Fructose-1,6-biphosphate Research Articles

The imbroglio of the physiological Cra effector clarified at last.

Owing to its role in controlling carbon and energy metabolism, the catabolite repressor/activator protein Cra has been one of the most studied prokaryotic regulators of the last 30 years. Yet, a key mechanistic detail of its biological function - i.e. the nature of the metabolic effector that rules its DNA-binding ability - has remained controversial. Despite the high affinity of Cra for fructose-1-phosphate (F1P), the prevailing view claimed that fructose-1,6-biphosphate (FBP) was the key physiological effector. Building on such responsiveness to FBP, Cra was proposed to act as a glycolytic flux sensor and central regulator of critical metabolic transactions. At the same time, data raised on the Cra protein of Pseudomonas putida ruled out that FBP could be an effector - but instead suggested that it was the unintentional carrier of a small contamination by F1P, the actual signal molecule. While these data on the P. putida Cra were received with skepticism - if not dismissal - by the community of the time, the paper by (Bley-Folly et al, 2018) now demonstrates beyond any reasonable doubt that the one and only effector of E. coli Cra is F1P and that every action of FBP on this regulator can be traced to its systematic mix with the authentic binder.

Determination of intracellular metabolites concentrations in Escherichia coli under nutrition stress using liquid chromatography-tandem mass spectrometry

Cells show a timely and appropriate physiological adjustment on all levels of cellular activities in response to nutrient stress. However, the regulations for cells under different carbon/nitrogen influxes are poorly understood. To unveil a fully metabolic regulatory profile, we applied a mass spectrometry based “bottom-up” approach to investigate the metabolic response of Escherichia coli to nutrient stress. A novel cell sample preparation procedure was developed to decrease the variation and leakage of intracellular metabolites. Volatile ion-pair reagent tributylamine was used to improve the retention and selectivity of charged metabolites on a C18 reversed-phase column. The growth rate and intracellular concentrations of 12 central carbon metabolites were measured systematically under various carbon/nitrogen influxes by manipulating titratable promoters. Fructose-1,6-biphosphate (FBP) concentration as a sensor of carbon influx was positively correlated with the growth rate, whereas α-ketoglutarate (αkg), served as a coordinator of carbon and nitrogen flux showed different dependence on growth rate between carbon limitation and nitrogen limitation. By integrating different behaviors of the metabolites with knowledge from previous reports, a scenario of feedback control under carbon and nitrogen limitations was proposed. Our findings revealed the key role of αkg in the coordination of carbon and nitrogen utilization under nutrition stress and highlighted the great potential of mass spectrometry based approach in deciphering the complex metabolic network.

Investigation of the Gracilaria gracilis (Gracilariales, Rhodophyta) proteome response to nitrogen limitation.

Inorganic nitrogen has been identified as the majorgrowth-limiting nutritional factor affecting Gracilaria gracilis populations in South Africa. Although the physiological mechanisms implemented by G.gracilis for adaption to low nitrogen environments have beeninvestigated, little is known about the molecularmechanisms of these adaptions. This studyprovidesthe first investigation of G.gracilis proteome changesin response to nitrogen limitation and subsequent recovery. A differential proteomics approach employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry was used to investigate G.gracilis proteome changes in response to nitrogen limitation and recovery. The putative identity of 22 proteins that changed significantly (P<0.05) in abundance in response to nitrogen limitation and recovery was determined. The identified proteins function in a range of biological processes including glycolysis, photosynthesis, ATPsynthesis, galactose metabolism, protein-refolding and biosynthesis, nitrogen metabolism and cytoskeleton remodeling. The identity of fructose 1,6 biphosphate (FBP) aldolase was confirmed by western blot analysis and the decreased abundance of FBP aldolase observed with two-dimensional gel electrophoresis was validated by enzyme assays and western blots. The identification of key proteins and pathways involved in the G.gracilis nitrogen stress response provide a better understanding of G.gracilis proteome responses to varying degrees of nitrogen limitation and is the first step in the identification of biomarkers for monitoring the nitrogen status of cultivated G.gracilis populations.

