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IUCrJ Nov 2023Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all...
Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of Mg is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.
Topics: Phosphopyruvate Hydratase; Mycobacterium tuberculosis; Protein Conformation; Models, Molecular; Catalysis
PubMed: 37860976
DOI: 10.1107/S2052252523008485 -
Lipids in Health and Disease Oct 2023Dysregulation of lipid metabolism is the most prominent metabolic alteration observed in obesity, cancer, and cardiovascular diseases. The present study aimed to explore...
BACKGROUND
Dysregulation of lipid metabolism is the most prominent metabolic alteration observed in obesity, cancer, and cardiovascular diseases. The present study aimed to explore the sex differences associated with lipid metabolism in urinary exosome proteins, and evaluate the correlation of urinary exosome proteins with serum lipid biomarkers.
METHODS
The key enzymes regulating lipid metabolism in healthy adults were screened using urinary exosome data. Urinary exosomes were isolated from 120 healthy subjects and the expression of urinary proteins was assessed by Western blotting and ELISA. The correlation between urinary protein concentrations and the levels of serum lipid biomarkers was analyzed using correlation analysis.
RESULTS
Three urinary exosome proteins, namely fatty acid synthase (FASN), phosphoenolpyruvate carboxykinase (PCK1), and ATP-citrate synthase (ACLY) were identified, and only FASN showed sex differences. Sex differences were also observed in the serum triglyceride (TG) levels. Healthy males had higher FASN levels than females, and a moderate positive correlation was found between FASN concentrations and serum TG levels in healthy males (r = 0.479, P < 0.05). FASN concentrations in different age groups were positively correlated with the level of serum TG (18 ~ 30 years, r = 0.502; 31 ~ 44 years, r = 0.587; 45 ~ 59 years, r = 0.654; all P < 0.05). In addition, FASN concentrations was positively related to the increase in serum TG levels (range:1.0 ~ 1.7 mmol/L; r = 0.574, P < 0.05).
CONCLUSIONS
Sex differences were observed in urinary exosome FASN protein levels in healthy adults. FASN protein levels positively correlated with increased serum TG levels. FASN may serve as a novel biomarker to evaluate fatty acid synthesis in the human body.
Topics: Humans; Male; Adult; Female; Sex Characteristics; Exosomes; Fatty Acid Synthases; Biomarkers; Lipids; Triglycerides; Fatty Acid Synthase, Type I
PubMed: 37858194
DOI: 10.1186/s12944-023-01936-7 -
Food & Nutrition Research 2023The fruits of L. are high in nutrients and have excellent health care function and developmental value. There are many management strategies available for diabetes and...
Antidiabetic and antihyperlipidemic activities of . extract and the regulation of Akt phosphorylation, gluconeogenesis, and peroxisome proliferator-activated receptor α in streptozotocin-induced diabetic mice.
BACKGROUND
The fruits of L. are high in nutrients and have excellent health care function and developmental value. There are many management strategies available for diabetes and hyperlipidemia. Nevertheless, there is a lack of an effective and nontoxic drug.
OBJECTIVE
The present study was designed to first screen four extracts of L. on insulin signaling target gene expression levels, including glucose transporter 4 (GLUT4) and p-Akt/t-Akt. The ethyl acetate extract of L. (EPE) exhibited the most efficient activity among the four extracts and was thus chosen to explore the antidiabetic and antihyperlipidemic activities in streptozotocin (STZ)-induced type 1 diabetic mice.
DESIGN
All mice (in addition to one control (CON) group) were administered STZ injections (intraperitoneal) for 5 consecutive days, and then STZ-induced mice were administered EPE (at 100, 200, or 400 mg/kg body weight), fenofibrate (Feno) (at 250 mg/kg body weight), glibenclamide (Glib) (at 10 mg/kg body weight), or vehicle by oral gavage once daily for 4 weeks. Finally, histological examination, blood biochemical parameters, and target gene mRNA expression levels were measured, and liver tissue was analyzed for the levels of malondialdehyde (MDA), a maker of lipid peroxidation.
