Key Enzyme Of Pentose Phosphate Pathway Research Articles

YAP ISGylation increases its stability and promotes its positive regulation on PPP by stimulating 6PGL transcription

Yes-associated protein (YAP) activation is crucial for tumor formation and development, and its stability is regulated by ubiquitination. ISGylation is a type of ubiquitination like post-translational modification, whereas whether YAP is ISGylated and how ISGylation influences YAP ubiquitination-related function remains uncovered. In addition, YAP can activate glucose metabolism by activating the hexosamine biosynthesis pathway (HBP) and glycolysis, and generate a large number of intermediates to promote tumor proliferation. However, whether YAP stimulates the pentose phosphate pathway (PPP), another tumor-promoting glucose metabolism pathway, and the relationship between this stimulation and ISGylation needs further investigation. Here, we found that YAP was ISGylated and this ISGylation inhibited YAP ubiquitination, proteasome degradation, interaction with-beta-transducin repeat containing E3 ubiquitin-protein ligase (βTrCP) to promote YAP stability. However, ISGylation-induced pro-YAP effects were abolished by YAP K497R (K, lysine; R, arginine) mutation, suggesting K497 could be the major YAP ISGylation site. In addition, YAP ISGylation promoted cell viability, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) tumor formation. YAP ISGylation also increased downstream genes transcription, including one of the key enzymes of PPP, 6-phosphogluconolactonase (6PGL). Mechanistically, YAP promoted 6PGL transcription by simultaneously recruiting SMAD family member 2 (SMAD2) and TEA domain transcription factor 4 (TEAD4) binding to the 6PGL promoter to activate PPP. In clinical lung adenocarcinoma (LUAD) specimens, we found that YAP ISGylation degree was positively associated with 6PGL mRNA level, especially in high glucose LUAD tissues compared to low glucose LUAD tissues. Collectively, this study suggested that YAP ISGylation is critical for maintaining its stability and further activation of PPP. Targeting ISGylated YAP might be a new choice for hyperglycemia cancer treatment.

Pharmacological HIF1 Inhibition Eliminates Downregulation of the Pentose Phosphate Pathway and Prevents Neuronal Apoptosis in Rat Hippocampus Caused by Severe Hypoxia.

The pentose phosphate pathway (PPP) of glucose metabolism in the brain serves as a primary source of NADPH which in turn plays a crucial role in multiple cellular processes, including maintenance of redox homeostasis and antioxidant defense. In our model of protective mild hypobaric hypoxia in rats (3MHH), an inverse correlation between hypoxia-inducible factor-1 (HIF1) activity and mRNA levels of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP, was observed. In the present study, it was demonstrated that severe hypobaric hypoxia (SH) induced short-term upregulation of HIF1 alpha-subunit (HIF1α) in the hippocampal CA1 subfield and decreased the activity of G6PD. The levels of NADPH were also reduced, promoting oxidative stress, triggering apoptosis, and neuronal loss. Injection of a HIF1 inhibitor (HIF1i), topotecan hydrochloride (5mg/kg, i.p.), before SH prevented the upregulation of HIF1α and normalized G6PD activity. In addition, HIF1i injection caused an increase in NADPH levels, normalization of total glutathione levels and of the cellular redox status as well as suppression of free-radical and apoptotic processes. These results demonstrate a new molecular mechanism of post-hypoxic cerebral pathology development which involves HIF1-dependent PPP depletion and support a recently suggested injurious role of HIF1 activation in the acute phase of cerebral hypoxia/ischemia. Application of PPP stimulators in early post-hypoxic/ischemic period might represent a promising neuroprotective strategy. Graphical abstract HIF1-dependent down-regulation of the pentose phosphate pathway contributes to the hypoxia-induced oxidative stress and neuronal apoptosis in the rat hippocampus.

Transketolase of Staphylococcus aureus in the Control of Master Regulators of Stress Response During Infection.

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. In the current study, we focused on one key PPP enzyme, transketolase (TKT). We showed that inactivation of the unique gene encoding TKT activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared with wild-type strain in early intracellular proliferation and in the ability to colonize kidneys. Transcriptional activity of the 2 master regulators sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription suggests that TKT-or a functional PPP-strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.

