TAp73 is a structural homologue of the pre-eminent tumour suppressor p53. However, unlike p53, TAp73 is rarely mutated, and instead is frequently overexpressed in human tumours. It remains unclear whether TAp73 affords an advantage to tumour cells and if so, what the underlying mechanism is. Here we show that TAp73 supports the proliferation of human and mouse tumour cells. TAp73 activates the expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). By stimulating G6PD, TAp73 increases PPP flux and directs glucose to the production of NADPH and ribose, for the synthesis of macromolecules and detoxification of reactive oxygen species (ROS). The growth defect of TAp73-deficient cells can be rescued by either enforced G6PD expression or the presence of nucleosides plus an ROS scavenger. These findings establish a critical role for TAp73 in regulating metabolism, and connect TAp73 and the PPP to oncogenic cell growth.
The circadian clock is a ubiquitous timekeeping system that organizes the behavior and physiology of organisms over the day and night. Current models rely on transcriptional networks that coordinate circadian gene expression of thousands of transcripts. However, recent studies have uncovered phylogenetically conserved redox rhythms that can occur independently of transcriptional cycles. Here we identify the pentose phosphate pathway (PPP), a critical source of the redox cofactor NADPH, as an important regulator of redox and transcriptional oscillations. Our results show that genetic and pharmacological inhibition of the PPP prolongs the period of circadian rhythms in human cells, mouse tissues, and fruit flies. These metabolic manipulations also cause a remodeling of circadian gene expression programs that involves the circadian transcription factors BMAL1 and CLOCK, and the redox-sensitive transcription factor NRF2. Thus, the PPP regulates circadian rhythms via NADPH metabolism, suggesting a pivotal role for NADPH availability in circadian timekeeping.
Ataxia telangiectasia (A‐T) is a human disease caused by ATM deficiency characterized among other symptoms by radiosensitivity, cancer, sterility, immunodeficiency and neurological defects. ATM controls several aspects of cell cycle and promotes repair of double strand breaks (DSBs). This probably accounts for most of A‐T clinical manifestations. However, an impaired response to reactive oxygen species (ROS) might also contribute to A‐T pathogenesis. Here, we show that ATM promotes an anti‐oxidant response by regulating the pentose phosphate pathway (PPP). ATM activation induces glucose‐6‐phosphate dehydrogenase (G6PD) activity, the limiting enzyme of the PPP responsible for the production of NADPH, an essential anti‐oxidant cofactor. ATM promotes Hsp27 phosphorylation and binding to G6PD, stimulating its activity. We also show that ATM‐dependent PPP stimulation increases nucleotide production and that G6PD‐deficient cells are impaired for DSB repair. These data suggest that ATM protects cells from ROS accumulation by stimulating NADPH production and promoting the synthesis of nucleotides required for the repair of DSBs. The DNA damage‐induced ATM kinase is linked to the metabolic pentose phosphate pathway, thus boosting biosynthesis of nucleotide precursors required for DNA repair and stimulating generation of the anti‐oxidant NADPH, which may explain neurological defects of ataxia telangiectasia patients lacking ATM function.
2,3‐ 13 C 2 ]glucose is a novel and specific tracer of the pentose phosphate pathway (PPP). 13 C NMR analysis of lactate after the administration of 2,3‐ 13 C 2 ]glucose informs PPP activity and glycolysis because 1,2‐ 13 C 2 ]lactate arises from glycolysis and 2,3‐ 13 C 2 ]lactate arises exclusively through the PPP. In rats receiving 2,3‐ 13 C 2 ]glucose, the PPP was more active in the fed versus fasted state in most organs. Both the PPP and glycolysis were substantially increased in hepatoma compared to liver. 2,3‐ 13 C 2 ]glucose and 13 C NMR simplify assessment of the PPP. The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as 1,2‐ 13 C 2 ]glucose rely on detection of 13 C in lactate and require assumptions to correct natural 13 C abundance. Here, we introduce a novel and specific tracer of the PPP, 2,3‐ 13 C 2 ]glucose. 13 C NMR analysis of the resulting isotopomers is informative because 1,2‐ 13 C 2 ]lactate arises from glycolysis and 2,3‐ 13 C 2 ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving 2,3‐ 13 C 2 ]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, 2,3‐ 13 C 2 ]glucose and 13 C NMR simplify assessment of the PPP.
