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The Plant Cell Jun 2024Organic carbon fixed in chloroplasts through the Calvin-Benson-Bassham Cycle can be diverted towards different metabolic fates, including cyoplasmic and mitochondrial...
Organic carbon fixed in chloroplasts through the Calvin-Benson-Bassham Cycle can be diverted towards different metabolic fates, including cyoplasmic and mitochondrial respiration, gluconeogenesis, and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known to exist in non-photosynthetic tissue. Here, we characterized a lower plastid glycolysis-gluconeogenesis pathway enabling the direct interconversion of glyceraldehyde-3-phosphate and phospho-enol-pyruvate in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis-gluconeogenesis, Enolase and bis-phospho-glycerate mutase (PGAM), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative 'omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that the cpEnolase and cpPGAM genes are expressed at elevated levels in high latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway.
PubMed: 38842420
DOI: 10.1093/plcell/koae168 -
Bioresource Technology Aug 2024Methane (CH) and carbon dioxide (CO) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH and CO...
Methane (CH) and carbon dioxide (CO) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH and CO biorefineries. This study demonstrated the synchronous incorporation of CH and CO into polyhydroxybutyrate (PHB) for the first time using C-labeling experiments in methanotrophs. By supplying substantial amounts of CO, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH and CO in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.
Topics: Methane; Carbon Dioxide; Hydroxybutyrates; Polyesters; Methylocystaceae; Carbon Isotopes
PubMed: 38838829
DOI: 10.1016/j.biortech.2024.130931 -
ACS Sensors Jun 2024The feasibility of hyperpolarized [2-C, 3-H]pyruvate for probing gluconeogenesis was investigated in this study. Whereas hyperpolarized [1-C]pyruvate has clear access...
The feasibility of hyperpolarized [2-C, 3-H]pyruvate for probing gluconeogenesis was investigated in this study. Whereas hyperpolarized [1-C]pyruvate has clear access to metabolic pathways that convert pyruvate to lactate, alanine, and bicarbonate, its utility for assessing pyruvate carboxylation and gluconeogenesis has been limited by technical challenges, including spectral overlap and an obscure enzymatic step that decarboxylates the labeled carbon. To achieve unambiguous detection of gluconeogenic products, the carbonyl carbon in pyruvate was labeled with C. To prolong the relaxation time, [2-C, 3-H]pyruvate was synthesized and dissolved with DO after dynamic nuclear polarization. The of [2-C, 3-H]pyruvate in DO could be improved by 76.9% (79.6 s at 1 T and 74.5 s at 3 T) as compared to [2-C]pyruvate in water. Hyperpolarized [2-C, 3-H]pyruvate with DO dissolution was applied to rat livers under normal feeding and fasting conditions. A gluconeogenic product, [2-C]phosphoenolpyruvate, was observed at 149.9 ppm from fasted rats only, highlighting the utility of [2-C, 3-H]pyruvate in detecting key gluconeogenic enzyme activities such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase .
Topics: Animals; Gluconeogenesis; Liver; Pyruvic Acid; Rats; Male; Rats, Sprague-Dawley; Carbon Isotopes
PubMed: 38838349
DOI: 10.1021/acssensors.4c00734 -
FASEB Journal : Official Publication of... Jun 2024Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates;...
Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.
Topics: Animals; Pyruvate Kinase; Mice; Cell Differentiation; Phosphorylation; Histones; Myoblasts; Transcription Factors; Thyroid Hormone-Binding Proteins; Humans; Chromosomal Proteins, Non-Histone; Thyroid Hormones; DNA-Binding Proteins; Isoenzymes
PubMed: 38837439
DOI: 10.1096/fj.202400784R -
ACS Infectious Diseases Jun 2024The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based...
The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant K12 (-R) compared with tetracycline-sensitive K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, -R and -R with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in -R was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.
Topics: Anti-Bacterial Agents; Tetracycline Resistance; Tetracycline; Membrane Potentials; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Escherichia coli; Escherichia coli K12; Proton-Motive Force; Microbial Sensitivity Tests; Escherichia coli Proteins; Metabolomics; Hydrogen-Ion Concentration; Proteomics
PubMed: 38836553
DOI: 10.1021/acsinfecdis.4c00158 -
Parasites, Hosts and Diseases May 2024Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and...
Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.
Topics: Animals; Phosphopyruvate Hydratase; Ixodidae; Cloning, Molecular; Amino Acid Sequence; Female; Molecular Sequence Data; Life Cycle Stages; Gene Silencing; Male; Phylogeny; Base Sequence; DNA, Complementary; Haemaphysalis longicornis
PubMed: 38835263
DOI: 10.3347/PHD.24015 -
Biophysical Chemistry Aug 2024We propose a detailed computational beta cell model that emphasizes the role of anaplerotic metabolism under glucose and glucose-glutamine stimulation. This model goes...