Transketolase (TKT) activity and nuclear localization promote hepatocellular carcinoma in a metabolic and a non-metabolic manner

BackgroundMetabolic reprogramming is one of the hallmarks of cancer cells. The pentose phosphate pathway (PPP), a branch of glycolysis, is an important metabolic pathway for the survival and biosynthesis of cancer cells. Transketolase (TKT) is a key enzyme in the non-oxidative phase of PPP. The mechanistic details of TKT in hepatocellular carcinoma (HCC) development remain unclear.MethodsTKT level and subcellular location were examined in HCC cell lines and tissue samples. We established the TKT overexpression and knocking-down stable cells in HCC cell lines. Proliferation, migration, viability and enzyme activity assays in vitro, tumor growth and metastasis assays in vivo were employed to test the effects of TKT on HCC development. GFP-tagged TKT truncations and mutants were used to locate the nuclear localization sequence (NLSs) of TKT. Cross-linking co-IP/MS was applied to identify the interaction proteins of nuclear TKT.ResultsWe showed that TKT increased the proliferation and migration of HCC cells, as well as the viability under oxidative stress in vitro and accelerated the growth and metastasis of HCC cells in vivo. We found as a key enzyme of PPP, TKT could promote the proliferation, cell cycle, migration and viability by regulating the metabolic flux. Moreover, it was firstly reported that unlike other key enzymes in PPP, TKT showed a strong nuclear localization in HCC cells. We found not only high TKT expression, but also its nuclear localization was a prediction for poor prognosis of HCC patients. We further identified the nuclear localization sequences (NLS) for TKT and demonstrated the NLS mutations decreased the pro-tumor function of TKT independent of the enzyme activity. Cross-linking Co-IP/MS showed that nuclear TKT interacted with kinases and transcriptional coregulators such as EGFR and MAPK3, which are associated with cell activation or stress response processes. EGF treatment significantly increased the viability and proliferation of HCC cells in the enzyme-inactivating mutation TKT-D155A overexpression cells but not in the NLS-D155A double mutant group, which could be blocked by EGFR inhibitor erlotinib treatment.ConclusionsOur research suggests that in addition to the metabolic manner, TKT can promote the development of HCC in a non-metabolic manner via its nuclear localization and EGFR pathway.

Adenosine A2B Receptor Controls Erythroid Lineage Commitment in Stress Erythropoiesis By Promoting Metabolic Reprogramming

Insufficient oxygen availability under stress conditions including hypoxia and anemia is a major stimulus for stress erythropoiesis. Adenosine is known to be induced under hypoxia and energy depletion. Increased adenosine signaling via its specific receptors regulates multiple cellular functions including anti-inflamation, anti-vascular leakage and vasodilation. However, its function in stress erythropoiesis and underlying mechanisms are enigmatic. Among four adenosine receptors, we report that adenosine A2B receptor (ADORA2B) is expressed at a significant higher level in megakaryocyte-erythroid progenitor (MEP) compared to common pluoripotent progenitors (CMP) or granulocyte-erythroid progenitor (GMP) in undifferentiated human CD34+. To determine the function role of ADORA2B in stress erythropoiesis, we generated erythroid Adora2b specific knockouts by crossing Adora2bf/f mice with EpoR-Cre+ mice. First, we demonstrated that EpoR specifically ablated ADORA2B gene only in MEP but not in CMP or GMP lineages. Next, we challenged EpoR-Cre+ mice (control) and Adora2bf/fEpoR-Cre+ mice (erythroid specific ablation of Adora2b genes) with hypoxia. We discovered that genetic deletion of ADORA2B at MEP stage blocked erythroid vs myeloid commitment under hypoxia-induced stress erythropoiesis. Further metabolic profiling revealed that ADORA2B activation regulated erythroid lineage commitment by promoting glucose uptake and erythroid metabolic reprogramming channelling glucose metabolism toward the pentose phosphate pathway (PPP) rather than glycolysis to generate more ribose phosphate as well as facilitate glutamine uptake to serve as a nitrogen donor for de novo nucleotide biosynthesis. Meanwhile, ADORA2B-stimulated glutaminolysis increased TCA cycle intermediates and thus increased energy production under stress erythropoiesis. We further demonstrated that ADORA2B on MEP is also important for erythroid commitment in response to PHZ-induced mouse model. Followup studies revealed that HIF-1a is induced in erythroid progenitors in a ADORA2B-dependent manner and ablation of HIF-1a only in MEP but not in CMP or GMP attenuated erythroid lineage commitment in both hypoxia-induced and anemia-induced stress erythropoiesis mouse models. Moreover, we showed that ADORA2B-triggered metabolic reprogramming depended on HIF-1a-preferentially upregulated gene expression of transporters for glucose and glutamine and key enyzmes of PPP and glutaminolysis over glycolytic enzymes. Similar to mouse studies, in cultured Epo-stimulated human CD34+ hematopoietic stem progenitor cells, enhancing ADORA2B signaling induced gene expression of the transporters for glucose and glutamine, key enzymes of PPP and glutaminolysis over glycolysis and thus controlled the commitment to erythrioid versus myeloid lineage and in turn promoted erythroid colony formation including BFU-E, CFU-E versus CFU-GM. Further studies showed that inhibition of HIF-1a by Chrysin significantly attenuated ADORA2B activation-induced upregulation of gene expression of the transporters of glucose and glutamine, metabolic enzymes and thus reduces erythroic commitment and BFU-E and CFU-E in Epo-stimualted CD34+ HPSCs. Overall, using multidisciplinary approaches including mouse genetics, metabolomics, isotopically labelled glucose and glutamine flux analysis, human CD34+ HPSCs and pharmacological studies, we provide both mouse and human evidence that ADORA2B is a missing cofactor controlling erythroid lineage commitment in stress erythropoiesis via HIF-1a-dependent upregulation of key genes to promote metabolic reprogramming. These findings add significant new insights to erythroid commitment and immediately provide new strategies for regulating stress erythropoiesis. DisclosuresNemkov:Omix Technologies inc: Equity Ownership.

