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Cell Metabolism Sep 2023Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8)....
Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues-K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.
Topics: Mice; Animals; Phosphoenolpyruvate; Diabetes Mellitus, Type 2; Proteins; Liver; Lysine; Glucose
PubMed: 37541251
DOI: 10.1016/j.cmet.2023.07.003 -
Cell Reports Mar 2023Aerobic glycolysis, a metabolic pathway essential for effector T cell survival and proliferation, regulates differentiation of autoimmune T helper (Th) 17 cells, but...
Aerobic glycolysis, a metabolic pathway essential for effector T cell survival and proliferation, regulates differentiation of autoimmune T helper (Th) 17 cells, but the mechanism underlying this regulation is largely unknown. Here, we identify a glycolytic intermediate metabolite, phosphoenolpyruvate (PEP), as a negative regulator of Th17 differentiation. PEP supplementation or inhibition of downstream glycolytic enzymes in differentiating Th17 cells increases intracellular PEP levels and inhibits interleukin (IL)-17A expression. PEP supplementation inhibits expression of signature molecules for Th17 and Th2 cells but does not significantly affect glycolysis, cell proliferation, or survival of T helper cells. Mechanistically, PEP binds to JunB and inhibits DNA binding of the JunB/basic leucine zipper transcription factor ATF-like (BATF)/interferon regulatory factor 4 (IRF4) complex, thereby modulating the Th17 transcriptional program. Furthermore, daily administration of PEP to mice inhibits generation of Th17 cells and ameliorates Th17-dependent autoimmune encephalomyelitis. These data demonstrate that PEP links aerobic glycolysis to the Th17 transcriptional program, suggesting the therapeutic potential of PEP for autoimmune diseases.
Topics: Mice; Animals; Phosphoenolpyruvate; Autoimmunity; Th17 Cells; Basic-Leucine Zipper Transcription Factors; Cell Differentiation; Encephalomyelitis, Autoimmune, Experimental; Mice, Inbred C57BL
PubMed: 36857180
DOI: 10.1016/j.celrep.2023.112205 -
International Journal of Molecular... Nov 2021Some metabolic pathways involve two different cell components, for instance, cytosol and mitochondria, with metabolites traffic occurring from cytosol to mitochondria... (Review)
Review
Some metabolic pathways involve two different cell components, for instance, cytosol and mitochondria, with metabolites traffic occurring from cytosol to mitochondria and vice versa, as seen in both glycolysis and gluconeogenesis. However, the knowledge on the role of mitochondrial transport within these two glucose metabolic pathways remains poorly understood, due to controversial information available in published literature. In what follows, we discuss achievements, knowledge gaps, and perspectives on the role of mitochondrial transport in glycolysis and gluconeogenesis. We firstly describe the experimental approaches for quick and easy investigation of mitochondrial transport, with respect to cell metabolic diversity. In addition, we depict the mitochondrial shuttles by which NADH formed in glycolysis is oxidized, the mitochondrial transport of phosphoenolpyruvate in the light of the occurrence of the mitochondrial pyruvate kinase, and the mitochondrial transport and metabolism of L-lactate due to the L-lactate translocators and to the mitochondrial L-lactate dehydrogenase located in the inner mitochondrial compartment.
Topics: Animals; Biological Transport; Gluconeogenesis; Glycolysis; Humans; Mitochondria; NAD; Phosphoenolpyruvate; Pyruvate Kinase
PubMed: 34884425
DOI: 10.3390/ijms222312620 -
Cells Dec 2019Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently...
Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.
Topics: Calcium; Calcium Signaling; Cell Line, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Cytosol; Gene Expression Regulation, Neoplastic; Glycolysis; HCT116 Cells; Humans; Interleukin-6; NFATC Transcription Factors; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (ATP); Proto-Oncogene Proteins c-myc
PubMed: 31861674
DOI: 10.3390/cells9010018 -
Microbiology and Molecular Biology... Jun 2014The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and... (Review)
Review
The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.
The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.
Topics: Bacteria; Bacterial Proteins; Biological Transport; Carbohydrate Metabolism; Phosphoenolpyruvate; Phosphorylation; Phosphotransferases; Protein Binding
PubMed: 24847021
DOI: 10.1128/MMBR.00001-14 -
American Journal of Physiology.... Jan 2023Acute exercise increases liver gluconeogenesis to supply glucose to working muscles. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of...