RESULTS
EPE treatment resulted in decreased levels of blood glucose, HbA1C, triglycerides (TGs), and total cholesterol and increased levels of insulin compared with the vehicle-treated STZ group. EPE treatment decreased blood levels of HbA1C and MDA but increased glutathione levels in liver tissue, implying that EPE exerts antioxidant activity and could prevent oxidative stress and diabetes. The EPE-treated STZ mice displayed an improvement in the sizes and numbers of insulin-expressing β cells. EPE treatment increased the membrane expression levels of skeletal muscular GLUT4, and also reduced hepatic mRNA levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase thereby inhibiting hepatic gluconeogenesis. This resulted in a net glucose lowering effect in EPE-treated STZ mice. Furthermore, EPE increased the expression levels of p-AMPK/t-AMPK in both the skeletal muscle and liver tissue compared with vehicle-treated STZ mice. EPE-treated STZ mice showed enhanced expression levels of fatty acid oxidation enzymes, including peroxisome proliferator-activated receptor α (PPARα), but reduced expression levels of lipogenic genes including fatty acid synthase, as well as decreased mRNA levels of sterol regulatory element binding protein 1c (SREBP1c), apolipoprotein-CIII (apo-CIII), and diacylglycerol acyltransferase-2 (DGAT2). This resulted in a reduction in plasma TG levels. EPE-treated STZ mice also showed reduced expression levels of PPAR γ. This resulted in decreased adipogenesis, fatty acid synthesis, and lipid accumulation within liver tissue, and consequently, lower TG levels in liver tissue and blood. Furthermore, EPE treatment not only displayed an increase in the Akt activation in liver tissue, but also in C2C12 myotube in the absence of insulin. These results implied that EPE acts as an activator of AMPK and /or as a regulator of the insulin (Akt) pathway.
CONCLUSIONS
Taken together, EPE treatment exhibited amelioration of the diabetic and hyperlipidemic state in STZ-induced diabetic mice.
PubMed: 37850072
DOI: 10.29219/fnr.v67.9854 -
Nature Communications Oct 2023Adiponectin is a secretory protein, primarily produced in adipocytes. However, low but detectable expression of adiponectin can be observed in cell types beyond...
Adiponectin is a secretory protein, primarily produced in adipocytes. However, low but detectable expression of adiponectin can be observed in cell types beyond adipocytes, particularly in kidney tubular cells, but its local renal role is unknown. We assessed the impact of renal adiponectin by utilizing male inducible kidney tubular cell-specific adiponectin overexpression or knockout mice. Kidney-specific adiponectin overexpression induces a doubling of phosphoenolpyruvate carboxylase expression and enhanced pyruvate-mediated glucose production, tricarboxylic acid cycle intermediates and an upregulation of fatty acid oxidation (FAO). Inhibition of FAO reduces the adiponectin-induced enhancement of glucose production, highlighting the role of FAO in the induction of renal gluconeogenesis. In contrast, mice lacking adiponectin in the kidney exhibit enhanced glucose tolerance, lower utilization and greater accumulation of lipid species. Hence, renal adiponectin is an inducer of gluconeogenesis by driving enhanced local FAO and further underlines the important systemic contribution of renal gluconeogenesis.
Topics: Animals; Male; Mice; Adiponectin; Gluconeogenesis; Glucose; Kidney; Liver; Mice, Knockout; Pyruvic Acid
PubMed: 37848446
DOI: 10.1038/s41467-023-42188-4 -
Animal Nutrition (Zhongguo Xu Mu Shou... Dec 2023Enhancing hepatic gluconeogenesis is one of the main modes of meeting the glucose requirement of dairy cows. This study attempted to determine whether the...
Enhancing hepatic gluconeogenesis is one of the main modes of meeting the glucose requirement of dairy cows. This study attempted to determine whether the gluconeogenesis precursor propionate had an effect on the expression of the main genes involved in gluconeogenesis in calf hepatocytes and elucidate the associated mechanisms. Calf hepatocytes were obtained from 5 healthy calves (1 d old; 30 to 40 kg) and exposed to 0-, 1-, 2.5-, or 5-mM sodium propionate (NaP), which is known to promote the expression of genes involved in the gluconeogenesis pathway, including fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase. With regard to the underlying mechanism, propionate promoted the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, hepatocyte nuclear factor 4, and forkhead box O1 (transcription factors that regulate the expression of hepatic gluconeogenic genes) by promoting mammalian target of rapamycin complex 1 (mTORC1), but inhibiting mTORC2 activity ( < 0.01). We also established a model of palmitic acid (PA)-induced hepatic injury in calf hepatocytes and found that PA could inhibit the gluconeogenic capacity of calf hepatocytes by suppressing the expression of gluconeogenic genes, inhibiting mTORC1, and promoting the activity of mTORC2 ( < 0.01). In contrast, NaP provided protection to calf hepatocytes by counteracting the inhibitory effect of PA on the gluconeogenic capacity of calf hepatocytes ( < 0.05). Collectively, these findings indicate that NaP enhances the gluconeogenic capacity of calf hepatocytes by regulating the mTOR pathway activity. Thus, in addition to improving the glucose production potential, propionate may have therapeutic potential for the treatment of hepatic injury in dairy cows.