Inhibiting 6-phosphogluconate dehydrogenase enhances chemotherapy efficacy in cervical cancer via AMPK-independent inhibition of RhoA and Rac1.

The oxidative pentose phosphate pathway (PPP) is essential for cancer metabolism and growth. However, the contribution of 6-phosphogluconate dehydrogenase (6PGD), a key enzyme of PPP, to cervical cancer development remains largely unknown. mRNA and protein levels of 6PGD were analyzed in cervical cancer cells and tissues derived from patients and compared to normal counterparts. Using cell culture system and xenograft mouse model, the functions of 6PGD in cervical cancer are determined and its molecular mechanism is analyzed. 6PGD inhibitor physcion and siRNA knockdown were used. In this work, we demonstrate that 6PGD is aberrantly upregulated and activated in cervical cancer cells and patient tissues compared to normal counterparts. Using different approaches and preclinical models, we show that 6PGD inhibition decreases growth and migration, and enhances chemosensitivity in cervical cancer. Mechanistically, inhibition of 6PGD activates AMP-activated protein kinase (AMPK) and decreases RhoA and Rac1 activities. AMPK depletion significantly reduces the effects of 6PGD inhibition in decreasing RhoA and Rac1 activities, growth and migration in cervical cancer cells. Our work is the first to demonstrate the aberrant expression of 6PGD and its predominant roles in cervical cancer cell growth and migration, via a AMPK-dependent activation. Our findings suggest 6PGD as a potential therapeutic target to enhance chemosensitivity in cervical cancer.

A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo

Pentose phosphate pathway (PPP) is a major glucose metabolism pathway, which has a fundamental role in cancer growth and metastasis. Even though PPP blockade has been pointed out as a very promising strategy against cancer, effective anti-PPP agents are not still available in the clinical setting. Here we demonstrate that the natural molecule polydatin inhibits glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of PPP. Polydatin blocks G6PD causing accumulation of reactive oxygen species and strong increase of endoplasmic reticulum stress. These effects are followed by cell cycle block in S phase, an about 50% of apoptosis, and 60% inhibition of invasion in vitro. Accordingly, in an orthotopic metastatic model of tongue cancer, 100 mg/kg polydatin induced an about 30% tumor size reduction with an about 80% inhibition of lymph node metastases and 50% reduction of lymph node size (p < 0.005). Polydatin is not toxic in animals up to a dose of 200 mg/kg and a phase II clinical trial shows that it is also well tolerated in humans (40 mg twice a day for 90 days). Thus, polydatin may be used as a reliable tool to limit human cancer growth and metastatic spread.

Association of transketolase polymorphisms with measures of polyneuropathy in patients with recently diagnosed diabetes.

