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Free Radical Biology & Medicine Mar 2024Due to insufficient and defective vascularization, the tumor microenvironment is often nutrient-depleted. LDHA has been demonstrated to play a tumor-promoting role by...
Due to insufficient and defective vascularization, the tumor microenvironment is often nutrient-depleted. LDHA has been demonstrated to play a tumor-promoting role by facilitating the glycolytic process. However, whether and how LDHA regulates cell survival in the nutrient-deficient tumor microenvironment is still unclear. Here, we sought to investigate the role and mechanism of LDHA in regulating cell survival and proliferation under energy stress conditions. Our results showed that the aerobic glycolysis levels, cell survival and proliferation of cervical cancer cells decreased significantly after inhibition of LDHA under normal culture condition while LDHA deficiency greatly inhibited glucose starvation-induced ferroptosis and promoted cell proliferation and tumor formation under energy stress conditions. Mechanistic studies suggested that glucose metabolism shifted from aerobic glycolysis to mitochondrial OXPHOS under energy stress conditions and LDHA knockdown increased accumulation of pyruvate in the cytosol, which entered the mitochondria and upregulated the level of oxaloacetate by phosphoenolpyruvate carboxylase (PC). Importantly, the increase in oxaloacetate production after absence of LDHA remarkably activated AMP-activated protein kinase (AMPK), which increased mitochondrial biogenesis and mitophagy, promoted mitochondrial homeostasis, thereby decreasing ROS level. Moreover, repression of lipogenesis by activation of AMPK led to elevated levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which effectively resisted ROS-induced cell ferroptosis and enhanced cell survival under energy stress conditions. These results suggested that LDHA played an opposing role in survival and proliferation of cervical cancer cells under energy stress conditions, and inhibition of LDHA may not be a suitable treatment strategy for cervical cancer.
Topics: Female; Humans; AMP-Activated Protein Kinases; Lactate Dehydrogenase 5; Oxaloacetates; Reactive Oxygen Species; Tumor Microenvironment; Uterine Cervical Neoplasms
PubMed: 38307156
DOI: 10.1016/j.freeradbiomed.2024.01.043 -
Plants (Basel, Switzerland) Nov 2023The genus sp. contains commercially important fleshy fruit-producing plants, including plantains and bananas, with a strong potential for providing food security and...
The genus sp. contains commercially important fleshy fruit-producing plants, including plantains and bananas, with a strong potential for providing food security and sources of revenue to farmers. Concerns with the quality of vegetative tissues along with the possibility of the transmission of phytopathogens makes the availability of healthy plantlets limited for farmers. Micropropagation of plantains offers an alternative to producing large numbers of plantlets. However, conventional methods of micropropagation have high production costs and are labor-intensive. Recently, the temporary immersion bioreactor (TIB) has emerged as an alternative to conventional micropropagation (CM) methods. Our work utilized SEM microscopy (scanning electron microscope) and molecular and biochemical tools (qRT-PCR and ICP-OES) to characterize and compare the morphological properties, elemental composition, and photosynthetic gene expression of plantains cultured on TIB. Additionally, morphological features of growth and propagation rates were analyzed to compare outputs obtained from TIB and CM. Results showed higher growth and multiplication rates for plantlets cultivated in TIB. Gene expression analysis of selected photosynthetic genes demonstrated high transcript abundance of phosphoenolpyruvate carboxylase () in plantain tissues obtained by TIB. Elemental composition analysis showed higher content of iron in plantains grown in TIB, suggesting a potential correlation with expression. These results demonstrate that micropropagation of sp. via the liquid medium in TIB is an efficient and low-cost approach in comparison with solid media in CM.
PubMed: 37960126
DOI: 10.3390/plants12213770 -
Scientific Reports Aug 2023Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary...
Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and K and k values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.
Topics: Paenibacillus; Bacillus; Mutagenesis, Site-Directed; Binding Sites
PubMed: 37591902
DOI: 10.1038/s41598-023-40072-1 -
Applied Microbiology and Biotechnology Oct 2023β-Arbutin is a plant-derived glycoside and widely used in cosmetic and pharmaceutical industries because of its safe and effective skin-lightening property as well as...
