scholarly journals Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 358
Author(s):  
Cecilia Ortíz ◽  
Francesca Moraca ◽  
Marc Laverriere ◽  
Allan Jordan ◽  
Niall Hamilton ◽  
...  
Keyword(s):  
De Novo ◽  
Pentose Phosphate ◽  
Small Subset ◽  
Cell Physiology ◽  
Single Digit ◽  
In Vitro Proliferation ◽  
African Trypanosomes ◽  
Thiol Redox ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH+ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding β-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit μM EC50 against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH+-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.

scholarly journals Defective carbohydrate metabolism in mice homozygous for the tubby mutation

2006 ◽  
Vol 27 (2) ◽  
pp. 131-140 ◽  
Author(s):  
Yun Wang ◽  
Kevin Seburn ◽  
Lawrence Bechtel ◽  
Bruce Y. Lee ◽  
Jin P. Szatkiewicz ◽  
...  
Keyword(s):  
Carbohydrate Metabolism ◽  
De Novo ◽  
Ketone Bodies ◽  
Late Onset ◽  
Acid Synthesis ◽  
Pentose Phosphate ◽  
Mrna Levels ◽  
Protein Levels ◽  
Energy Needs ◽  
Glucose 6 Phosphate Dehydrogenase

Tub is a member of a small gene family, the tubby-like proteins (TULPs), with predominant expression in neurons. Mice carrying a mutation in Tub develop retinal and cochlear degeneration as well as late-onset obesity with insulin resistance. During behavioral and metabolic testing, we found that homozygous C57BL/6J- Tub tub mice have a lower respiratory quotient than C57BL/6J controls before the onset of obesity, indicating that tubby homozygotes fail to activate carbohydrate metabolism and instead rely on fat metabolism for energy needs. In concordance with this, tubby mice show higher excretion of ketone bodies and accumulation of glycogen in the liver. Quantitation of liver mRNA levels shows that, during the transition from light to dark period, tubby mice fail to induce glucose-6-phosphate dehydrogenase ( G6pdh), the rate-limiting enzyme in the pentose phosphate pathway that normally supplies NADPH for de novo fatty acid synthesis and glutathione reduction. Reduced G6PDH protein levels and enzymatic activity in tubby mice lead accordingly to lower levels of NADPH and reduced glutathione (GSH), respectively. mRNA levels for the lipolytic enzymes acetyl-CoA synthetase and carnitine palmitoyltransferase are increased during the dark cycle and decreased during the light period, and several citric acid cycle genes are dysregulated in tubby mice. Examination of hypothalamic gene expression showed high levels of preproorexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. We hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the moderate obesity observed in tubby mice.

scholarly journals Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro

Reproduction ◽  
2002 ◽  
pp. 675-681 ◽  
Author(s):  
P Cetica ◽  
L Pintos ◽  
G Dalvit ◽  
M Beconi
Keyword(s):  
Enzymatic Activity ◽  
Pentose Phosphate Pathway ◽  
In Vitro Maturation ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
Key Enzymes ◽  
Glucose 6 Phosphate Dehydrogenase

Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.

scholarly journals Immunometabolic Endothelial Phenotypes: Integrating Inflammation and Glucose Metabolism

2021 ◽  
Author(s):  
Wusheng Xiao ◽  
William M Oldham ◽  
Carnen Priolo ◽  
Arvind K Pandey ◽  
Joseph Loscalzo
Keyword(s):  
Inflammatory Mediators ◽  
Nuclear Factor Κb ◽  
Pentose Phosphate ◽  
Pyruvate Dehydrogenase Kinase ◽  
Extracellular Flux ◽  
Pharmacological Blockade ◽  
Factor Α ◽  
Glucose 6 Phosphate Dehydrogenase