Shunting of glycolytic intermediates into the pentose phosphate pathway has been suggested to protect from hyperglycaemia-induced microvascular damage. We hypothesized that genetic variability in the gene encoding transketolase, a key pentose phosphate pathway enzyme, contributes to early nerve dysfunction in recent-onset diabetes. In this cross-sectional study, we assessed nine single nucleotide polymorphisms (SNPs) in the transketolase gene, plasma methylglyoxal concentrations, and clinical and quantitative measures of peripheral nerve function in 165 type 1 and 373 type 2 diabetic patients with a diabetes duration up to 1year. The Total Symptom Score was associated with transketolase SNPs rs7648309, rs62255988, and rs7633966, while peroneal motor nerve conduction velocity (MNCV) correlated only with rs7648309 (P < 0.01). Cold thermal detection threshold (TDT) (foot) was associated with transketolase SNPs rs11130362 and rs7648309, while warm TDT (hand) correlated with rs62255988 and rs7648309 (P < 0.01). After Bonferroni correction, the correlations of transketolase SNP rs7648309 with Total Symptom Score and rs62255988 with warm TDT (hand) remained statistically significant. Among subgroups, men with type 2 diabetes showed the strongest associations. No associations were observed between each of the nine tagged transketolase SNPs and plasma methylglyoxal concentrations. The observed associations of genetic variation in transketolase enzyme with neuropathic symptoms and reduced thermal sensation in recent-onset diabetes suggest a role of pathways metabolizing glycolytic intermediates in early diabetic neuropathy. Copyright © 2016 John Wiley & Sons, Ltd.

Statistical analysis of pentose phosphate pathway genes from eubacteria and eukarya reveals translational selection as a major force in shaping codon usage pattern.

Comparative analysis of metabolic pathways among widely diverse species provides an excellent opportunity to extract information about the functional relation of organisms and pentose phosphate pathway exemplifies one such pathway. A comparative codon usage analysis of the pentose phosphate pathway genes of a diverse group of organisms representing different niches and the related factors affecting codon usage with special reference to the major forces influencing codon usage patterns was carried out. It was observed that organism specific codon usage bias percolates into vital metabolic pathway genes irrespective of their near universality. A clear distinction in the codon usage pattern of gram positive and gram negative bacteria, which is a major classification criterion for bacteria, in terms of pentose phosphate pathway was an important observation of this study. The codon utilization scheme in all the organisms indicates the presence of translation selection as a major force in shaping codon usage. Another key observation was the segregation of the H. sapiens genes as a separate cluster by correspondence analysis, which is primarily attributed to the different codon usage pattern in this genus along with its longer gene lengths. We have also analyzed the amino acid distribution comparison of transketolase protein primary structures among all the organisms and found that there is a certain degree of predictability in the composition profile except in A. fumigatus and H. sapiens, where few exceptions are prominent. In A. fumigatus, a human pathogen responsible for invasive aspergillosis, a significantly different codon usage pattern, which finally translated into its amino acid composition model portraying a unique profile in a key pentose phosphate pathway enzyme transketolase was observed.

Enhancement of cytidine production by coexpression of gnd, zwf, and prs genes in recombinant Escherichia coli CYT15

Cytidine is a precursor of several antiviral drugs. The pentose phosphate pathway (PPP) is primarily responsible for NADPH and 5-phospho-α-D-ribose 1-diphosphate as an important precursor of cytidine biosynthesis in Escherichia coli. To enhance cytidine production, we obtained the recombinant E. coli CYT15-gnd-prs-zwf that co-expressed the prs, zwf, and gnd genes encoding phosphoribosylpyrophosphate synthetase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (three key enzymes in PPP) respectively. In fermentation experiments, strain CYT15-gnd-prs-zwf produced 735mg cytidine/l using glucose as substrate, which was approx. 128% higher than the cytidine production by the parental strain (CYT15). Co-expression of zwf, gnd, and prs decreased growth (3.2%) slightly and increased glucose uptake (72%). This is the first study to report increased cytidine production by increasing metabolic flux through the PPP in E. coli.

The 6‐phosphogluconate Dehydrogenase Genes Are Responsive to Abiotic Stresses in Rice

