Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway

After inhibition of cytochrome c oxidase by nitric oxide, astrocytes maintain energy production by upregulating glycolysis--a response which does not seem to be available to neurons. Here, we show that in astrocytes, after inhibition of respiration by nitric oxide, there is a rapid, cyclic GMP-independent increase in the activity of 6-phosphofructo-1-kinase (PFK1), a master regulator of glycolysis, and an increase in the concentration of its most powerful positive allosteric activator, fructose-2,6-bisphosphate (F2,6P(2)). In neurons, nitric oxide failed to alter F2,6P(2) concentration or PFK1 activity. This failure could be accounted for by the much lower amount of 6-phosphofructo-2-kinase (PFK2, the enzyme responsible for F2,6P(2) biosynthesis) in neurons. Indeed, full activation of neuronal PFK1 was achieved by adding cytosol from nitric oxide-treated astrocytes. Furthermore, using the small interfering RNA (siRNA) strategy, we demonstrated that the rapid activation of glycolysis by nitric oxide is dependent on phosphorylation of the energy charge-sensitive AMP-activated protein kinase, resulting in activation of PFK2 and protection of cells from apoptosis. Thus the virtual absence of PFK2 in neurons may explain their extreme sensitivity to energy depletion and degeneration.     

Glucuronoxylomannan from Cryptococcus neoformans down-regulates the enzyme 6-phosphofructo-1-kinase of macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.     

Nature of the rat brain 6-phosphofructo-1-kinase isozymes

The complex nature of the brain 6-phosphofructo-1-kinase isozymes was examined by elution with a discontinuous gradient from QAE (quaternary aminoethyl)-Sephadex. In the first wash (150 mM NaCl), where the rat muscle 6-phosphofructo-1-kinase isozyme (M4) eluted, about 40% of the total brain 6-phosphofructo-1-kinase activity washed through without exhibiting a sharp peak. In the second elution (300 mM NaCl), the remaining activity eluted in a sharp peak that preceded where the major rat liver 6-phosphofructo-1-kinase isozyme (L4) eluted. Enzyme activity in brain extracts or purified brain isozymes was titrated above 90% with M4 anti-IgG and 20% with L4 anti-IgG. A purification procedure was developed which resulted in a recovery of 70 to 80% of the original enzyme activity in brain 100,000 X g supernatant fluids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on slab gels and detection by silver staining indicated that three components were present with apparent molecular weights of 87,500, 85,000, and 80,000. The 85,000- and 80,000-dalton components corresponded to the subunits of M4 and L4, respectively. The third component (C type) was thought to be an actual subunit since it exhibited the highest molecular weight and was present in an exhaustively washed immunoprecipitate of the purified brain isozymes. From 10 different purifications of the brain enzyme, the subunit distributions of the liver, muscle, and C-type subunit were 1.4 +/- 0.2, 4.9 +/- 0.5, and 3.9 +/- 0.3, respectively. A comparison of the kinetic properties of purified liver, muscle, and brain isozymes clearly demonstrated that all three preparations had quantitatively different regulatory properties. All three subunits were present in different regions of the brain, and region-specific changes in total activity and the relative amounts of each subunit were observed. This study suggests that brain 6-phosphofructo-1-kinase is a complex mixture of homotetramers and hybrids which are composed of different amounts of the three subunits.     

Posttranslational modification of 6-phosphofructo-1-kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.     

Insulin-mediated regulation of heart atrial and ventricular 6-phosphofructo-1-kinase

Atrial 6-phosphofructo-1-kinase activity from the hearts of diabetic rats was decreased by 50%, but ventricular 6-phosphofructo-1-kinase activity was found not to be insulin-sensitive. This decrease in atrial 6-phosphofructo-1-kinase activity during diabetes was characterized by diminished levels of all three types of 6-phosphofructo-1-kinase subunits. As shown by immunological titration and column chromatography, the population of native 6-phosphofructo-1-kinase isozymes in the ventricles was not measurably affected during insulin deprivation. However, the atrial isozyme population in diabetic rat heart appeared to contain, on a relative basis, higher levels of the isozymic forms containing the L-type subunit. Measurement of the levels of this subunit indicated that in diabetic atria it was less affected than the other subunits. In the ventricles, insulin deficiency did not promote significant losses of fructose-2,6-P2; but, in diabetic rats, the atrial levels of this activator were decreased by 80% and subsequently restored by insulin treatment. These data suggest that any insulin-mediated effects on ventricular 6-phosphofructo-1-kinase activity and resultant effects on ventricular glycolysis do not appear to be exerted through changes in enzyme concentration, but probably through changes in modulators other than fructose-2,6-P2. In contrast to the ventricles, it appears that insulin exerts its effects on atrial 6-phosphofructo-1-kinase activity and, in part, influences atrial glycolysis through alteration of fructose-2,6-P2 levels, enzyme concentration, and isozymic content.     

