 and 2.22 ± 0.14 U/1010 spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/1010 spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Isocitrato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Fosfofrutoquinases/metabolismo , Capacitação Espermática/efeitos dos fármacos , Espermatozoides/enzimologia , Animais , Bicarbonatos/farmacologia , Feminino , Líquido Folicular/fisiologia , Isocitrato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/antagonistas & inibidores , Masculino , Fosfofrutoquinases/antagonistas & inibidores , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Sus scrofa , Tartronatos/farmacologiaRESUMO
Torrent ducks inhabit fast-flowing rivers in the Andes from sea level to altitudes up to 4500â m. We examined the mitochondrial physiology that facilitates performance over this altitudinal cline by comparing the respiratory capacities of permeabilized fibers, the activities of 16 key metabolic enzymes and the myoglobin content in muscles between high- and low-altitude populations of this species. Mitochondrial respiratory capacities (assessed using substrates of mitochondrial complexes I, II and/or IV) were higher in highland ducks in the gastrocnemius muscle - the primary muscle used to support swimming and diving - but were similar between populations in the pectoralis muscle and the left ventricle. The heightened respiratory capacity in the gastrocnemius of highland ducks was associated with elevated activities of cytochrome oxidase, phosphofructokinase, pyruvate kinase and malate dehydrogenase (MDH). Although respiratory capacities were similar between populations in the other muscles, highland ducks had elevated activities of ATP synthase, lactate dehydrogenase, MDH, hydroxyacyl CoA dehydrogenase and creatine kinase in the left ventricle, and elevated MDH activity and myoglobin content in the pectoralis. Thus, although there was a significant increase in the oxidative capacity of the gastrocnemius in highland ducks, which correlates with improved performance at high altitudes, the variation in metabolic enzyme activities in other muscles not correlated to respiratory capacity, such as the consistent upregulation of MDH activity, may serve other functions that contribute to success at high altitudes.
Assuntos
Altitude , Patos/fisiologia , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Músculos Peitorais/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Migração Animal/fisiologia , Animais , Creatina Quinase/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ventrículos do Coração/metabolismo , Lactato Desidrogenases/metabolismo , Malato Desidrogenase/metabolismo , Mitocôndrias/fisiologia , Mioglobina/metabolismo , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , América do SulRESUMO
During the transition from a healthy state to a cancerous one, cells alter their metabolism to increase proliferation. The underlying metabolic alterations may be caused by a variety of different regulatory events on the transcriptional or post-transcriptional level whose identification contributes to the rational design of therapeutic targets. We present a mechanistic strategy capable of inferring enzymatic regulation from intracellular metabolome measurements that is independent of the actual mechanism of regulation. Here, enzyme activities are expressed by the space of all feasible kinetic constants (k-cone) such that the alteration between two phenotypes is given by their corresponding kinetic spaces. Deriving an expression for the transformation of the healthy to the cancer k-cone we identified putative regulated enzymes between the HeLa and HaCaT cell lines. We show that only a few enzymatic activities change between those two cell lines and that this regulation does not depend on gene transcription but is instead post-transcriptional. Here, we identify phosphofructokinase as the major driver of proliferation in HeLa cells and suggest an optional regulatory program, associated with oxidative stress, that affects the activity of the pentose phosphate pathway.
Assuntos
Enzimas/metabolismo , Metaboloma , Linhagem Celular , Proliferação de Células , Eletroforese Capilar , Regulação da Expressão Gênica , Células HeLa , Humanos , Cinética , Espectrometria de Massas , Fosfofrutoquinases/metabolismoRESUMO
Glycolytic enzymes, such as hexokinase and phosphofructokinase, have been reported to be upregulated in many cancer types. Here, we evaluated these two enzymes in 54 breast cancer samples collected from volunteers subjected to mastectomy, and the results were correlated with the prognosis markers commonly used. We found that both enzymes positively correlate with the major markers for invasiveness and aggressiveness. For invasiveness, the enzymes activities increase in parallel to the tumor size. Moreover, we found augmented activities for both enzymes when the samples were extirpated from patients presenting lymph node involvement or occurrence of metastasis. For aggressiveness, we stained the samples for the estrogen and progesterone receptors, HER-2, p53 and Ki-67. The enzyme activities positively correlated with all markers but Ki-67. Finally, we conclude that these enzymes are good markers for breast cancer prognosis.