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Journal of Chemical Information and... Apr 2024Phosphoenolpyruvate carboxylase (PEPC) is used in plant metabolism for fruit maturation or seed development as well as in the C4 and crassulacean acid metabolism (CAM)...
Phosphoenolpyruvate carboxylase (PEPC) is used in plant metabolism for fruit maturation or seed development as well as in the C4 and crassulacean acid metabolism (CAM) mechanisms in photosynthesis, where it is used for the capture of hydrated CO (bicarbonate). To find the yet unknown binding site of bicarbonate in this enzyme, we have first identified putative binding sites with nonequilibrium molecular dynamics simulations and then ranked these sites with alchemical free energy calculations with corrections of computational artifacts. Fourteen pockets where bicarbonate could bind were identified, with three having realistic binding free energies with differences with the experimental value below 1 kcal/mol. One of these pockets is found far from the active site at 14 Å and predicted to be an allosteric binding site. In the two other binding sites, bicarbonate is in direct interaction with the magnesium ion; neither sequence alignment nor the study of mutant K606N allowed to discriminate between these two pockets, and both are good candidates as the binding site of bicarbonate in phosphoenolpyruvate carboxylase.
Topics: Phosphoenolpyruvate Carboxylase; Bicarbonates; Molecular Dynamics Simulation; Binding Sites; Thermodynamics; Protein Conformation
PubMed: 38533570
DOI: 10.1021/acs.jcim.3c01830 -
BMC Biotechnology Sep 2023Biofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for...
Comparison of the effects of nitrogen-, sulfur- and combined nitrogen- and sulfur-deprivations on cell growth, lipid bodies and gene expressions in Chlamydomonas reinhardtii cc5373-sta6.
BACKGROUND
Biofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for biofuel production research. This study compares the effects of nitrogen-, sulfur-, and nitrogen and sulfur- deprivations on the C. reinhardtii starchless mutant cc5373-sta6. Specifically, it compares growth, lipid body accumulation, and expression levels of acetyl-CoA carboxylase (ACC) and phosphoenolpyruvate carboxylase (PEPC).
RESULTS
Among nutrient-deprived cells, TAP-S cells showed significantly higher total chlorophyll, cell density, and protein content at day 6 (p < 0.05). Confocal analysis showed a significantly higher number of lipid bodies in cells subjected to nutrient deprivation than in the control over the course of six days; N deprivation for six days significantly increased the size of lipid bodies (p < 0.01). In comparison with the control, significantly higher ACC expression was observed after 8 and 24 h of NS deprivation and only after 24 h with N deprivation. On the other hand, ACC and PEPC expression at 8 and 24 h of S deprivation was not significantly different from that in the control. A significantly lower PEPC expression was observed after 8 h of N and NS deprivation (p < 0.01), but a significantly higher PEPC expression was observed after 24 h (p < 0.01).
CONCLUSIONS
Based on our findings, it would be optimum to cultivate cc5373-sta6 cells in nutrient deprived conditions (-N, -S or -NS) for four days; whereby there is cell growth, and both a high number of lipid bodies and a larger size of lipid bodies produced.
Topics: Lipid Droplets; Chlamydomonas reinhardtii; Biofuels; Acetyl-CoA Carboxylase; Nitrogen; Sulfur; Gene Expression
PubMed: 37684579
DOI: 10.1186/s12896-023-00808-3 -
ChemMedChem Mar 2024Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis...
Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure-based design of AG-946, an activator of PK isoforms, including red blood cell-specific forms of PK (PKR). This was designed to have a pseudo-C2-symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off-targets observed from the original scaffold. AG-946 (1) demonstrated activation of human wild-type PK (half-maximal activation concentration [AC ]=0.005 μM) and a panel of mutated PK proteins (K410E [AC =0.0043 μM] and R510Q [AC =0.0069 μM]), (2) displayed a significantly longer half-time of activation (>150-fold) compared with 6-(3-methoxybenzyl)-4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thieno[2',3':4,5]pyrrolo[2,3-d]pyridazin-5-one, and (3) stabilized PKR R510Q, an unstable mutant PKR enzyme, and preserved its catalytic activity under increasingly denaturing conditions. As a potent, oral, small-molecule allosteric activator of wild-type and mutant PKR, AG-946 was advanced to human clinical trials.