Steady-state hydrogen peroxide induces glycolysis in Staphylococcus aureus and Pseudomonas aeruginosa.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from human pathogens Staphylococcus aureus and Pseudomonas aeruginosa can be readily inhibited by reactive oxygen species (ROS)-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment with H2O2 rapidly induces glycolysis and the pentose phosphate pathway (PPP). We conducted transcriptome sequencing (RNA-seq) analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which revealed profound transcriptional changes, including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces metabolic levels of glycolysis and the PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Both GapR and HexR may directly sense oxidative stresses, such as menadione.

Dual regulation of mast cell degranulation through IgE receptor-mediated modulation of M2-type pyruvate kinase

It was reported that mast cell degranulation is inversely related to the enzymatic activity of M₂-type pyruvate kinase (M₂PK). This study shows that activation of high-affinity IgE receptor (FcεRI) evokes a sequential dual regulation of M₂PK, i.e., an immediate decrement followed by slow phase increment of enzymatic activities. Changes in the activities of M₂PK and mast cell degranulation showed similar time course after antigenic stimulation of FcεRI. The immediate inhibition of M₂PK involved tyrosine phosphorylation, and subsequently led to a cellular accumulation of glycolytic intermediates, including fructose 1,6-biphosphate (FBP), a feedforward activator of M₂PK. As the cellular levels of FBP were increased, both the enzymatic acitivity of M₂PK and mast cell degranulation slowly returned to near basal levels. A-Raf, when exogenously introduced into RBL-2H3 cells, phosphorylated M₂PK on the serine residues, elevated enzyme activities of M₂PK, and resulted in the inhibition of degranulation. These results suggest that dual regulation of M₂PK which involves the phosphorylation of M₂PK and accumulation of a feedforward activator of M₂PK plays important roles in the control of mast cell degranulation.

Relationship Between Ischemia/Reperfusion Injury and the Stimulus of Fibrogenesis in an Experimental Model: Comparison Among Different Preservation Solutions

Background and Aims Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis. Materials and Methods Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor. Results AST was significantly lower during cold storage with UW than with the older media ( P = .001); ALT was lower in the FBP group ( P = .023) and LDH was lower in the FBP and SNAC groups ( P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups ( P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 ( P = .004). Renin levels were significantly lower in the FBP group ( P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor. Conclusion In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Fructose 1-6 Bisphosphate Versus University of Wisconsin Solution for Rat Liver Preservation: Does FBP Prevent Early Mitochondrial Injury?

Background Fructose 1,6-biphosphate (FBP) has been shown to exert therapeutic effects in models of ischemia-reperfusion in organs other than the liver. This study compared FBP and University of Wisconsin (UW) solution during cold storage and reperfusion, among mitochondria of adult male Wistar rat livers. Methods Adult male Wistar rats were assigned to two groups according to the preservation solution used; UW or FBP Aspartate transaminase (AST), alanine transferase (ALT); and lactic dehydrogenase (LDH) were measured in samples of the storage solution obtained at 2, 4 and 6 hours of preservation. After 6 hours of cold storage, we reperfused the liver, taking blood samples to measure AST, ALT, LDH, and throbarbituric acid reactive substances (TBARS). Hepatic fragments were processed for histologic analysis; for determinations of TBARS, catalase, and nitric oxide as well as for mitochondrial evaluation by infrared spectroscopy. Results During cold preservation, levels of AST and LDH in the storage solution were lower among the FBP group, but after reperfusion, serum levels of AST, ALT, and LDH were higher in this group, as was catalase activity. TBARS and nitric oxide were comparable between the groups. In the UW group there was a higher amide I/amide II ratio than in the FBP group, suggesting an abnormal protein structure of the mitochondrial membrane. No signs of preservation injury were observed in any liver biopsy, but sinusoidal congestion was present in livers preserved with FBP. Conclusion FBP showed a protective effect for preservation during cold storage seeming to protect the mitochondrial membrane although it did not prevent reperfusion injury.

CcpA‐mediated repression of Clostridium difficile toxin gene expression

The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile.

