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1.
Biomed Res Int ; 2020: 3858465, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104690

RESUMO

The cytosolic isozyme of phosphoenolpyruvate carboxykinase (PCK1) was the first rate-limiting enzyme in the gluconeogenesis pathway, which exerted a critical role in maintaining the blood glucose levels. PCK1 has been established to be involved in various physiological and pathological processes, including glucose metabolism, lipid metabolism, diabetes, and tumorigenesis. Nonetheless, the association of PCK1 with aging process and the detailed underlying mechanisms of PCK1 on aging are still far to be elucidated. Hence, we herein constructed the PCK1-deficient (pck1Δ) and PCK1 overexpression (PCK1 OE) Saccharomyces cerevisiae. The results unveiled that PCK1 deficiency significantly shortened the replicative lifespan (RLS) in the S. cerevisiae, while overexpression of PCK1 prolonged the RLS. Additionally, we noted that the ROS level was significantly enhanced in PCK1-deficient strain and decreased in PCK1 OE strain. Then, a high throughput analysis by deep sequencing was performed in the pck1Δ and wild-type strains, in an attempt to shed light on the effect of PCK1 on the lifespan of aging process. The data showed that the most downregulated mRNAs were enriched in the regulatory pathways of glucose metabolism. Fascinatingly, among the differentially expressed mRNAs, PFK1 was one of the most upregulated genes, which was involved in the glycolysis process and ROS generation. Thus, we further constructed the pfk1Δpck1Δ strain by deletion of PFK1 in the PCK1-deficient strain. The results unraveled that pfk1Δpck1Δ strain significantly suppressed the ROS level and restored the RLS of pck1Δ strain. Taken together, our data suggested that PCK1 deficiency enhanced the ROS level and shortened the RLS of S. cerevisiae via PFK1.


Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Fosfoenolpiruvato Carboxiquinase (ATP) , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Int J Parasitol ; 48(12): 955-968, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30176233

RESUMO

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.


Assuntos
Carbono/metabolismo , Glicólise , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Encéfalo/parasitologia , Modelos Animais de Doenças , Deleção de Genes , Gluconeogênese , Glutamina/metabolismo , Hexoquinase/deficiência , Análise do Fluxo Metabólico , Camundongos , Fosforilação Oxidativa , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Virulência
3.
Mol Genet Metab ; 113(3): 161-70, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24863970

RESUMO

The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Receptores de N-Metil-D-Aspartato/genética , Síndrome de Smith-Magenis/diagnóstico , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Síndrome de Smith-Magenis/genética , Transativadores
4.
Appl Environ Microbiol ; 78(19): 7120-3, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22798371

RESUMO

A Planctomyces limnophilus mutant generated using the EZ-Tn5 transposome was found to possess an insertion within pckA, encoding phosphoenolpyruvate carboxykinase. Disruption of pckA expression and elimination of enzymatic activity resulted in poor growth in glucose-free medium, demonstrating a gluconeogenic role for pckA in P. limnophilus.


Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional/métodos , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Planctomycetales/enzimologia , Meios de Cultura/química , Gluconeogênese , Glucose/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Planctomycetales/genética , Planctomycetales/crescimento & desenvolvimento , Planctomycetales/metabolismo
5.
J Nutr ; 139(12): 2257-65, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19812223

RESUMO

Obesity and type 2 diabetes are growing problems worldwide in adults and children. In this study, we focused on understanding the patterning of insulin resistance as a result of altered perinatal nutrition. We analyzed mice in which the binding site for PPARgamma was deleted from the promoter of the cytosolic phosphoenolpyruvate carboxykinase gene (Pck1) (PPARE(-/-)). We analyzed pups from dams with the same genotype as well as fostered and cross-fostered pups. Pck1 expression and triglyceride concentration in the milk were measured. The PPARE mutation reduced Pck1 expression in white adipose tissue (WAT) to 2.2% of wild type (WT) and reduced Pck1 expression in whole mammary gland tissue to 1% of WT. The female PPARE(-/-) mice had reduced lipid storage in mammary gland adipocytes and in WAT, resulting in a 40% reduction of milk triglycerides during lactation. Pups from PPARE(-/-) dams had insulin resistance as early as 14 d after birth, a condition that persisted into adulthood. WT pups fostered by PPARE(-/-) dams had lower body weights and plasma insulin concentrations compared with WT pups reared by WT dams. PPARE(-/-) pups fostered by WT dams had improved glucose clearance compared with pups raised by PPARE(-/-) dams. PPARE(+/-) and PPARE(-/-) dams also patterned newborn pups for reduced growth and insulin resistance in utero. Thus, the in utero environment and altered nutrition during the perinatal period cause epigenetic changes that persist into adulthood and contribute to the development of insulin resistance.


Assuntos
Adipócitos/enzimologia , Tecido Adiposo/enzimologia , Resistência à Insulina/genética , Glândulas Mamárias Animais/enzimologia , Leite/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Triglicerídeos/metabolismo , Adipócitos/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Adulto , Animais , Cruzamentos Genéticos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Gravidez
6.
Biotechnol Bioeng ; 84(2): 129-44, 2003 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-12966569

RESUMO

The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C(6)]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed.


Assuntos
Escherichia coli/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Acetilcoenzima A/metabolismo , Nucleotídeos de Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Aminoácidos/química , Aminoácidos/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Isótopos de Carbono/metabolismo , Ácidos Carboxílicos/metabolismo , Divisão Celular/fisiologia , Ciclo do Ácido Cítrico , Metabolismo Energético , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glucose/metabolismo , Glicólise , Hidrólise , Isocitrato Desidrogenase/metabolismo , Isocitrato Liase/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Monossacarídeos/metabolismo , Mutação , Oxigênio/metabolismo , Via de Pentose Fosfato , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Ciclização de Substratos
7.
Biotechnol Bioeng ; 77(1): 61-72, 2002 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-11745174

RESUMO

Biomass yields for several null mutants in Saccharomyces cerevisiae were successfully predicted with a metabolic network model. Energetic parameters of the model were obtained from growth data in C-limited aerobic chemostat cultures of the corresponding wild-type strain, which exhibited a P/O ratio of 1.46, a non-growth-related maintenance of 56 mmol ATP/C-mol biomass/h, and a growth-related requirement of 655 mmol ATP/C-mol biomass. Biomass yields and carbon uptake rates were modeled for different mutants incapacitated in their glyoxylate cycle and their gluconeogenesis. Biomass yields were calculated for different feed ratios of glucose to ethanol, and decreases for higher ethanol fractions were correctly predicted for mutants with deletions of the malate synthase, the isocitrate lyase, or the phosphoenolpyruvate carboxykinase. The growth of the fructose- 1,6-bisphosphatase deletion mutant was anticipated less accurate, but the tendency was modeled correctly.


Assuntos
Gluconeogênese/genética , Glioxilatos/metabolismo , Modelos Genéticos , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Aerobiose/genética , Biomassa , Carbono/metabolismo , Metabolismo Energético/genética , Etanol/metabolismo , Glucose/metabolismo , Isocitrato Liase/deficiência , Isocitrato Liase/genética , Malato Sintase/deficiência , Malato Sintase/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/deficiência , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
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