Topics: Humans; Pyruvate Kinase; Adenosine Triphosphate; Allosteric Site; Binding Sites; Pyruvic Acid
PubMed: 38109501
DOI: 10.1002/cmdc.202300559 -
BMC Complementary Medicine and Therapies Dec 2023Artichoke (Cynara scolymus L.) is a typical element of a traditional Mediterranean diet and has potential health advantages for insulin resistance (IR) and type 2...
Artichoke (Cynara scolymus L.) water extract alleviates palmitate-induced insulin resistance in HepG2 hepatocytes via the activation of IRS1/PI3K/AKT/FoxO1 and GSK-3β signaling pathway.
BACKGROUND
Artichoke (Cynara scolymus L.) is a typical element of a traditional Mediterranean diet and has potential health advantages for insulin resistance (IR) and type 2 diabetes mellitus (T2DM). This study aims to evaluate the effect and underlying mechanism of artichoke water extract (AWE) on palmitate (PA)-induced IR in human hepatocellular carcinoma (HepG2) cells.
METHODS
The effect of AWE on cell viability was determined using CCK8 assay. Cellular glucose uptake, glucose consumption, glucose production, and glycogen content were assessed after AWE treatment. The gene expression and protein levels were examined by real-time polymerase chain reaction (qRT-PCR) and western blotting.
RESULTS
The results showed that AWE dose-dependently increased cell viability in IR HepG2 cells (P < 0.01). AWE treatment significantly promoted glucose uptake and consumption, decreased glucose production, and increased the cellular glycogen content in IR HepG2 cells (P < 0.01). Mechanistically, AWE elevated the phosphorylation and total protein levels of major insulin signaling molecules in IR HepG2 cells, which resulted in a decrease in the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and the inhibition of glycogen synthase (GS) phosphorylation in IR HepG2 cells. Furthermore, the protective effect of AWE on IR HepG2 cells might be ascribed to the inhibition of the endoplasmic reticulum (ER) stress.
CONCLUSION
We conclude that AWE may improve glucose metabolism by regulating IRS1/PI3K/AKT/FoxO1 and GSK-3β signaling associated with the inhibition of ER stress in IR HepG2 cells induced by PA.
Topics: Humans; Insulin Resistance; Cynara scolymus; Glycogen Synthase Kinase 3 beta; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Diabetes Mellitus, Type 2; Palmitates; Signal Transduction; Hepatocytes; Glucose; Glycogen; Insulin Receptor Substrate Proteins
PubMed: 38102588
DOI: 10.1186/s12906-023-04275-3 -
Frontiers in Molecular Biosciences 2023When is exposed to changing environmental conditions, the expression of many genes is regulated at the transcriptional level. We reported previously that the...
When is exposed to changing environmental conditions, the expression of many genes is regulated at the transcriptional level. We reported previously that the enterococcal MafR protein causes genome-wide changes in the transcriptome. Here we show that MafR activates directly the transcription of the gene, which encodes a hypothetical protein of 111 amino acid residues. We have identified the promoter and demonstrated that MafR enhances the efficiency of this promoter by binding to a DNA site that contains the -35 element. Moreover, our analysis of the OG1RF_10478 protein AlphaFold model indicates high similarity to 1) structures of EIIB components of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system, and 2) structures of receiver domains that are found in response regulators of two-component signal transduction systems. However, unlike typical EIIB components, OG1RF_10478 lacks a Cys or His residue at the conserved phosphorylation site, and, unlike typical receiver domains, OG1RF_10478 lacks a conserved Asp residue at the position usually required for phosphorylation. Different from EIIB components and receiver domains, OG1RF_10478 contains an insertion between residues 10 and 30 that, according to ColabFold prediction, may serve as a dimerization interface. We propose that OG1RF_10478 could participate in regulatory functions by protein-protein interactions.