Protective effects of exogenous fructose-1,6-biphosphate during small bowel transplantation in rats

Background We assessed the effect of adding exogenous fructose-1,6-biphosphate (F16BP) to the preservation solution (University of Wisconsin storage solution) used during an experimental procedure of small bowel transplantation in rats. Methods We studied levels of the nucleotides hypoxanthine/xanthine and adenosine in tissue after cold ischemia, as well as histologic changes and associated deleterious processes such as bacterial translocation produced by the reperfusion associated with the transplantation. Results The groups of rats treated with F16BP showed the lowest levels of hypoxanthine/xanthine and uric acid, the highest levels of adenosine, and the lowest levels of histologic damage and lactate dehydrogenase release to the bloodstream. Consumption of intestinal hypoxanthine during reperfusion was lowest in the groups treated with F16BP, as was the incidence of bacterial translocation. Conclusions This study shows a protective effect of exogenous F16BP added to University of Wisconsin solution during experimental intestinal transplantation in rats. This protective effect, reflected by decreased intestinal damage and bacterial translocation, was related to a decrease in adenosine triphosphate depletion during cold ischemia before intestinal transplantation, and to the reduced availability of xanthine oxidase substrates for free radical generation during reperfusion.

Regulation of the central glycolytic genes in Bacillus subtilis: binding of the repressor CggR to its single DNA target sequence is modulated by fructose-1,6-bisphosphate.

Glycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA, pgk, tpi, pgm and eno. They are transcribed in a hexacistronic operon together with cggR, the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN6TGTCN4CGGGACN6TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G+C, Gram-positive bacterial species.

Fructose-1,6-biphosphate prevents excitotoxic neuronal cell death in the neonatal mouse brain

The excitotoxic cascade may represent an important pathway leading to brain damage and cerebral palsy. Brain lesions induced in newborn mice by ibotenate (acting on N-methyl- d-aspartate receptors) and by S-bromowillardiine (acting on alpha-3-amino-hydroxy-5-methyl-4-isoxazole propionic acid and kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage. Fructose 1,6-biphosphate (FBP) is a high-energy glycolytic pathway intermediate which, in therapeutic doses, is non-toxic and neuroprotective in hypoxic–ischemic models of brain injury. Mechanisms of action include modulation of intracellular calcium through phospholipase C (PLC) activation. The goal of this study was to determine the neuroprotective effects of FBP in a mouse model of neonatal excitotoxic brain injury. Mice that received intraperitoneal FBP had a significant reduction in size of ibotenate-induced (80% reduction) or S-bromowillardiine-induced (40% reduction) cortical plate lesions when compared with control animals. Studies of fragmented DNA and cleaved caspase 3 confirmed the survival promoting effects of FBP. FBP had no detectable effect on excitotoxic white matter lesions. The effects of FBP were antagonized by co-administration of PLC, protein kinase C or mitogen-associated protein kinase inhibitors but not by protein kinase A inhibitor. A moderate, transient cooling of pups immediately after the insult extended the therapeutic window for FBP, as FBP administered 24 h after ibotenate was still significantly neuroprotective in these pups. This data extends the neuroprotective profile of FBP in neonatal brain injury and identifies gray matter lesions involving N-methyl- d-aspartate receptors as a major target for this promising drug.

A modelling study of feedforward activation in human erythrocyte glycolysis

Though feedforward activation (FA) is a little known principle of control in metabolic networks, there is one well-known example; namely, the activation of pyruvate kinase (PK) by fructose-1,6-biphosphate (FBP) in glycolysis. The effects of this activation on the enzyme’s kinetics are well characterised, but its possible role in glycolytic control has not been determined, and, experimentally, there is as yet no direct way of modifying the enzyme to remove just the FBP activation without affecting other aspects of the enzyme’s kinetics. Given this limitation, we used a detailed numerical simulation of human erythrocyte glycolysis to simulate the effects of selective removal of the activation of PK by FBP on steady-state metabolite concentrations and on the dynamic response of glycolytic flux to a sudden increase of the cell’s demand for ATP. Our modelling results predict that in the absence of FA steady-state levels of metabolites within the activation loop, i.e. from FBP to phosphoenolpyruvate, would be four- to thirteen-fold higher than normal, whereas levels of ATP and metabolites outside the loop, i.e. glucose-6-phosphate, fructose-6-phosphate and pyruvate, would be lower than normal. Existing clinical evidence in a patient with haemolytic anaemia, correlated with a lack of activation of PK by FBP (Paglia D.E., Valentine W.N., Holbrook C.T., Brockway R., Blood (1983) 62 972–979), is consistent with this prediction. In response to changing demand for ATP, the model predicts that the corresponding change of glycolytic flux would entail changes of metabolite concentrations in the absence of FA, but that in its presence the levels of metabolites within the activation loop remain essentially unperturbed. Thus, our results suggest that by stabilising metabolite pools in the face of variable glycolytic flux, FA may serve to avoid perturbations of the oxygen affinity of haemoglobin (sensitive to the levels of 2,3-phosphoglycerate) and of cell osmolality that would otherwise occur during variations in the cell’s demand for ATP. In addition, by significantly raising the steady-state setpoint of intermediate metabolite pools, the productivity index (ratio of glycolytic flux to total metabolites in the pathway) of glycolysis would fall almost four-fold in the absence of forward activation.