β-Arbutin is a plant-derived glycoside and widely used in cosmetic and pharmaceutical industries because of its safe and effective skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. In recent years, microbial fermentation has become a highly promising method for the production of β-arbutin. However, this method suffers from low titer and low yield, which has become the bottleneck for its widely industrial application. In this study, we used β-arbutin to demonstrate methods for improving yields for industrial-scale production in Escherichia coli. First, the supply of precursors phosphoenolpyruvate and uridine diphosphate glucose was improved, leading to a 4.6-fold increase in β-arbutin production in shaking flasks. The engineered strain produced 36.12 g/L β-arbutin with a yield of 0.11 g/g glucose in a 3-L bioreactor. Next, based on the substrate and product's structural similarity, an endogenous O-acetyltransferase was identified as responsible for 6-O-acetylarbutin formation for the first time. Eliminating the formation of byproducts, including 6-O-acetylarbutin, tyrosine, and acetate, resulted in an engineered strain producing 43.79 g/L β-arbutin with a yield of 0.22 g/g glucose in fed-batch fermentation. Thus, the yield increased twofold by eliminating byproducts formation. To the best of our knowledge, this is the highest titer and yield of β-arbutin ever reported, paving the way for the industrial production of β-arbutin. This study demonstrated a systematic strategy to alleviate undesirable byproduct accumulation and improve the titer and yield of target products. KEY POINTS: • A systematic strategy to improve titer and yield was showed • Genes responsible for 6-O-acetylarbutin formation were firstly identified • 43.79 g/L β-arbutin was produced in bioreactor, which is the highest titer so far.
Topics: Arbutin; Bioreactors; Fermentation; Escherichia coli; Glucose; Metabolic Engineering
PubMed: 37597019
DOI: 10.1007/s00253-023-12706-x -
Carbohydrate Polymers Dec 2023Walnut green husk polysaccharides (WGP) are isolated from the walnut green husk with a mean molecular weight of 12.77 kDa. The structural characterization revealed by...
Walnut green husk polysaccharides (WGP) are isolated from the walnut green husk with a mean molecular weight of 12.77 kDa. The structural characterization revealed by methylation and NMR analysis indicated that WGP might consist of →4-α-D-Galp-(1→, α-D-Galp (1→, and →2)-α-L-Rhap-(1→. Previous studies have been demonstrated that WGP effectively prevented liver injury and modulated gut microbiota in high fructose-treated mice and high fat diet-treated rats. In this study, we found for the first time that WGP presenting outstanding protective effects on liver inflammation and gluconeogenesis dysfunction induced by ochratoxin A (OTA) in mice. Firstly, WGP decreased oxidative stress, down-regulated the expression of inflammatory factors and inhibited the TLR4/p65/IκBα pathway in the liver. Then, WGP reversed OTA-induced lower phosphoenolpyruvate carboxyl kinase (PEPCK), and glucose 6-phosphatase (G6PC) activities in the liver. Furthermore, WGP increased the diversity of gut microbiota and the abundance of beneficial bacteria, especially Lactobacillus and Akkermansia. Importantly, the results of fecal microbiota transplantation (FMT) experiment further confirmed that gut microbiota involved in the protective effects of WGP on liver damage induced by OTA. Our results indicated that the protective effect of WGP on liver inflammation and gluconeogenesis dysfunction caused by OTA may be due to the regulation of gut microbiota.
Topics: Mice; Rats; Animals; Gluconeogenesis; Juglans; Gastrointestinal Microbiome; Liver; Polysaccharides; Inflammation
PubMed: 37839834
DOI: 10.1016/j.carbpol.2023.121362 -
Journal of Experimental Botany Nov 2023Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4...
Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3-C4 intermediates'. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phosphoenolpyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments.
Topics: Phylogeny; Carbon; Brassicaceae; Carbon Dioxide; Photosynthesis; Glycine; Plant Leaves
PubMed: 37392176
DOI: 10.1093/jxb/erad250 -
Talanta Aug 2024Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate...
Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry.
Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP-metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.
Topics: Tandem Mass Spectrometry; Animals; Humans; Hydrophobic and Hydrophilic Interactions; Edetic Acid; Mice; Chromatography, Liquid; Rats; Phosphoenolpyruvate; HEK293 Cells; Hep G2 Cells; Rats, Sprague-Dawley; Male
PubMed: 38692044
DOI: 10.1016/j.talanta.2024.126134 -
European Journal of Immunology Dec 2023Viral infections can result in metabolism rewiring of host cells, which in turn affects the viral lifecycle. Phosphoenolpyruvate (PEP), a metabolic intermediate in the...
Viral infections can result in metabolism rewiring of host cells, which in turn affects the viral lifecycle. Phosphoenolpyruvate (PEP), a metabolic intermediate in the glycolytic pathway, plays important roles in several biological processes including anti-tumor T cell immunity. However, whether PEP might participate in modulating viral infection remains largely unknown. Here, we demonstrate that PEP generally inhibits viral replication via upregulation of apoptosis-associated tyrosine kinase (AATK) expression. Targeted metabolomic analyses have shown that the intracellular level of PEP was increased upon viral infection. PEP treatment significantly restricted viral infection and hence declined subsequent inflammatory response both in vitro and in vivo. Besides, PEP took inhibitory effect on the stage of viral replication and also decreased the mortality of mice with viral infection. Mechanistically, PEP significantly promoted the expression of AATK. Knockdown of AATK led to enhanced viral replication and consequent increased levels of cytokines. Moreover, AATK deficiency disabled the antiviral effect of PEP. Together, our study reveals a previously unknown role of PEP in broadly inhibiting viral replication by promoting AATK expression, highlighting the potential application of activation or upregulation of the PEP-AATK axis in controlling viral infections.