PubMed: 38192335
DOI: 10.3389/fmolb.2023.1294974 -
Metabolites Sep 2023Obesity is a multifactorial disorder that is remarkably heterogeneous. It presents itself in a variety of phenotypes that can be metabolically unhealthy or healthy,... (Review)
Review
Obesity is a multifactorial disorder that is remarkably heterogeneous. It presents itself in a variety of phenotypes that can be metabolically unhealthy or healthy, associate with no or multiple metabolic risk factors, gain extreme body weight (super-responders), as well as resist obesity despite the obesogenic environment (non-responders). Progression to obesity is ultimately linked to the overall net energy balance and activity of different metabolic fluxes. This is particularly evident from variations in fatty acids oxidation, metabolic fluxes through the pyruvate-phosphoenolpyruvate-oxaloacetate node, and extracellular accumulation of Krebs cycle metabolites, such as citrate. Patterns of fat accumulation with a focus on visceral and ectopic adipose tissue, microbiome composition, and the immune status of the gastrointestinal tract have emerged as the most promising targets that allow personalization of obesity and warrant further investigations into the critical issue of a wider and long-term weight control. Advances in understanding the biochemistry mechanisms underlying the heterogenous obesity phenotypes are critical to the development of targeted strategies to maintain healthy weight.
PubMed: 37755296
DOI: 10.3390/metabo13091016 -
Microbiology Spectrum Sep 2023Glutathione (GSH) is an essential component of the glutaredoxin (Grx) system, and it is synthesized by the enzyme glutathione synthase GshF in . GSH plays a crucial role...
Glutathione (GSH) is an essential component of the glutaredoxin (Grx) system, and it is synthesized by the enzyme glutathione synthase GshF in . GSH plays a crucial role in regulating virulence by modifying the virulence factors LLO and PrfA. In this study, we investigated the involvement of GshF in oxidative tolerance and intracellular infection. Our findings revealed that the deletion of resulted in a significant reduction in bacterial growth when exposed to diamide and copper ions stress. More importantly, this deletion also impaired the efficiency of invasion and proliferation in macrophages and mice organs. Furthermore, GshF influenced global transcriptional profiles, including carbohydrate and amino acid metabolism, particularly those related to the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) genes , under oxidative stress conditions. In the wild-type strain, the transcription of was notably downregulated in response to copper ions and diamide stress compared to normal conditions. However, in the absence of , the transcripts of were upregulated in response to these stress conditions. Notably, the deletion of () enhanced oxidative stress tolerance to copper ions, whereas overexpression of reduced this resistance. In conclusion, our study provides the first evidence that GshF plays a crucial role in bacterial antioxidation through the regulation of .IMPORTANCE has developed various mechanisms to withstand oxidative stress, including the thioredoxin and glutaredoxin systems. However, the specific role of the glutathione synthase GshF, responsible for synthesizing GSH in , in oxidative tolerance remains unclear. This study aimed to elucidate the relationship between GshF and oxidative tolerance in by examining the efficiency of invasion and proliferation in macrophages and mice organs, as well as analyzing global transcriptional profiles under oxidative stress conditions. The results revealed that GshF plays a significant role in ' response to oxidative stress. Notably, GshF acts to suppress the transcription of phosphoenolpyruvate-carbohydrate phosphotransferase system genes , among which () was identified as the most critical gene for resisting oxidative stress. These findings enhance our understanding of how adapts to its environment and provide valuable insights for investigating the environmental adaptation mechanisms of other pathogenic bacteria.
PubMed: 37668404
DOI: 10.1128/spectrum.02365-23 -
Plants (Basel, Switzerland) Jul 2023Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A...