AbstractGlucose‐6‐phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) and 6‐phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) are both key enzymes of the pentose phosphate pathway (PPP). The OsG6PDH1 and Os6PGDH1 genes encoding cytosolic G6PDH and cytosolic 6PGDH were isolated from rice (Oryza sativa L.). We have shown that Os6PGDH1 gene was up‐regulated by salt stress. Here we reported the isolation and characterization of Os6PGDH2 from rice, which encode the plastidic counterpart of 6PGDH. Genomic organization analysis indicated that OsG6PDH1 and OsG6PDH2 genes contain multiple introns, whereas two Os6PGDH1 and Os6PGDH2 genes have no introns in their translated regions. In a step towards understanding the functions of the pentose phosphate pathway in plants in response to various abiotic stresses, the expressions of four genes in the rice seedlings treated by drought, cold, high salinity and abscisic acid (ABA) were investigated. The results show that OsG6PDH1 and OsG6PDH2 are not markedly regulated by the abiotic stresses detected. However, the transcript levels of both Os6PGDH1 and Os6PGDH2 are up‐regulated in rice seedlings under drought, cold, high salinity and ABA treatments. Meanwhile, the enzyme activities of G6PDH and 6PGDH in the rice seedlings treated by various abiotic stresses were investigated. Like the mRNA expression patterns, G6PDH activity remains constant but the 6PGDH increases steadily during the treatments. Taken together, we suggest that the pentose phosphate pathway may play an important role in rice responses to abiotic stresses and the second key enzyme of PPP, 6PGDH, may function as a regulator controlling the efficiency of the pathway under abiotic stresses.(Handling editor: Kang Chong)

Fructose‐1,6‐diphosphate reduces cellular ROS level through up‐regulation of pentose phosphate pathway in BV‐2 microglia.

Fructose-1,6-disphosphate(F1,6DP), a glycolytic intermediate, is known to have protective activities against ischemia/reperfusion injury and septic shock induced by bacterial lipopolysacharide(LPS). In the previous report, we have shown that F1,6DP could reduce LPS-induced iNOS expression and NO production in BV-2 mouse microglia. In consideration of the importance of ROS contribution to LPS-stimulated iNOS expression and NO production in BV-2 cells, we hypothesized that F1,6DP's anti-inflammatory effects would be due to ROS inhibition by up-regulation of pentose phosphate pathway(PPP), which is one of the most important antioxidative mechanisms. Upon investigation, we confirmed that LPS-induced ROS production in BV-2 cells was significantly reduced by F1,6DP(10mM). We found that F1,6DP enhanced LPS-induced activity of glucose-6-phopsphate dehydrogenase(G6PD), the key enzyme of PPP, even in a comparatively short time in the presence or absence of glucose. Interestingly, F1,6DP further increased LPS-stimulated G6PD expression in the levels of mRNA and protein. In consideration of PPP as one of the most important ROS scavenging mechanism, these data suggest that F1,6DP can reduce the cellular ROS generation by increasing metabolic flux through PPP, that is, by providing the substrate for G6PD, and by its up-regulating effect on G6PD expression. The research was supported by the “GRRC” Project of Gyeonggi Provincial Government, Republic of Korea.

Molecular Ordering in HIV-induced Apoptosis: OXIDATIVE STRESS, ACTIVATION OF CASPASES, AND CELL SURVIVAL ARE REGULATED BY TRANSALDOLASE

Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.

Effect of adrenaline and phorbol myristate acetate or bacterial lipopolysaccharide on stimulation of pathways of macrophage glucose, glutamine and O2 metabolism. Evidence for cyclic AMP-dependent protein kinase mediated inhibition of glucose-6-phosphate dehydrogenase and activation of NADP+-dependent 'malic' enzyme.

Adrenaline has recently been shown to stimulate both glucose metabolism and H2O2 release by macrophages but the activity of the key pentose phosphate pathway enzyme, glucose-6-phosphate dehydrogenase (which generates the NADPH crucial for the reduction of molecular oxygen), was reduced under these conditions [Costa Rosa, Safi, Cury and Curi (1992) Biochem. Pharmacol. 44, 2235-2241]. We report here that adrenaline activates another NADPH-producing enzyme, NADP(+)-dependent 'malic' enzyme, while also inhibiting glucose-6-phosphate dehydrogenase, via cyclic AMP-dependent protein kinase (PKA) activation. Regulation of glucose-6-phosphate dehydrogenase activity by PKA has not been reported elsewhere. The sparing of some glucose from pentose phosphate pathway consumption may be important in the provision of glycerol 3-phosphate which in the macrophage may be required for new phospholipid synthesis. Glutamine oxidation was also stimulated by adrenaline thus providing increased substrate (malate) for NADP(+)-dependent 'malic' enzyme and therefore shifting some of the burden of NADPH production from glucose to glutamine metabolism. We also report a novel synergistic effect of adrenaline and some bacterial products and/or gamma-interferon in stimulating secretory and metabolic pathways in macrophages which may be a part of a larger network of signals that lead to enhanced macrophage activity.