Fructose-1,6-biphosphate in rat intestinal preconditioning: involvement of nitric oxide

BACKGROUND AND AIMSInhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by nitric oxide (NO) in intestinal preconditioning could modify the rate of formation of glycolytic intermediates. Fructose-1,6-biphosphate (F16BP) is a glycolytic intermediate that...

Energy metabolism in hypoxic astrocytes: protective mechanism of fructose-1,6-bisphosphate.

The protective effects of fructose-1,6-biphosphate (FBP) during hypoxia/ischemia are thought to result from uptake and utilization of FBP as a substrate for glycolysis or from stimulation of glucose metabolism. To test these hypotheses, we measured CO2 and lactate production from [6-14C]glucose, [1-14C]glucose, and [U-14C]FBP in normoxic and hypoxic cultured astrocytes with and without FBP present. FBP had little effect on CO2 production by glycolysis, but increased CO2 production by the pentose phosphate pathway. Labeled FBP produced very small amounts of CO2. Lactate production from [1-, and 6-14C]glucose increased similarly during hypoxic hypoxia; the increase was independent of added FBP. Labeled lactate from [U-14C]FBP was minimal. We conclude that exogenous FBP is not used by astrocytes as a substrate for glycolysis and that FBP alters glucose metabolism.

Molecular cloning, nucleotide sequence and fine‐structural analysis of the Corynebacterium glutamicum fda gene: structural comparison of C. glutamicum fructose‐1,6‐biphosphate aldolase to class I and class II aldolases

The Corynebacterium glutamicum fda gene encoding fructose-1,6-biphosphate (FBP) aldolase has been isolated by complementation of an Escherichia coli mutant. The nucleotide sequence of a 3371 bp chromosomal fragment containing the C. glutamicum fda gene was determined. The N-terminal amino acid sequence of C. glutamicum FBP aldolase identified the correct initiation site for the fda gene, and a molecular weight of 37,092 was predicted for the fda polypeptide. S1 nuclease mapping identified the transcriptional start site, and Northern hybridization analysis indicated that the fda gene encodes a single 1.3 kb transcript. The primary structure of C. glutamicum FBP aldolase shows strong homology to class II FBP aldolases. Conservation of primary structure was observed between class I and class II aldolases, but several residues essential for catalytic activity in class I aldolases were absent from class II aldolases.

Pyruvate kinase of mussel foot ( Mytilus edulis L.)—Kinetic properties. Effectors

1. 1. Pyruvate kinase of mussel foot shows positive cooperativity with respect to phosphoenolpyruvate as substrate. 2. 2. The enzyme is subject to allosteric regulation by fructose-1,6-biphosphate (FbP), alanine and adenosine triphosphate (ATP). 3. 3. The activation of the enzyme by fructose-1,6-biphosphate as well as its inhibition by alanine and ATP are pH-dependent.

Starch formation in isolated pea chloroplasts

Incorporation of 14CO 2 into an insoluble product (starch) by isolated pea ( Pisum sativum) chloroplasts in relation to adenylates, inorganic pyrophosphate (PPi) and other compounds has been investigated in some detail. Formation of starch was favoured in the presence of PPi and catalytic amounts of adenylates (ADP or ATP) while 3-phosphoglyceric acid (PGA), ribose-5-phosphate (R5P), or fructose 1,6-biphosphate (F-B-P) had zero or very little effect. it is suggested that the effect of adenylates and PPi appears as a consequence of more rapid exchange of these compounds across the chloroplast envelope in pea than that observed in spinach chloroplasts.