Protein phosphatase 2A (PP2A) is a heterotrimeric conserved serine/threonine phosphatase complex that includes catalytic, scaffolding, and regulatory subunits. The 3 A subunits, 17 B subunits, and 5 C subunits that are encoded by the Arabidopsis genome allow 255 possible PP2A holoenzyme combinations. The regulatory subunits are crucial for substrate specificity and PP2A complex localization and are classified into the B, B', and B" non-related families in land plants. In Arabidopsis, the close homologs B'η, B'θ, B'γ, and B'ζ are further classified into a subfamily of B' called B'η. Previous studies have suggested that mitochondrial targeted PP2A subunits (B'ζ) play a role in energy metabolism and plant innate immunity. Potentially, the PP2A-B'ζ holoenzyme is involved in the regulation of the mitochondrial succinate/fumarate translocator, and it may affect the enzymes involved in energy metabolism. To investigate this hypothesis, the interactions between PP2A-B'ζ and the enzymes involved in the mitochondrial energy flow were investigated using bimolecular fluorescence complementation in tobacco and onion cells. Interactions were confirmed between the B'ζ subunit and the Krebs cycle proteins succinate/fumarate translocator (mSFC1), malate dehydrogenase (mMDH2), and aconitase (ACO3). Additional putative interacting candidates were deduced by comparing the enriched phosphoproteomes of wild type and B'ζ mutants: the mitochondrial regulator Arabidopsis pentatricopeptide repeat 6 (PPR6) and the two metabolic enzymes phosphoenolpyruvate carboxylase (PPC3) and phosphoenolpyruvate carboxykinase (PCK1). Overall, this study identifies potential PP2A substrates and highlights the role of PP2A in regulating energy metabolism in mitochondria.
PubMed: 37447147
DOI: 10.3390/plants12132586 -
Plant Physiology and Biochemistry : PPB Aug 2023Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO assimilation than mesophyll cells. However, it is unknown...
Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out aC-HCO labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the C-enrichment in pyruvate, succinate and glutamate. Only one C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.
Topics: Malates; Carbon Dioxide; Mesophyll Cells; Phosphoenolpyruvate Carboxylase; Citrates
PubMed: 37413941
DOI: 10.1016/j.plaphy.2023.107862 -
Animal Bioscience Nov 2023Cows that are nursing get around 80% of their glucose from liver gluconeogenesis. Propionate, a significant precursor of liver gluconeogenesis, can regulate the key...
OBJECTIVE
Cows that are nursing get around 80% of their glucose from liver gluconeogenesis. Propionate, a significant precursor of liver gluconeogenesis, can regulate the key genes involved in hepatic gluconeogenesis expression, but its precise effects on the activity of enzymes have not yet been fully elucidated. Therefore, the aim of this study was to investigate the effects of propionate on the activity, gene expression, and protein abundance of the key enzymes involved in the gluconeogenesis of dairy cow hepatocytes.
METHODS
The hepatocytes were cultured and treated with various concentrations of sodium propionate (0, 1.25, 2.50, 3.75, and 5.00 mM) for 12 h. Glucose content in the culture media was determined by an enzymatic coloring method. The activities of gluconeogenesis related enzymes were determined by enzyme linked immunosorbent assay kits, and the levels of gene expression and protein abundance of the enzymes were detected by real-time quantitative polymerase chain reaction and Western blot, respectively.
RESULTS
Propionate supplementation considerably increased the amount of glucose in the culture medium compared to the control (p<0.05); while there was no discernible difference among the various treatment concentrations (p>0.05). The activities of cytoplasmic phosphoenolpyruvate carboxylase (PEPCK1), mitochondrial phosphoenolpyruvate carboxylase (PEPCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC) were increased with the addition of 2.50 and 3.75 mM propionate; the gene expressions and protein abundances of PEPCK1, PEPCK2, PC, and G6PC were increased by 3.75 mM propionate addition.
CONCLUSION
Propionate encouraged glucose synthesis in bovine hepatocytes, and 3.75 mM propionate directly increased the activities, gene expressions and protein abundances of PC, PEPCK1, PEPCK2, and G6PC in bovine hepatocytes, providing a theoretical basis of propionate-regulating gluconeogenesis in bovine hepatocytes.
PubMed: 37402451
DOI: 10.5713/ab.23.0061