Rationale: Specific mechanisms linking inflammation and metabolic re-programming, two hallmarks of many pathobiological processes, remain incompletely defined. Objective: To delineate the integrative regulatory actions governing inflammation and metabolism in endothelial cells (ECs). Methods and Results: Metabolomic profiling, glucose labeling and tracing, and Seahorse extracellular flux analyses revealed that the inflammatory mediators, tumor necrosis factor α (TNFα) and lipopolysaccharide (LPS), extensively reprogram cellular metabolism, and particularly enhance glycolysis, mitochondrial oxidative phosphorylation (OXPHOS), and the pentose phosphate pathway (PPP) in primary human arterial ECs. Mechanistically, the enhancement in glycolysis and PPP is mediated by activation of the nuclear factor-κB (NF-κB)-6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3 (PFKFB3) axis and upregulation of glucose 6-phosphate dehydrogenase (G6PD), respectively; while enhanced OXPHOS was attributed to suppression of the forkhead box O1 (FOXO1)-pyruvate dehydrogenase kinase 4 (PDK4) axis. Restoration of the FOXO1-PDK4 axis attenuated the TNFα- or LPS-induced increase in OXPHOS but worsened inflammation in vitro, whereas enhancement of OXPHOS by pharmacological blockade of PDKs attenuated inflammation in mesenteric vessels of LPS-treated mice. Notably, suppression of G6PD expression or its activity potentiated the metabolic shift to glycolysis and/or endothelial inflammation, while inhibition of the NF-κB-PFKFB3 signaling, conversely, blunted the increased glycolysis and/or inflammation in in vitro and in vivo sepsis models. Conclusions: These results indicate that inflammatory mediators modulate the metabolic fates of glucose, and that stimulation of glycolysis promotes inflammation, whereas enhancement of OXPHOS and the PPP suppresses inflammation in the endothelium. Characterization of these immunometabolic phenotypes may have implications for the pathogenesis and treatment of many cardiovascular diseases.

The Compability with Enzyme Activity of Unusual Organic Osmolytes from Mangrove Red Algae

1996 ◽  
Vol 23 (5) ◽  
pp. 577 ◽  
Author(s):  
U Karsten ◽  
KD Barrow ◽  
O Nixdorf ◽  
RJ King
Keyword(s):  
Red Algae ◽  
Citric Acid Cycle ◽  
Compatible Solutes ◽  
Pentose Phosphate ◽  
Organic Osmolytes ◽  
Maximum Solubility ◽  
Organic Osmolyte ◽  
Negative Effect ◽  
Glucose 6 Phosphate Dehydrogenase

The effects of organic osmolytes synthesised and accumulated by red algae from mangrove habitats were investigated on the in vitro activities of two major enzymes, one of the citric acid cycle (malate dehydrogenase, MDH) and one of the oxidative pentose phosphate pathway (glucose-6- phosphate dehydrogenase, G6PDH). These enzymes were extracted from the mangrove algae Bostrychia tenella, Caloglossa leprieurii, Catenella nipae and Stictosiphonia hookeri. In each case, activity of the enzymes was inhibited with increasing NaCl concentrations up to 600 mM . In contrast, equimolar concentrations of mannitol (the major osmolyte in C. leprieurii), sorbitol (the major osmolyte in B. Tenella and S. hookeri) and a heteroside mixture (of which floridoside is the major osmolyte in C. nipae) did not inhibit enzyme function. Dulcitol, the second most important organic osmolyte in B. tenella, exerted no negative effect at its maximum solubility of 180 rnM on the salt-sensitive MDH. These data are all consistent with the proposed function of these organic compounds as compatible solutes.

scholarly journals Glucose-6-Phosphate Dehydrogenase Regulation in Anoxia Tolerance of the Freshwater Crayfish Orconectes virilis

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Benjamin Lant ◽  
Kenneth B. Storey
Keyword(s):  
Protein Kinase ◽  
Antioxidant Defense ◽  
Pentose Phosphate ◽  
Kinetic Properties ◽  
Freshwater Crayfish ◽  
Orconectes Virilis ◽  
Rate Determining Step ◽  
Flow Through ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PDH), the enzyme which catalyzes the rate determining step of the pentose phosphate pathway (PPP), controls the production of nucleotide precursor molecules (R5P) and powerful reducing molecules (NADPH) that support multiple biosynthetic functions, including antioxidant defense. G6PDH from hepatopancreas of the freshwater crayfish (Orconectes virilis) showed distinct kinetic changes in response to 20 h anoxic exposure. Km values for both substrates decreased significantly in anoxic crayfish; Km NADP+ dropped from 0.015±0.008 mM to 0.012±0.008 mM, and Km G6P decreased from 0.13±0.02 mM to 0.08±0.007 mM. Two lines of evidence indicate that the mechanism involved is reversible phosphorylation. In vitro incubations that stimulated protein kinase or protein phosphatase action mimicked the effects on anoxia on Km values, whereas DEAE-Sephadex chromatography showed the presence of two enzyme forms (low- and high-phosphate) whose proportions changed during anoxia. Incubation studies implicated protein kinase A and G in mediating the anoxia-responsive changes in G6PDH kinetic properties. In addition, the amount of G6PDH protein (measured by immunoblotting) increased by ∼60% in anoxic hepatopancreas. Anoxia-induced phosphorylation of G6PDH could contribute to modifying carbon flow through the PPP under anoxic conditions, potentially maintaining NADPH supply for antioxidant defense during prolonged anoxia-induced hypometabolism.