The pentose phosphate pathway (PPP) plays a critical role in macromolecule biosynthesis and maintaining cellular redox homoeostasis in rapidly proliferating cells. Upregulation of the PPP has been shown in several types of cancer. However, how the PPP is regulated to confer a selective growth advantage on cancer cells is not well understood. Here we show that glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, is dynamically modified with an O-linked beta-N-acetylglucosamine sugar in response to hypoxia. Glycosylation activates G6PD activity and increases glucose flux through the PPP, thereby providing precursors for nucleotide and lipid biosynthesis, and reducing equivalents for antioxidant defense. Blocking glycosylation of G6PD reduces cancer cell proliferation in vitro and impairs tumor growth in vivo. Importantly, G6PD glycosylation is increased in human lung cancers. Our findings reveal a mechanistic understanding of how O-glycosylation directly regulates the PPP to confer a selective growth advantage to tumours.
Haem oxygenase (HO)-1/carbon monoxide (CO) protects cancer cells from oxidative stress, but the gas-responsive signalling mechanisms remain unknown. Here we show using metabolomics that CO-sensitive methylation of PFKFB3, an enzyme producing fructose 2,6-bisphosphate (F-2,6-BP), serves as a switch to activate phosphofructokinase-1, a rate-limiting glycolytic enzyme. In human leukaemia U937 cells, PFKFB3 is asymmetrically di-methylated at R131 and R134 through modification by protein arginine methyltransferase 1. HO-1 induction or CO results in reduced methylation of PFKFB3 in varied cancer cells to suppress F-2,6-BP, shifting glucose utilization from glycolysis toward the pentose phosphate pathway. Loss of PFKFB3 methylation depends on the inhibitory effects of CO on haem-containing cystathionine beta-synthase (CBS). CBS modulates remethylation metabolism, and increases NADPH to supply reduced glutathione, protecting cells from oxidative stress and anti-cancer reagents. Once the methylation of PFKFB3 is reduced, the protein undergoes polyubiquitination and is degraded in the proteasome. These results suggest that the CO/ CBS-dependent regulation of PFKFB3 methylation determines directional glucose utilization to ensure resistance against oxidative stress for cancer cell survival.
Oleaginous microbes represent an attractive means of converting a diverse range of feedstocks into oils that can be transesterified to biodiesel. However, the mechanism of lipid overproduction in these organisms is incompletely understood, hindering the development of strategies for engineering superior biocatalysts for "single-cell oil" production. In particular, it is unclear which pathways are used to generate the large quantities of NADPH required for overproduction of the highly reduced fatty acid species. While early studies implicated malic enzyme as having a key role in production of lipogenic NADPH in oleaginous fungi, several recent reports have cast doubts as to whether malic enzyme may contribute to production of lipogenic NADPH in the model oleaginous yeast Yarrowia lipolytica. To address this problem we have used C-13-Metabolic Flux Analysis to estimate the metabolic flux distributions during lipid accumulation in two Y. lipolytica strains; a control strain and a previously published engineered strain capable of producing lipids at roughly twice the yield. We observe a dramatic rearrangement of the metabolic flux distribution in the engineered strain which supports lipid overproduction. The NADPH-producing flux through the oxidative Pentose Phosphate Pathway is approximately doubled in the engineered strain in response to the roughly two-fold increase in fatty acid biosynthesis, while the flux through malic enzyme does not differ significantly between the two strains. Moreover, the estimated rate of NADPH production in the oxidative Pentose Phosphate Pathway is in good agreement with the estimated rate of NADPH consumption in fatty acid biosynthesis in both strains. These results suggest the oxidative Pentose Phosphate Pathway is the primary source of lipogenic NADPH in Y. Iipolytica. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Integrity of human skin is endangered by exposure to UV irradiation and chemical stressors, which can provoke a toxic production of reactive oxygen species (ROS) and oxidative damage. Since oxidation of proteins and metabolites occurs virtually instantaneously, immediate cellular countermeasures are pivotal to mitigate the negative implications of acute oxidative stress. We investigated the short-term metabolic response in human skin fibroblasts and keratinocytes to H2O2 and UV exposure. In time-resolved metabolomics experiments, we observed that within seconds after stress induction, glucose catabolism is routed to the oxidative pentose phosphate pathway (PPP) and nucleotide synthesis independent of previously postulated blocks in glycolysis (i. e., of GAPDH or PKM2). Through ultra-short C-13 labeling experiments, we provide evidence for multiple cycling of carbon backbones in the oxidative PPP, potentially maximizing NADPH reduction. The identified metabolic rerouting in oxidative and non-oxidative PPP has important physiological roles in stabilization of the redox balance and ROS clearance.
