Daniel Amador-Noguez

Evidence for an Alternative Glycolytic Pathway in Rapidly Proliferating Cells

Glucose Metabolism Revisited Cancer cells are revved up to reproduce rapidly and typically consume glucose rapidly by glycolysis. Why then do cancer cells express an isoform of a rate-limiting enzyme in glycolysis, pyruvate kinase M2, which has decreased activity? Vander Heiden et al. (p. 1492) propose that consequent accumulation of phosphoenolpyruvate, with the help of an enzymatic activity that remains to be characterized, can lead to phosphate transfer to phosphoglycerate mutase, another glycolytic enzyme, providing the cell with a different way to make pyruvate. This may allow cancer cells to produce pyruvate without generating excess adenosine triphosphate, which can act through feedback to inhibit glycolyis. Characterization of cancer cell metabolism provides evidence for a previously uncharacterized metabolic pathway. Proliferating cells, including cancer cells, require altered metabolism to efficiently incorporate nutrients such as glucose into biomass. The M2 isoform of pyruvate kinase (PKM2) promotes the metabolism of glucose by aerobic glycolysis and contributes to anabolic metabolism. Paradoxically, decreased pyruvate kinase enzyme activity accompanies the expression of PKM2 in rapidly dividing cancer cells and tissues. We demonstrate that phosphoenolpyruvate (PEP), the substrate for pyruvate kinase in cells, can act as a phosphate donor in mammalian cells because PEP participates in the phosphorylation of the glycolytic enzyme phosphoglycerate mutase (PGAM1) in PKM2-expressing cells. We used mass spectrometry to show that the phosphate from PEP is transferred to the catalytic histidine (His11) on human PGAM1. This reaction occurred at physiological concentrations of PEP and produced pyruvate in the absence of PKM2 activity. The presence of histidine-phosphorylated PGAM1 correlated with the expression of PKM2 in cancer cell lines and tumor tissues. Thus, decreased pyruvate kinase activity in PKM2-expressing cells allows PEP-dependent histidine phosphorylation of PGAM1 and may provide an alternate glycolytic pathway that decouples adenosine triphosphate production from PEP-mediated phosphotransfer, allowing for the high rate of glycolysis to support the anabolic metabolism observed in many proliferating cells.

Systems-Level Metabolic Flux Profiling Elucidates a Complete, Bifurcated Tricarboxylic Acid Cycle in Clostridium acetobutylicum

Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/alpha-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organisms one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

Alterations in xenobiotic metabolism in the long‐lived Little mice

Our previous microarray expression analysis of the long‐lived Little mice (Ghrhrlit/lit) showed a concerted up‐regulation of xenobiotic detoxification genes. Here, we show that this up‐regulation is associated with a potent increase in resistance against the adverse effects of a variety of xenobiotics, including the hepatotoxins acetaminophen and bromobenzene and the paralyzing agent zoxazolamine. The classic xenobiotic receptors Car (Constitutive Androstane Receptor) and Pxr (Pregnane X Receptor) are considered key regulators of xenobiotic metabolism. Using double and triple knockout/mutant mouse models we found, however, that Car and Pxr are not required for the up‐regulation of xenobiotic genes in Little mice. Our results suggest instead that bile acids and the primary bile acid receptor Fxr (farnesoid X receptor) are likely mediators of the up‐regulation of xenobiotic detoxification genes in Little mice. Bile acid levels are considerably elevated in the bile, serum, and liver of Little mice. We found that treatment of wild‐type animals with cholic acid, one of the major bile acids elevated in Little mice, mimics in large part the up‐regulation of xenobiotic detoxification genes observed in Little mice. Additionally, the loss of Fxr had a major effect on the expression of the xenobiotic detoxification genes up‐regulated in Little mice. A large fraction of these genes lost or decreased their high expression levels in double mutant mice for Fxr and Ghrhr. The alterations in xenobiotic metabolism in Little mice constitute a form of increased stress resistance and may contribute to the extended longevity of these mice.

Gene expression profile of long-lived Ames dwarf mice and Little mice.

Ames dwarf mice (Prop1df/df) and Little mice (Ghrhrlit/lit) are used as models of delayed aging and show significant increases in lifespan (50% and 25%, respectively) when compared with their wild‐type siblings. To gain further insight into the molecular basis for the extended longevity of these mice, we used oligonucleotide microarrays to measure levels of expression of over 14 000 RNA transcripts in liver during normal aging at 3, 6, 12 and 24 months. We found that the Prop1df/df and Ghrhrlit/lit genotypes produce dramatic alterations in gene expression, which are predominantly maintained at all ages. We found 1125 genes to be significantly affected by the Prop1df/df genotype and 1152 genes were significantly affected by the Ghrhrlit/lit genotype; 547 genes were present in both gene lists and showed parallel changes in gene expression, suggesting common mechanisms for the extended longevity in these mutants. Some of the functional gene classes most affected in these mutants included: amino acid metabolism, TCA cycle, mitochondrial electron transport, fatty acid, cholesterol and steroid metabolism, xenobiotic metabolism and oxidant metabolism. We found that the Prop1df/df genotype, and to a minor extent the Ghrhrlit/lit genotype, also produced complex alterations in age‐dependent changes in gene expression as compared with wild‐type mice. In some cases these alterations reflected a partial delay or deceleration of age‐related changes in gene expression as seen in wild‐type mice but they also introduced age‐related changes that are unique for each of these mutants and not present in wild‐type mice.