scholarly journals Thiol Redox Sensitivity of Two Key Enzymes of Heme Biosynthesis and Pentose Phosphate Pathways: Uroporphyrinogen Decarboxylase and Transketolase

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Brian McDonagh ◽  
José Rafael Pedrajas ◽  
C. Alicia Padilla ◽  
José Antonio Bárcena
Keyword(s):  
Redox Regulation ◽  
Tumour Progression ◽  
Pentose Phosphate ◽  
Heme Biosynthesis ◽  
Label Free ◽  
Uroporphyrinogen Decarboxylase ◽  
Cell Extracts ◽  
Thiol Redox

Uroporphyrinogen decarboxylase (Hem12p) and transketolase (Tkl1p) are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP). The redox properties of both Hem12p and Tkl1p fromSaccharomyces cerevisiaewere investigated using proteomic techniques (SRM and label-free quantification) and biochemical assays in cell extracts andin vitrowith recombinant proteins. Thein vivoanalysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.

scholarly journals Combining tubercidin and cordycepin scaffolds results in highly active candidates to treat late-stage sleeping sickness

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Fabian Hulpia ◽  
Dorien Mabille ◽  
Gustavo D. Campagnaro ◽  
Gabriela Schumann ◽  
Louis Maes ◽  
...  
Keyword(s):  
Late Stage ◽  
De Novo ◽  
Treatment Options ◽  
Pool Analysis ◽  
Sleeping Sickness ◽  
Adenosine Kinase ◽  
Nucleoside Analogues ◽  
Nucleoside Transporter ◽  
African Trypanosomes

AbstractAfrican trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3’-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3’-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3’-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.

scholarly journals Inhibition of the pentose phosphate shunt by lead: a potential mechanism for hemolysis in lead poisoning

Blood ◽  
1984 ◽  
Vol 63 (3) ◽  
pp. 518-524 ◽  
Author(s):  
NA Lachant ◽  
A Tomoda ◽  
KR Tanaka
Keyword(s):  
Lead Poisoning ◽  
Lead Acetate ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Pentose Phosphate ◽  
Mechanism Of Inhibition ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Oxidant Sensitivity ◽  
Clinical Lead Poisoning ◽  
Pentose Phosphate Shunt

Abstract Recent investigations have disclosed a decrease in pentose phosphate shunt activity in hereditary pyrimidine 5′-nucleotidase deficiency. Clinical lead poisoning is associated with an acquired decrease in pyrimidine 5′-nucleotidase activity. The current investigations were undertaken (1) to determine if pentose shunt activity was decreased in erythrocytes exposed to lead, and (2) to compare the mechanism of inhibition to that seen in hereditary pyrimidine 5′-nucleotidase deficiency. Normal erythrocytes incubated with lead acetate in vitro demonstrated increased Heinz body formation, decreased reduced glutathione, a positive ascorbate cyanide test, and a reversible suppression of pentose shunt activity in the intact erythrocyte. Lead acetate added to normal red cell hemolysates markedly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase. The mean Kis of lead for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) for G6PD were 1.5 microM and 2.1 microM, respectively, which is within the range of intraerythrocytic lead concentrations found in clinical lead poisoning. Magnesium enhanced the ability of lead to inhibit G6PD. Thus, the shortened erythrocyte survival in lead poisoning appears to be due, in part, to increased oxidant sensitivity secondary to inhibition of G6PD and the pentose shunt. The mechanism of shunt inhibition is, in part, similar to that seen in hereditary pyrimidine 5′-nucleotidase deficiency.

scholarly journals Forced expression of miR-143 and -145 in cardiomyocytes induces cardiomyopathy with a reductive redox shift

2020 ◽  
Vol 25 (1) ◽  
Author(s):  
Kota Ogawa ◽  
Akiko Noda ◽  
Jun Ueda ◽  
Takehiro Ogata ◽  
Rumiko Matsuyama ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
De Novo ◽  
Mrna Level ◽  
Pentose Phosphate ◽  
Related Factor ◽  
Potent Inducer ◽  
Model Studies ◽  
Reductive Stress ◽  
Underlying Mechanisms