Acute exercise increases liver gluconeogenesis to supply glucose to working muscles. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of gluconeogenesis. This functional coupling between liver gluconeogenesis and lipid oxidation has been proposed to underlie the ability of regular exercise to enhance liver mitochondrial oxidative metabolism and decrease liver steatosis in individuals with nonalcoholic fatty liver disease. Herein we tested whether repeated bouts of increased hepatic gluconeogenesis are necessary for exercise training to lower liver lipids. Experiments used diet-induced obese mice lacking hepatic phosphoenolpyruvate carboxykinase 1 (KO) to inhibit gluconeogenesis and wild-type (WT) littermates. H/C metabolic flux analysis quantified glucose and mitochondrial oxidative fluxes in untrained mice at rest and during acute exercise. Circulating and tissue metabolite levels were determined during sedentary conditions, acute exercise, and refeeding postexercise. Mice also underwent 6 wk of treadmill running protocols to define hepatic and extrahepatic adaptations to exercise training. Untrained KO mice were unable to maintain euglycemia during acute exercise resulting from an inability to increase gluconeogenesis. Liver triacylglycerides were elevated after acute exercise and circulating β-hydroxybutyrate was higher during postexercise refeeding in untrained KO mice. In contrast, exercise training prevented liver triacylglyceride accumulation in KO mice. This was accompanied by pronounced increases in indices of skeletal muscle mitochondrial oxidative metabolism in KO mice. Together, these results show that hepatic gluconeogenesis is dispensable for exercise training to reduce liver lipids. This may be due to responses in ketone body metabolism and/or metabolic adaptations in skeletal muscle to exercise. Exercise training reduces hepatic steatosis partly through enhanced hepatic terminal oxidation. During acute exercise, hepatic gluconeogenesis is elevated to match the heightened rate of muscle glucose uptake and maintain glucose homeostasis. It has been postulated that the hepatic energetic stress induced by elevating gluconeogenesis during acute exercise is a key stimulus underlying the beneficial metabolic responses to exercise training. This study shows that hepatic gluconeogenesis is not necessary for exercise training to lower liver lipids.
Topics: Mice; Animals; Phosphoenolpyruvate; Glucose; Liver; Gluconeogenesis; 3-Hydroxybutyric Acid
PubMed: 36351254
DOI: 10.1152/ajpendo.00222.2022 -
FEBS Letters Feb 2023Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate...
Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reaction of decarboxylation and phosphorylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) and CO playing mainly a gluconeogenic role in green algae. We found two PEPCK isoforms in Chlamydomonas reinhardtii and we cloned, purified and characterised both enzymes. ChlrePEPCK1 is more active as decarboxylase than ChlrePEPCK2. ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylalanine and malate, while ChlrePEPCK2 is monomeric and it is regulated by citrate, phenylalanine and glutamine. We postulate that the two PEPCK isoforms found originate from alternative splicing of the gene or regulated proteolysis of the enzyme. The presence of these two isoforms would be part of a mechanism to finely regulate the biological activity of PEPCKs.
Topics: Phosphoenolpyruvate; Chlamydomonas reinhardtii; Phosphoenolpyruvate Carboxykinase (ATP); Protein Isoforms; Phenylalanine; Citrates
PubMed: 36708098
DOI: 10.1002/1873-3468.14590 -
Proceedings of the National Academy of... Aug 2021(Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic...
(Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.
Topics: Antitubercular Agents; Drug Resistance, Microbial; Drug Tolerance; Humans; Hypoxia; Mycobacterium tuberculosis; Phosphoenolpyruvate; Tuberculosis
PubMed: 34426499
DOI: 10.1073/pnas.2105800118 -
Applied and Environmental Microbiology Aug 2013Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase...
Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.
Topics: Escherichia coli; Industrial Microbiology; Metabolic Engineering; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (ATP); Phosphoenolpyruvate Carboxylase; Recombinant Proteins; Succinic Acid
PubMed: 23747698
DOI: 10.1128/AEM.00826-13 -
Journal of Advanced Research Dec 2022Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis.
INTRODUCTION
Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis.
OBJECTIVES
This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus.
METHODS
We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function.
RESULTS
The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%.
CONCLUSION
Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.
Topics: Brassica napus; Gene Expression Regulation, Plant; Phosphoenolpyruvate; Plant Oils; Seeds; Chloroplasts
PubMed: 35907629
DOI: 10.1016/j.jare.2022.07.008