Evolution of allosteric citrate binding sites on 6-phosphofructo-1-kinase

As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1) level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A) as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V) of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.     

citrate inhibition-resistant form of 6-phosphofructo-1-kinase from Aspergillus niger

Two forms of Aspergillus niger 6-phosphofructo-1-kinase (PFK1) have been described recently, the 85-kDa native enzyme and 49-kDa shorter fragment that is formed from the former by posttranslational modification. So far, kinetic characteristics have never been determined on the enzyme purified to near homogeneity. For the first time, kinetic parameters were determined for individual enzymes with respect to citrate inhibition. The native 85-kDa enzyme was found to be moderately inhibited by citrate, with the Ki value determined to be 1.5 mM, in the system with 5 mM Mg2+ ions, while increasing magnesium concentrations relieved the negative effect of citrate. An identical inhibition coefficient was determined also in the presence of ammonium ions, although ammonium acted as a strong activator of enzyme activity. On the other hand, the shorter fragment of PFK1 proved to be completely resistant to inhibition by citrate. Allosteric citrate binding sites were most probably lost after the truncation of the C-terminal part of the native protein, in which region some binding sites for inhibitor are known to be located. At near physiological conditions, characterized by low fructose-6-phosphate concentrations, a much higher efficiency of the shorter fragment was observed during an in vitro experiment. Since the enzyme became more susceptible to the positive control by specific ligands, while the negative control was lost after posttranslational modification, the shorter PFK1 fragment seems to be the enzyme most responsible for generating undisturbed metabolic flow through glycolysis in A. niger cells.     

Regulation of mammalian muscle type 6-phosphofructo-1-kinase and its implication for the control of the metabolism

Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism.     

Herpes simplex virus type 1 infection activates the Epstein-Barr virus replicative cycle via a CREB-dependent mechanism

The reactivation of latent Epstein-Barr virus (EBV) to lytic replication is important in pathogenesis and requires virus-host cellular interactions. However, the mechanism underlying the reactivation of EBV is not yet fully understood. In the present study, herpes simplex virus type 1 (HSV-1) was shown to induce the reactivation of latent EBV by triggering BZLF1 expression. The BZLF1 promoter (Zp) was not activated by HSV-1 essential glycoprotein-induced membrane fusion. Nevertheless, Zp was activated within 6 h post HSV-1 infection in virus entry-dependent and replication-independent manners. Using a panel of Zp deletion mutants, HSV-1 was shown to promote Zp through a cyclic adenosine monophosphate (cAMP) response element (CRE) located in ZII. The phosphorylated cAMP response element-binding (phos-CREB) protein, the cellular transactivator that binds to CRE, also increased after HSV-1 infection. By transient transfection, cAMP-dependent protein kinase A and HSV-1 US3 protein were found to be capable of activating Zp in CREB- and CRE-dependent manners. The relationship between EBV activation and HSV-1 infection revealed a possible common mechanism that stimulated latent EBV into lytic cycles in vivo.     

Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver.

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.     

Stereospecificity of the fructose 2,6-bisphosphate site of muscle 6-phosphofructo-1-kinase

We explored the stereospecificity of the fructose 2,6-bisphosphate site of rabbit muscle 6-phosphofructo-1-kinase by determination of the activation constants (Ka) of several structurally locked analogues of this potent metabolic regulator. Under the assay conditions used, the Ka of fructose 2,6-bisphosphate was 0.12 microM. The most effective synthetic analogues and their Ka's were 2,5-anhydro-D-mannitol 1,6-bisphosphate (2.9 microM), 1,4-butanediol bisphosphate (6.6 microM), hexitol 1,6-bisphosphate (40 microM), and 2,5-anhydro-D-glucitol 1,6-bisphosphate (47 microM). Ten other bisphosphate compounds were much less effective as activators of the enzyme. These findings indicate that, unlike its active site, this allosteric site of 6-phosphofructo-1-kinase does not require the furanose ring. Its basic requirement seems to be a compound with two phosphate groups approximately 9 A apart. Although the free hydroxy groups of the activator do not seem to be essential, their presence enhances appreciably the affinity of the ligand for this regulatory site.     

Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.

Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.     

C-terminal modification of 6-phosphofructo-1-kinase from Saccharomyces cerevisiae and its influence on enzyme structure and activity

Studies on limited proteolysis of 6-phosphofructo-1-kinase (Pfk-1) from Saccharomyces cerevisiae led to the suggestion that the C-terminal part of the alpha-subunit must contribute to the stabilisation of the octameric enzyme structure. To analyse the role of the C-terminus in vivo, the respective terminus of one of both types of subunits of Pfk-1 was sequentially truncated or extended. These modifications resulted in a decrease of the protein level of the mutated subunit and of the specific enzyme activity in the cell-free extract as well as in changes of the kinetic properties. Size exclusion HPLC demonstrated that the modified subunit is still able to assemble with the native counterpart generating an enzymatically active hetero-octamer. On the basis of our results we assume that the C-termini are important for the three-dimensional structure of the subunits determining their susceptibility to proteolysis and the ability to assembly to an active, oligomeric Pfk-1.     

Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress.

Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte.     

Herpes simplex virus 1 infection activates poly(ADP-ribose) polymerase and triggers the degradation of poly(ADP-ribose) glycohydrolase

Herpes simplex virus 1 infection triggers multiple changes in the metabolism of host cells, including a dramatic decrease in the levels of NAD(+). In addition to its role as a cofactor in reduction-oxidation reactions, NAD(+) is required for certain posttranslational modifications. Members of the poly(ADP-ribose) polymerase (PARP) family of enzymes are major consumers of NAD(+), which they utilize to form poly(ADP-ribose) (PAR) chains on protein substrates in response to DNA damage. PAR chains can subsequently be removed by the enzyme poly(ADP-ribose) glycohydrolase (PARG). We report here that the HSV-1 infection-induced drop in NAD(+) levels required viral DNA replication, was associated with an increase in protein poly(ADP-ribosyl)ation (PARylation), and was blocked by pharmacological inhibition of PARP-1/PARP-2 (PARP-1/2). Neither virus yield nor the cellular metabolic reprogramming observed during HSV-1 infection was altered by the rescue or further depletion of NAD(+) levels. Expression of the viral protein ICP0, which possesses E3 ubiquitin ligase activity, was both necessary and sufficient for the degradation of the 111-kDa PARG isoform. This work demonstrates that HSV-1 infection results in changes to NAD(+) metabolism by PARP-1/2 and PARG, and as PAR chain accumulation can induce caspase-independent apoptosis, we speculate that the decrease in PARG levels enhances the auto-PARylation-mediated inhibition of PARP, thereby avoiding premature death of the infected cell.     

Purification and kinetic characterization of 6-phosphofructo-1-kinase from the liver of gilthead sea bream (Sparus aurata)

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.     

Studies on the in vitro phosphorylation of 6-phosphofructo-1-kinase from rat liver

Rat hepatic 6-phosphofructo-1-kinase (ATP:d-fructose-6-phosphate 1-phosphotransferase) was purified to homogeneity and its phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase examined. Up to 4 mol of phosphate could be incorporated per mole of tetrameric enzyme, and the phosphate was incorporated into seryl residues. Phosphorylation did not alter the affinity of the enzyme for fructose 6-phosphate or fructose 2,6-bisphosphate. The rate of phosphorylation was enhanced by allosteric activators of 6-phosphofructo-1-kinase such as AMP and fructose 2,6-bisphosphate, and it was decreased by the allosteric inhibitors ATP and H⁺. The phosphopeptide region of the enzyme subunit was susceptible to limited proteolysis by trypsin. Removal of the phosphopeptide did not affect the subunit molecular weight nor the maximum activity of the enzyme, but it enhanced the apparent affinity of the enzyme for both fructose 6-phosphate and fructose 2,6-bisphosphate. It is concluded that the phosphopeptide region of the enzyme subunit is an important determinant of the affinity of the enzyme for its substrate as well as for the allosteric activator fructose 2,6-bisphosphate.