RESUMO
Recent studies in Diffuse Midline Gliomas (DMG) demonstrated a strong connection between epigenome dysregulation and metabolic rewiring. Here, we evaluated the value of targeting a glycolytic protein named Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) in H3.3K27M DMG. We observed that the viability of H3.3K27M cells is dramatically reduced by PFK15, a potent inhibitor of PFKFB3. Furthermore, PFKFB3 inhibition induced apoptosis and G2/M arrest. Interestingly, CRISPR-Knockout of the K27M mutant allele has a synergistic effect on the observed phenotype. Altogether, we identified PFKFB3 as a new target for H3.3K27M DMG, making PFK15 a potential candidate for future animal studies and clinical trials.
Assuntos
Glioma , Histonas , Fosfofrutoquinase-2 , Humanos , Glioma/metabolismo , Glioma/patologia , Glioma/genética , Fosfofrutoquinase-2/metabolismo , Fosfofrutoquinase-2/genética , Histonas/metabolismo , Histonas/genética , Linhagem Celular Tumoral , Criança , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Apoptose , Mutação , Glicólise/efeitos dos fármacosRESUMO
The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a critical engine that supports glucose catabolism. PFKFB3 produces the signaling molecule fructose-2,6-biphosphate (F2,6BP), which activates the second gatekeeper in glycolysis, 6-phosphofructo-1-kinase (PFK-1), and favors the Warburg phenotype. Transcriptional and post-transcriptional processes regulate the abundance and phosphorylation of PFKFB3 in cells, and its activation has been implicated in the progression of several types of cancer. PFKFB3 is important for sustaining glycolysis in the tumorigenesis environment even under unfavorable conditions, thereby promoting metabolic reprogramming, cell proliferation, DNA repair, and drug resistance. Despite its heterogeneous phenotype, breast cancer has unique characteristics that drive the constitutive and inducible expression of PFKFB3 in this opportunistic glycolytic shift. This enzyme is a point of convergence of multiple exogenous and endogenous growth-promoting and oncogenic signaling pathways, especially kinase cascades. The present review summarizes advances in in vitro and in vivo therapy studies that focus on PFKFB3 and the interplay between hormone receptor status and the underlying essential signal transduction system in breast cancer metabolic remodeling.
Assuntos
Neoplasias da Mama , Fosfofrutoquinase-2 , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Glicólise , Humanos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.
Assuntos
Penaeidae/enzimologia , Penaeidae/genética , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Brânquias/metabolismo , Hipóxia/enzimologia , Hipóxia/genética , Ácido Láctico/metabolismo , Modelos Moleculares , Fosfofrutoquinase-2/química , Filogenia , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
NEW FINDINGS: What is the central question of this study? How does an acute session of exercise affect food intake of male Wistar rats? What is the main finding and its importance? Food intake in male Wistar rats is decreased in the first hour after physical exercise independent of the intensity. Moreover, high-intensity exercise potentiates the anorexic effect of peripheral glucose administration. This work raises new feeding-related targets that would explain how exercise drives body weight loss. ABSTRACT: Obesity has emerged as a critical metabolic disorder in modern society. An adequate lifestyle with a well-oriented programme of diet and physical exercise (PE) can prevent or potentially even cure obesity. Additionally, PE might lead to weight loss by increasing energy expenditure and decreasing hunger perception. In this article, we hypothesize that an acute exercise session would potentiate the glucose inhibitory effects on food intake in male Wistar rats. Our data show that moderate- or high-intensity PE significantly decreased food intake, although no changes in the expression of feeding-related neuropeptide in the arcuate nucleus of the hypothalamus were found. Exercised animals demonstrated a reduced glucose tolerance and increased blood insulin concentration. Intraperitoneal administration of glucose decreased food intake in control animals. In the animals submitted to moderate-intensity PE, the decrease in food intake promoted by glucose was similar to controls; however, an interaction was observed when glucose was injected in the high-intensity PE group, in which food intake was significantly lower than the effect produced by glucose alone. A different pattern of expression was observed for the monocarboxylate transporter isoforms (MCT1, 2 and 4) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFBP3) in the hypothalamus, which was dependent on the exercise intensity. In conclusion, PE decreases food intake independently of the intensity. However, an interaction between PE and the anorexic effect of glucose is only observed when a high-intensity exercise is performed. These data show an essential role of exercise intensity in the modulation of the glucose inhibitory effect on food intake.
Assuntos
Ingestão de Alimentos/fisiologia , Glucose/farmacologia , Condicionamento Físico Animal/fisiologia , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Energia/fisiologia , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fosfofrutoquinase-2/metabolismo , Ratos , Ratos WistarRESUMO
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
The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the physiological relevance of this mechanism. As for the regulation of PDC, analysis of pyruvate dehydrogenase kinase (Pdk) expression showed that FSH increases Pdk3 and decreases Pdk4 mRNA levels while bFGF increases the expression of all Pdks. In addition, we showed that bFGF increases phosphorylated PDC levels and that bFGF-stimulated lactate production is partially inhibited in the presence of a PDK inhibitor. Altogether, these results add new information regarding novel molecular mechanisms involved in hormonal regulation of lactate production in Sertoli cells. Considering that lactate is essential for the production of energy in spermatocytes and spermatids, these mechanisms might be relevant in maintaining spermatogenesis and male fertility.
Assuntos
Hormônios/fisiologia , Ácido Láctico/metabolismo , Células de Sertoli/metabolismo , Animais , Células Cultivadas , Fertilidade , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hormônio Foliculoestimulante/farmacologia , Masculino , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Ratos Sprague-Dawley , EspermatogêneseRESUMO
Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 â 2I â 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened ß-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 µM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the ß-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the ß-meander module is not affected by monomerizing mutations.
Assuntos
Proteínas de Escherichia coli/química , Fosfofrutoquinase-2/química , Dobramento de Proteína , Multimerização Proteica , Sequência de Aminoácidos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutação , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.
Assuntos
Temperatura Baixa , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Fosfofrutoquinase-2/química , Desnaturação Proteica , Solventes/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Escherichia coli/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fosfofrutoquinase-2/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Solventes/metabolismoRESUMO
Folding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N(2)â2Iâ2U). This is characterized by a strong coupling between the subunits' tertiary structure and the integrity of the dimer interface because "I" represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N(2)â2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins.
Assuntos
Temperatura Baixa , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Temperatura Alta , Fosfofrutoquinase-2/química , Fosfofrutoquinase-2/metabolismo , Desdobramento de Proteína , Dicroísmo Circular , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Guanidina/farmacologia , Luz , Desnaturação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Desdobramento de Proteína/efeitos dos fármacos , Espalhamento de RadiaçãoRESUMO
Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K(0.5) for fructose-6-P and a decrease in the apparent k(cat) as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n(H) of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.