PubMed: 37841648
DOI: 10.1016/j.aninu.2023.07.001 -
International Journal of Molecular... Sep 2023RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes....
RNA polymerase III (RNAP III) holoenzyme activity and the processing of its products have been linked to several metabolic dysfunctions in lower and higher eukaryotes. Alterations in the activity of RNAP III-driven synthesis of non-coding RNA cause extensive changes in glucose metabolism. Increased RNAP III activity in the strain is lethal when grown on a non-fermentable carbon source. This lethal phenotype is suppressed by reducing tRNA synthesis. Neither the cause of the lack of growth nor the underlying molecular mechanism have been deciphered, and this area has been awaiting scientific explanation for a decade. Our previous proteomics data suggested mitochondrial dysfunction in the strain. Using model mutant strains (with increased tRNA abundance) and (with reduced tRNA abundance), we collected data showing major changes in the TCA cycle metabolism of the mutants that explain the phenotypic observations. Based on C flux data and analysis of TCA enzyme activities, the present study identifies the flux constraints in the mitochondrial metabolic network. The lack of growth is associated with a decrease in TCA cycle activity and downregulation of the flux towards glutamate, aspartate and phosphoenolpyruvate (PEP), the metabolic intermediate feeding the gluconeogenic pathway. , the strain that is unable to increase tRNA synthesis due to a mutation in the C128 subunit, has increased TCA cycle activity under non-fermentable conditions. To summarize, cells with non-optimal activity of RNAP III undergo substantial adaptation to a new metabolic state, which makes them vulnerable under specific growth conditions. Our results strongly suggest that balanced, non-coding RNA synthesis that is coupled to glucose signaling is a fundamental requirement to sustain a cell's intracellular homeostasis and flexibility under changing growth conditions. The presented results provide insight into the possible role of RNAP III in the mitochondrial metabolism of other cell types.
Topics: Saccharomyces cerevisiae; Mitochondria; Homeostasis; RNA, Transfer; RNA, Untranslated
PubMed: 37834211
DOI: 10.3390/ijms241914763 -
Microbial Cell Factories Oct 2023Phenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids,...
BACKGROUND
Phenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p-coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p-coumaric acid from glucose.
RESULTS
Heterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p-coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p-coumaric acid accumulation. A maximum titer of 661 mg/L p-coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C. glutamicum strain previously engineered for the conversion of p-coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p-coumaric acid supplementation.
CONCLUSIONS
The utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p-coumaric acid with C. glutamicum. Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants.
Topics: Resveratrol; Corynebacterium glutamicum; Tryptophan; Plants; Glucose; Polyphenols; Phenylalanine; Metabolic Engineering
PubMed: 37833813
DOI: 10.1186/s12934-023-02222-y -
PloS One 2023To evaluated the effects melatonin (MT) on the sugar and acid metabolism of early-ripening peach fruits, the concentration of 150 μmol/L MT was sprayed on the leaves of...
To evaluated the effects melatonin (MT) on the sugar and acid metabolism of early-ripening peach fruits, the concentration of 150 μmol/L MT was sprayed on the leaves of peach trees. MT increased the contents of total soluble sugar and sucrose in peach fruits during the whole ripening period, and increased the contents of glucose and sorbitol at the mature stage. During the whole ripening period, MT also increased the activities of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase and the relative expression levels of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase genes, while decreased the activity of sorbitol oxidase and the relative expression level of sorbitol dehydrogenase to some extent. Moreover, MT decreased the contents of total organic acid, malic acid, and citric acid at mature stage. At mature stage, MT decreased the activities of citrate synthetase and phosphoenolpyruvate carboxylase and the relative expression levels of citrate synthetase and phosphoenolpyruvate carboxylase genes, while increased the relative expression levels of Nicotinamide adenine dinucleotide phosphate (NADP+)-malic enzyme, malate dehydrogenase, and aconitase genes. Therefore, MT promotes the sugar accumulation and organic acid degradation in early-ripening peach fruits.