The reaction sequences of central metabolism, glycolysis and the pentose phosphate pathway provide essential precursors for nucleic acids, amino acids and lipids. However, their evolutionary origins are not yet understood. Here, we provide evidence that their structure could have been fundamentally shaped by the general chemical environments in earth's earliest oceans. We reconstructed potential scenarios for oceans of the prebiotic Archean based on the composition of early sediments. We report that the resultant reaction milieu catalyses the interconversion of metabolites that in modern organisms constitute glycolysis and the pentose phosphate pathway. The 29 observed reactions include the formation and/or interconversion of glucose, pyruvate, the nucleic acid precursor ribose‐5‐phosphate and the amino acid precursor erythrose‐4‐phosphate, antedating reactions sequences similar to that used by the metabolic pathways. Moreover, the Archean ocean mimetic increased the stability of the phosphorylated intermediates and accelerated the rate of intermediate reactions and pyruvate production. The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. The reactions were particularly sensitive to ferrous iron Fe( II ), which is understood to have had high concentrations in the Archean oceans. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron‐rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world. Modern cells possess a sophisticated metabolic network, but its origins remain largely unknown. Reconstructing scenarios of the Archean ocean, we observe chemical reactions reminiscent of modern metabolic sequences, indicating that metabolism could be of prebiotic origin. Metabolites of glycolysis and the pentose phosphate undergo non‐enzymatic interconversion reactions. Metal ions abundantly found in sediments of the prebiotic Archean ocean, predominantly F e( II ), catalyse additional sugar phosphate interconversion reactions. Reactions catalysed by the Archean ocean metals resemble enzyme‐catalysed reactions found in the modern glycolytic and pentose phosphate pathways. The observed reactions are accelerated and gain specificity in conditions simulating the Archean ocean. Modern cells possess a sophisticated metabolic network, but its origins remain largely unknown. Reconstructing scenarios of the Archean ocean, we observe chemical reactions reminiscent of modern metabolic sequences, indicating that metabolism could be of prebiotic origin.
The efficient use of hemicellulose in the plant cell wall is critical for the economic conversion of plant biomass to renewable fuels and chemicals. Previously, the yeast Saccharomyces cerevisiae has been engineered to convert the hemicellulose-derived pentose sugars xylose and arabinose to D-xylulose-5-phosphate for conversion via the pentose phosphate pathway (PPP). However, efficient pentose utilization requires PPP optimization and may interfere with its roles in NADPH and pentose production. Here, we developed an alternative xylose utilization pathway that largely bypasses the PPP. In the new pathway, D-xylulose is converted to D-xylulose-1-phosphate, a novel metabolite to S. cerevisiae, which is then cleaved to glycolaldehyde and dihydroxyacetone phosphate. This synthetic pathway served as a platform for the biosynthesis of ethanol and ethylene glycol. The use of D-xylulose-1-phosphate as an entry point for xylose metabolism opens the way for optimizing chemical conversion of pentose sugars in S. cerevisiae in a modular fashion.