We propose a detailed computational beta cell model that emphasizes the role of anaplerotic metabolism under glucose and glucose-glutamine stimulation. This model goes beyond the traditional focus on mitochondrial oxidative phosphorylation and ATP-sensitive K channels, highlighting the predominant generation of ATP from phosphoenolpyruvate in the vicinity of K channels. It also underlines the modulatory role of HO as a signaling molecule in the first phase of glucose-stimulated insulin secretion. In the second phase, the model emphasizes the critical role of anaplerotic pathways, activated by glucose stimulation via pyruvate carboxylase and by glutamine via glutamate dehydrogenase. It particularly focuses on the production of NADPH and glutamate as key enhancers of insulin secretion. The predictions of the model are consistent with empirical data, highlighting the complex interplay of metabolic pathways and emphasizing the primary role of glucose and the facilitating role of glutamine in insulin secretion. By delineating these crucial metabolic pathways, the model provides valuable insights into potential therapeutic targets for diabetes.
Topics: Glutamine; Glucose; Insulin; Insulin Secretion; Models, Biological; Humans; Insulin-Secreting Cells; Animals; Pyruvate Carboxylase; Hydrogen Peroxide; Adenosine Triphosphate
PubMed: 38833963
DOI: 10.1016/j.bpc.2024.107270 -
Vaccines Apr 2024BCG vaccination affects other diseases beyond tuberculosis by unknown-potentially immunomodulatory-mechanisms. Recent studies have shown that BCG vaccination...
BCG vaccination affects other diseases beyond tuberculosis by unknown-potentially immunomodulatory-mechanisms. Recent studies have shown that BCG vaccination administered during overt type 1 diabetes (T1D) improved glycemic control and affected immune and metabolic parameters. Here, we comprehensively characterized Ghanaian T1D patients with or without routine neonatal BCG vaccination to identify vaccine-associated alterations. Ghanaian long-term T1D patients ( = 108) and matched healthy controls ( = 214) were evaluated for disease-related clinical, metabolic, and immunophenotypic parameters and compared based on their neonatal BCG vaccination status. The majority of study participants were BCG-vaccinated at birth and no differences in vaccination rates were detected between the study groups. Notably, glycemic control metrics, i.e., HbA1c and IDAA1c, showed significantly lower levels in BCG-vaccinated as compared to unvaccinated patients. Immunophenotype comparisons identified higher expression of the T cell activation marker CD25 on CD8 T cells from BCG-vaccinated T1D patients. Correlation analysis identified a negative correlation between HbA1c levels and CD25 expression on CD8 T cells. In addition, we observed fractional increases in glycolysis metabolites (phosphoenolpyruvate and 2/3-phosphoglycerate) in BCG-vaccinated T1D patients. These results suggest that neonatal BCG vaccination is associated with better glycemic control and increased activation of CD8 T cells in T1D patients.
PubMed: 38793703
DOI: 10.3390/vaccines12050452 -
Communications Biology May 2024Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains...
Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG.
Topics: Proteostasis; Gluconeogenesis; Humans; Endothelial Cells; Phosphoenolpyruvate Carboxykinase (GTP); Human Umbilical Vein Endothelial Cells; Glucose; Autophagy; Unfolded Protein Response; Phosphoenolpyruvate Carboxykinase (ATP)
PubMed: 38783087
DOI: 10.1038/s42003-024-06186-6 -
International Journal of Biological... Jun 2024It is well-accepted that the liver plays a vital role in the metabolism of glucose and its homeostasis. Dysregulated hepatic glucose production and utilization, leads to...
It is well-accepted that the liver plays a vital role in the metabolism of glucose and its homeostasis. Dysregulated hepatic glucose production and utilization, leads to type 2 diabetes (T2DM). In the current study, RNA sequencing and qRT-PCR analysis of nanoformulation-treated T2DM mice (TG group) revealed beneficial crosstalk of PCK-1 silencing with other pathways involved in T2DM. The comparison of precise genetic expression profiles of the different experimental groups showed significantly improved hepatic glucose, fatty acid metabolism and several other T2DM-associated crucial markers after the nanoformulation treatment. As a result of these improvements, we observed a significant acceleration in wound healing and improved insulin signaling in vascular endothelial cells in the TG group as compared to the T2DM group. Enhanced phosphorylation of PI3K/Akt pathway proteins in the TG group resulted in increased angiogenesis as observed by the increased expression of endothelial cell markers (CD31, CD34) thereby improving endothelial dysfunctions in the TG group. Additionally, therapeutic nanoformulation has been observed to improve the inflammatory cytokine profile in the TG group. Overall, our results demonstrated that the synthesized therapeutic nanoformulation referred to as GPR8:PCK-1 holds the potential in ameliorating hyperglycemia-associated complications such as delayed wound healing in diabetes.
Topics: Animals; Mice; Diabetes Mellitus, Type 2; Wound Healing; RNA, Small Interfering; Homeostasis; Glucose; Male; Diabetes Mellitus, Experimental; Signal Transduction; Liver; Phosphoenolpyruvate Carboxykinase (GTP)
PubMed: 38772464
DOI: 10.1016/j.ijbiomac.2024.132504