Abstract Background Animal model studies show that reductive stress is involved in cardiomyopathy and myopathy, but the exact physiological relevance remains unknown. In addition, the microRNAs miR-143 and miR-145 have been shown to be upregulated in cardiac diseases, but the underlying mechanisms associated with these regulators have yet to be explored. Methods We developed transgenic mouse lines expressing exogenous miR-143 and miR-145 under the control of the alpha-myosin heavy chain (αMHC) promoter/enhancer. Results The two transgenic lines showed dilated cardiomyopathy-like characteristics and early lethality with markedly increased expression of miR-143. The expression of hexokinase 2 (HK2), a cardioprotective gene that is a target of miR-143, was strongly suppressed in the transgenic hearts, but the in vitro HK activity and adenosine triphosphate (ATP) content were comparable to those observed in wild-type mice. In addition, transgenic complementation of HK2 expression did not reduce mortality rates. Although HK2 is crucial for the pentose phosphate pathway (PPP) and glycolysis, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was unexpectedly higher in the hearts of transgenic mice. The expression of gamma-glutamylcysteine synthetase heavy subunit (γ-GCSc) and the in vitro activity of glutathione reductase (GR) were also higher, suggesting that the recycling of GSH and its de novo biosynthesis were augmented in transgenic hearts. Furthermore, the expression levels of glucose-6-phosphate dehydrogenase (G6PD, a rate-limiting enzyme for the PPP) and p62/SQSTM1 (a potent inducer of glycolysis and glutathione production) were elevated, while p62/SQSTM1 was upregulated at the mRNA level rather than as a result of autophagy inhibition. Consistent with this observation, nuclear factor erythroid-2 related factor 2 (Nrf2), Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) were activated, all of which are known to induce p62/SQSTM1 expression. Conclusions Overexpression of miR-143 and miR-145 leads to a unique dilated cardiomyopathy phenotype with a reductive redox shift despite marked downregulation of HK2 expression. Reductive stress may be involved in a wider range of cardiomyopathies than previously thought.

343 DISTRIBUTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND IN VITRO MATURATION OF PORCINE OOCYTE - CUMULUS COMPLEXES FROM SMALL AND MEDIUM FOLLICLES

2007 ◽  
Vol 19 (1) ◽  
pp. 287
Author(s):  
Y. Ishida ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Critical Role ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Pentose Phosphate ◽  
G6pd Activity ◽  
Cresyl Blue ◽  
Key Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose metabolism through the pentose phosphate pathway (PPP) plays a critical role in meiotic maturation and fertilization. However, the relationship between the distribution of a PPP key enzyme, glucose-6-phosphate dehydrogenase (G6PD), in cumulus–oocyte complexes (COCs) and the in vitro maturation (IVM) of the oocytes is not clear. In the present study, we examined the distribution of G6PD, the morphological characteristics in OCCs derived from small (d2 mm in diameter) and medium (3 to 6 mm in diameter) follicles, and the rate of oocyte maturation. Porcine COCs were collected from small or medium follicles of slaughterhouse ovaries. The COCs were cultured in a maturation medium, BSA-free NCSU37 supplemented with 10% porcine follicular fluid, eCG, and hCG, for 20 h and then in the absence of hormones for 24 h. To determine the distribution of G6PD, the COCs were treated with 13 �M brilliant cresyl blue (BCB) in TL-HEPES-PVA for 90 min. Results from 3–6 replicates were analyzed by ANOVA and Duncan&apos;s multiple range test. The mean diameters for COCs collected from small follicles (136.7 &micro;m for the outer zona and 103.1 &micro;m for ooplasm) were significantly less than for those derived from medium follicles (164.1 &micro;m and 122.0 &micro;m, respectively). G6PD activity was detected in the cumulus cells for most of the COCs derived from medium follicles, but it was not significantly different from that of COCs derived from small follicles. In the second group of COCs, very little G6PD activity was found in both the cumulus cells and the oocytes (34.7 &plusmn; 11.5&percnt; and 18.0 &plusmn; 6.7&percnt; in COCS derived from small and medium follicles, respectively). After stimulation by eCG and hCG, the percentages of COCS in which G6PD activity was detected in the cumulus cells, but not in the oocytes, were 56.2 &plusmn; 23.8&percnt; and 72.9 &plusmn; 6.1&percnt; for small and medium follicles, respectively. The percentage of oocytes at the metaphase II stage (53&percnt; and 63.9&percnt; in oocytes from small and medium follicles, respectively) was higher for the COCs that showed higher G6PD activity in their cumulus cells. In conclusion, although no statistical differences were detected in the distribution of G6PD between COCs from small and medium follicles, due to a large variation, a higher percentage of mature oocytes seems to be the result of COCs where the G6PD activity is detected in the cumulus cells, but not in the oocyte, during IVM.

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