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1.  Autophagy is Required for Glucose Homeostasis and Lung Tumor Maintenance 
Cancer discovery  2014;4(8):914-927.
Macroautophagy (autophagy hereafter) recycles intracellular components to sustain mitochondrial metabolism that promotes the growth, stress tolerance and malignancy of lung cancers, suggesting that autophagy inhibition may have antitumor activity. To assess the functional significance of autophagy in both normal and tumor tissue, we conditionally deleted the essential autophagy gene, autophagy-related-7, Atg7, throughout adult mice. Here we report that systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2–3 months. Moreover, upon fasting, autophagy-deficient mice suffered fatal hypoglycemia. Prior autophagy ablation did not alter the efficiency of non-small-cell lung cancer (NSCLC) initiation by activation of oncogenic KrasG12D and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with pre-existing NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign disease (oncocytomas). This anti-tumor activity occurred prior to destruction of normal tissues, suggesting that, acute autophagy inhibition may be therapeutically beneficial in cancer.
PMCID: PMC4125614  PMID: 24875857
autophagy; fasting; hypoglycemia; cancer; oncocytoma
2.  Quantitative flux analysis reveals folate-dependent NADPH production 
Nature  2014;510(7504):298-302.
ATP is the dominant energy source in animals for mechanical and electrical work (e.g., muscle contraction, neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defense and reductive biosynthesis1. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway (oxPPP), with malic enzyme sometimes also important. While the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analyzed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labeled substrates into NADPH, and combine this approach with carbon labeling and mathematical modeling to measure cytosolic NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxPPP. Surprisingly a nearly comparable contribution comes from serine-driven one-carbon metabolism, where oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. Since folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and GSH/GSSG ratios and increased cell sensitivity to oxidative stress. Thus, while the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.
PMCID: PMC4104482  PMID: 24805240
3.  Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate 
Cancer & Metabolism  2014;2:23.
Cell growth requires fatty acids for membrane synthesis. Fatty acids are assembled from 2-carbon units in the form of acetyl-CoA (AcCoA). In nutrient and oxygen replete conditions, acetyl-CoA is predominantly derived from glucose. In hypoxia, however, flux from glucose to acetyl-CoA decreases, and the fractional contribution of glutamine to acetyl-CoA increases. The significance of other acetyl-CoA sources, however, has not been rigorously evaluated. Here we investigate quantitatively, using 13C-tracers and mass spectrometry, the sources of acetyl-CoA in hypoxia.
In normoxic conditions, cultured cells produced more than 90% of acetyl-CoA from glucose and glutamine-derived carbon. In hypoxic cells, this contribution dropped, ranging across cell lines from 50% to 80%. Thus, under hypoxia, one or more additional substrates significantly contribute to acetyl-CoA production. 13C-tracer experiments revealed that neither amino acids nor fatty acids are the primary source of this acetyl-CoA. Instead, the main additional source is acetate. A large contribution from acetate occurs despite it being present in the medium at a low concentration (50–500 μM).
Acetate is an important source of acetyl-CoA in hypoxia. Inhibition of acetate metabolism may impair tumor growth.
Electronic supplementary material
The online version of this article (doi:10.1186/2049-3002-2-23) contains supplementary material, which is available to authorized users.
PMCID: PMC4322440  PMID: 25671109
Acetate; Acetyl-CoA; Cancer metabolism; Fatty acids; Hypoxia; Lipogenesis; Mass spectrometry; Palmitate; 13C-tracing
4.  Liquid chromatography – high resolution mass spectrometry analysis of fatty acid metabolism 
Analytical chemistry  2011;83(23):9114-9122.
We present a liquid chromatography – mass spectrometry (LC-MS) method for long-chain and very-long-chain fatty acid analysis, and its application to 13C-tracer studies of fatty acid metabolism. Fatty acids containing 14 to 36 carbon atoms are separated by C8 reversed-phase chromatography using a water-methanol gradient with tributylamine as ion pairing agent, ionized by electrospray, and analyzed by a stand-alone orbitrap mass spectrometer. The median limit of detection is 5 ng/ml with a linear dynamic range of 100-fold. Ratios of unlabeled to 13C-labeled species are quantitated precisely and accurately (average relative standard deviation 3.2% and deviation from expectation 2.3%). In samples consisting of fatty acids saponified from cultured mammalian cells, 45 species are quantified, with average intraday relative standard deviations for independent biological replicates of 11%. The method enables quantitation of molecular ion peaks for all labeled forms of each fatty acid. Different degrees of 13C-labeling from glucose and glutamine correspond to fatty acid uptake from media, de novo synthesis, and elongation. To exemplify the utility of the method, we examined isogenic cell lines with and without activated Ras oncogene expression. Ras increases the abundance and alters the labeling patterns of saturated and monounsaturated very-long-chain fatty acids, with the observed pattern consistent with Ras leading to enhanced activity of ELOVL4 or an enzyme with similar catalytic activity. This LC-MS method and associated isotope tracer techniques should be broadly applicable to investigating fatty acid metabolism.