Topics: Fruit; Sugars; Carbohydrate Metabolism; Melatonin; Phosphoenolpyruvate Carboxylase; beta-Fructofuranosidase; Carbohydrates; Citrates; Ligases
PubMed: 37831703
DOI: 10.1371/journal.pone.0292959 -
Frontiers in Molecular Biosciences 2023Chagas disease (ChD), caused by , is endemic in American countries and an estimated 8 million people worldwide are chronically infected. Currently, only two drugs are...
Chagas disease (ChD), caused by , is endemic in American countries and an estimated 8 million people worldwide are chronically infected. Currently, only two drugs are available for therapeutic use against and their use is controversial due to several disadvantages associated with side effects and low compliance with treatment. Therefore, there is a need to search for new tripanocidal agents. Natural products have been considered a potential innovative source of effective and selective agents for drug development to treat infection. Recently, our research group showed that hexanic extract from (CFHEX) exhibits anti-parasitic activity against all stages of parasite, being apoptosis the main cell death mechanism in both epimastigotes and trypomastigotes stages. With the aim of deepening the understanding of the mechanisms of death induced by CFHEX, the metabolic alterations elicited after treatment using a multiplatform metabolomics analysis (RP/HILIC-LC-QTOF-MS and GC-QTOF-MS) were performed. A total of 154 altered compounds were found significant in the treated parasites corresponding to amino acids (Arginine, threonine, cysteine, methionine, glycine, valine, proline, isoleucine, alanine, leucine, glutamic acid, and serine), fatty acids (stearic acid), glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine), sulfur compounds (trypanothione) and carboxylic acids (pyruvate and phosphoenolpyruvate). The most affected metabolic pathways were mainly related to energy metabolism, which was found to be decrease during the evaluated treatment time. Further, exogenous compounds of the triterpene type (betulinic, ursolic and pomolic acid) previously described in were found inside the treated parasites. Our findings suggest that triterpene-type compounds may contribute to the activity of CFHEX by altering essential processes in the parasite.
PubMed: 37818099
DOI: 10.3389/fmolb.2023.1206074 -
Applied and Environmental Microbiology Oct 2023The foodborne bacterial pathogen exhibits remarkable survival capabilities under challenging conditions, severely threatening food safety and human health. The orphan...
The foodborne bacterial pathogen exhibits remarkable survival capabilities under challenging conditions, severely threatening food safety and human health. The orphan regulator DegU is a pleiotropic regulator required for bacterial environmental adaptation. However, the specific mechanism of how DegU participates in oxidative stress tolerance remains unknown in . In this study, we demonstrate that DegU suppresses carbohydrate uptake under stress conditions by altering global transcriptional profiles, particularly by modulating the transcription of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS)-related genes, such as , , and . Specifically, in the absence of , the transcripts of are significantly upregulated and those of are significantly downregulated in response to copper ion-induced stress. Overexpression of significantly increases bacterial growth , while overexpression of leads to a decrease in growth. We further demonstrate that DegU directly senses oxidative stress, downregulates transcription, and upregulates transcription. Additionally, through an electrophoretic mobility shift assay, we demonstrate that DegU directly regulates the transcription of and by binding to specific regions within their respective promoter sequences. Notably, the putative pivotal DegU binding sequence for is located from 38 to 68 base pairs upstream of the transcription start site (TSS), whereas for , it is mapped from 36 to 124 base pairs upstream of the TSS. In summary, we elucidate that DegU plays a significant role in suppressing carbohydrate uptake in response to oxidative stress through the direct regulation of and .ImportanceUnderstanding the adaptive mechanisms employed by in harsh environments is of great significance. This study focuses on investigating the role of DegU in response to oxidative stress by examining global transcriptional profiles. The results highlight the noteworthy involvement of DegU in this stress response. Specifically, DegU acts as a direct sensor of oxidative stress, leading to the modulation of gene transcription. It downregulates transcription while it upregulates transcription through direct binding to their promoters. Consequently, these regulatory actions impede bacterial growth, providing a defense mechanism against stress-induced damage. These findings gained from this study may have broader implications, serving as a reference for studying adaptive mechanisms in other pathogenic bacteria and aiding in the development of targeted strategies to control and ensure food safety.
Topics: Humans; Listeria monocytogenes; Bacterial Proteins; Gene Expression Regulation, Bacterial; Carbohydrates; Oxidative Stress
PubMed: 37787570
DOI: 10.1128/aem.01017-23