Stoichiometry of site-specific lysine acetylation in an entire proteome.

Background: Lysine acetylation sites have been mapped, but information on stoichiometry is lagging. Results: We developed and utilized the first direct, unbiased method for quantifying site-specific acetylation stoichiometry of a proteome without antibody enrichment. Conclusion: High stoichiometry is associated with central metabolism, transcription, and translation. Loss of deacetylase CobB affects site-specific and global acetylation stoichiometry, altering acetyl-CoA metabolism. Significance: Stoichiometry provides functional insight into protein acetylation. Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD+-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

Metabolome Remodeling during the Acidogenic-Solventogenic Transition in Clostridium acetobutylicum

ABSTRACT The fermentation carried out by the biofuel producer Clostridium acetobutylicum is characterized by two distinct phases. Acidogenesis occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). Solventogenesis initiates as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol). Using metabolomics, isotope tracers, and quantitative flux modeling, we have mapped the metabolic changes associated with the acidogenic-solventogenic transition. We observed a remarkably ordered series of metabolite concentration changes, involving almost all of the 114 measured metabolites, as the fermentation progresses from acidogenesis to solventogenesis. The intracellular levels of highly abundant amino acids and upper glycolytic intermediates decrease sharply during this transition. NAD(P)H and nucleotide triphosphates levels also decrease during solventogenesis, while low-energy nucleotides accumulate. These changes in metabolite concentrations are accompanied by large changes in intracellular metabolic fluxes. During solventogenesis, carbon flux into amino acids, as well as flux from pyruvate (the last metabolite in glycolysis) into oxaloacetate, decreases by more than 10-fold. This redirects carbon into acetyl coenzyme A, which cascades into solventogenesis. In addition, the electron-consuming reductive tricarboxylic acid (TCA) cycle is shutdown, while the electron-producing oxidative (clockwise) right side of the TCA cycle remains active. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources (carbon and reducing power) from biomass production into solvent production.

Metabolite concentrations, fluxes, and free energies imply efficient enzyme usage

In metabolism, available free energy is limited and must be divided across pathway steps to maintain ΔG negative throughout. For each reaction, ΔG is log-proportional both to a concentration ratio (reaction quotient-to-equilibrium constant) and to a flux ratio (backward-to-forward flux). Here we use isotope labeling to measure absolute metabolite concentrations and fluxes in Escherichia coli, yeast, and a mammalian cell line. We then integrate this information to obtain a unified set of concentrations and ΔG for each organism. In glycolysis, we find that free energy is partitioned so as to mitigate unproductive backward fluxes associated with ΔG near zero. Across metabolism, we observe that absolute metabolite concentrations and ΔG are substantially conserved, and that most substrate (but not inhibitor) concentrations exceed the associated enzyme binding site affinity. The observed conservation of metabolite concentrations is consistent with an evolutionary drive to utilize enzymes efficiently given thermodynamic and osmotic constraints.

Ultrasensitive regulation of anapleurosis via allosteric activation of PEP carboxylase.

Anapleurosis is the filling of the TCA cycle with four-carbon units. The common substrate for both anapleurosis and glucose phosphorylation in bacteria is the terminal glycolytic metabolite, phosphoenolpyruvate (PEP). Here we show that E. coli quickly and almost completely turns off PEP consumption upon glucose removal. The resulting build-up of PEP is used to quickly import glucose if it becomes re-available. The switch-like termination of anapleurosis results from depletion of fructose-1,6-bisphosphate (FBP), an ultrasensitive allosteric activator of PEP carboxylase. E. coli expressing an FBP-insensitive point mutant of PEP carboxylase grow normally on steady glucose. However, they fail to build-up PEP upon glucose removal, grow poorly on oscillating glucose, and suffer from futile cycling at the PEP node on gluconeogenic substrates. Thus, bacterial central carbon metabolism is intrinsically programmed with ultrasensitive allosteric regulation to enable rapid adaptation to changing environmental conditions.

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

BackgroundClostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum.ResultsUsing a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis.ConclusionsC. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

Steady-State Metabolite Concentrations Reflect a Balance between Maximizing Enzyme Efficiency and Minimizing Total Metabolite Load

Steady-state metabolite concentrations in a microorganism typically span several orders of magnitude. The underlying principles governing these concentrations remain poorly understood. Here, we hypothesize that observed variation can be explained in terms of a compromise between factors that favor minimizing metabolite pool sizes (e.g. limited solvent capacity) and the need to effectively utilize existing enzymes. The latter requires adequate thermodynamic driving force in metabolic reactions so that forward flux substantially exceeds reverse flux. To test this hypothesis, we developed a method, metabolic tug-of-war (mTOW), which computes steady-state metabolite concentrations in microorganisms on a genome-scale. mTOW is shown to explain up to 55% of the observed variation in measured metabolite concentrations in E. coli and C. acetobutylicum across various growth media. Our approach, based strictly on first thermodynamic principles, is the first method that successfully predicts high-throughput metabolite concentration data in bacteria across conditions.