Topics: Mice; Animals; Phosphoenolpyruvate; Glycolysis; Virus Diseases
PubMed: 37724936
DOI: 10.1002/eji.202350536 -
Nutrients Dec 2023Type 2 diabetes mellitus (T2DM) has become a worldwide public health problem. Epimedin C is considered one of the most important flavonoids in , a famous edible herb in...
Type 2 diabetes mellitus (T2DM) has become a worldwide public health problem. Epimedin C is considered one of the most important flavonoids in , a famous edible herb in China and Southeast Asia that is traditionally used in herbal medicine to treat diabetes. In the present study, the therapeutic potential of epimedin C against T2DM was ascertained using a mouse model, and the mechanism underlying the hypoglycemic activity of epimedin C was explored using a label-free proteomic technique for the first time. Levels of fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), and oral glucose tolerance, as well as contents of malondialdehyde (MDA) and low-density lipoprotein cholesterol (LDL-C) in the 30 mg·kg epimedin C group (EC30 group), were significantly lower than those in the model control group (MC group) ( < 0.05), while the contents of hepatic glycogen, insulin, and high-density lipoprotein cholesterol (HDL-C), as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the EC30 group were notably higher than those in the MC group ( < 0.05). The structures of liver cells and tissues were greatly destroyed in the MC group, whereas the structures of cells and tissues were basically complete in the EC30 group, which were similar to those in the normal control group (NC group). A total of 92 differentially expressed proteins (DEPs) were enriched in the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In the EC30 vs. MC groups, the expression level of cytosolic phosphoenolpyruvate carboxykinase (Pck1) was down-regulated, while the expression levels of group XIIB secretory phospholipase A2-like protein (Pla2g12b), apolipoprotein B-100 (Apob), and cytochrome P450 4A14 (Cyp4a14) were up-regulated. According to the KEGG pathway assay, Pck1 participated in the gluconeogenesis and insulin signaling pathways, and Pla2g12b, Apob, and Cyp4a14 were the key proteins in the fat digestion and fatty acid degradation pathways. Pck1, Pla2g12b, Apob, and Cyp4a14 seemed to play important roles in the prevention and treatment of T2DM. In summary, epimedin C inhibited Pck1 expression to maintain FBG at a relatively stable level, promoted Pla2g12b, Apob, and Cyp4a14 expressions to alleviate liver lipotoxicity, and protected liver tissues and cells from oxidant stress possibly by its phenolic hydroxyl groups.
Topics: Humans; Hypoglycemic Agents; Diabetes Mellitus, Type 2; Proteomics; Flavonoids; Insulin; Phosphoenolpyruvate Carboxykinase (ATP); Cholesterol
PubMed: 38201855
DOI: 10.3390/nu16010025 -
Scientific Reports Dec 2023Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by complex lung pathogenesis affecting approximately three million people...
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by complex lung pathogenesis affecting approximately three million people worldwide. While the molecular and cellular details of the IPF mechanism is emerging, our current understanding is centered around the lung itself. On the other hand, many human diseases are the products of complex multi-organ interactions. Hence, we postulate that a dysfunctional crosstalk of the lung with other organs plays a causative role in the onset, progression and/or complications of IPF. In this study, we employed a generative computational approach to identify such inter-organ mechanism of IPF. This approach found unexpected molecular relatedness of IPF to neoplasm, diabetes, Alzheimer's disease, obesity, atherosclerosis, and arteriosclerosis. Furthermore, as a potential mechanism underlying this relatedness, we uncovered a putative molecular crosstalk system across the lung and the liver. In this inter-organ system, a secreted protein, kininogen 1, from hepatocytes in the liver interacts with its receptor, bradykinin receptor B1 in the lung. This ligand-receptor interaction across the liver and the lung leads to the activation of calmodulin pathways in the lung, leading to the activation of interleukin 6 and phosphoenolpyruvate carboxykinase 1 pathway across these organs. Importantly, we retrospectively identified several pre-clinical and clinical evidence supporting this inter-organ mechanism of IPF. In conclusion, such feedforward and feedback loop system across the lung and the liver provides a unique opportunity for the development of the treatment and/or diagnosis of IPF. Furthermore, the result illustrates a generative computational framework for machine-mediated synthesis of mechanisms that facilitates and complements the traditional experimental approaches in biomedical sciences.
Topics: Humans; Retrospective Studies; Idiopathic Pulmonary Fibrosis; Lung
PubMed: 38081956
DOI: 10.1038/s41598-023-49281-0