PMCID: PMC3230881  PMID: 22004349
elongase; exactive; fatty acids; high resolution mass spectrometry; lipids; liquid chromatography-mass spectrometry; mass isotopomer distribution analysis; tracer studies; very-long-chain fatty acids
5.  Teaching the design principles of metabolism 
Nature chemical biology  2012;8(6):497-501.
Learning metabolism inevitably involves memorizing pathways. The teacher’s challenge is to motivate memorization and to help students progress beyond it. To this end, students should be taught a few fundamental chemical reaction mechanisms and how these are repeatedly used to achieve pathway goals. Pathway knowledge should then be reinforced through quantitative problems that emphasize the relevance of metabolism to bioengineering and medicine.
PMCID: PMC4084555  PMID: 22596190
6.  Metabolite turns master regulator 
Nature  2013;500(7462):283-284.
The phenomenon of catabolite repression enables microorganisms to use their favourite carbon source first. New work reveals α-ketoacids as key effectors of this process, with their levels regulating gene expression.
PMCID: PMC4084598  PMID: 23925111
7.  Quantitation of Cellular Metabolic Fluxes of Methionine 
Analytical chemistry  2014;86(3):1583-1591.
Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosyn-thesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with 13C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography−mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools.
PMCID: PMC4060246  PMID: 24397525
8.  LC-MS Data Processing with MAVEN: A Metabolomic Analysis and Visualization Engine 
MAVEN is an open-source software program for interactive processing of LC-MS-based metabolomics data. MAVEN enables rapid and reliable metabolite quantitation from multiple reaction monitoring data or high-resolution full-scan mass spectrometry data. It automatically detects and reports peak intensities for isotope-labeled metabolites. Menu-driven, click-based navigation allows visualization of raw and analyzed data. Here we provide a User Guide for MAVEN. Step-by-step instructions are provided for data import, peak alignment across samples, identification of metabolites that differ strongly between biological conditions, quantitation and visualization of isotope-labeling patterns, and export of tables of metabolite-specific peak intensities. Together, these instructions describe a workflow that allows efficient processing of raw LC-MS data into a form ready for biological analysis.
PMCID: PMC4055029  PMID: 22389014
metabolomics; liquid chromatography-mass spectrometry; pathway visualization and mapping; stable isotope labeling; metabolic flux analysis; kinetic flux profiling
10.  Chemical-Genetics of Rapamycin-Insensitive TORC2 in S. cerevisiae 
Cell reports  2013;5(6):1725-1736.
Current approaches for identifying synergistic targets use cell culture models with combinations of clinically available drugs to see if the combined effect of the combination is better than predicted by their individual efficacy. New techniques are needed to systematically and rationally identify targets and pathways that have a high potential as synergistic targets. In this study, we create a tool to screen and identify molecular targets that may synergize with new inhibitors of TOR (Target of Rapamycin), a conserved protein that is a major integrator of cell proliferation signals in the nutrient-signaling pathway. While clinical results from TORC1 inhibition using rapamycin analogs (that only inhibit TORC1) have been disappointing, trials using inhibitors that also target TORC2 have been promising. To understand the molecular basis for this increased therapeutic efficacy and to discover secondary targets that may have potential in targeted combination therapy, we engineered TOR2 in S. cerevisiae to accept an orthogonal inhibitor in order to create the first chemical tool to selectively inhibit TORC2. We used this tool to create a Chemical Epistasis Mini-Array Profile, or ChE-MAP, by measuring interactions between the chemically inhibited TOR2 kinase and a diverse library of deletion mutants. The ChE-MAP identified known TOR components and distinguished between TORC1 (assessed using rapamycin) and TORC2 dependent functions. Results showed a novel TORC2-specific interaction with the pentose phosphate pathway (PPP). We used global metabolic profiling to show that that TORC2 inhibition led to decreases in metabolites specific to the PPP and confirmed that TOR2 was regulating this process using metabolic flux analysis. Regulation of the PPP is a previously unappreciated role for TORC2 that may suggest a role for the complex in balancing the high energy demand required for ribosome biogenesis.
PMCID: PMC4007695  PMID: 24360963
11.  Metabolomic analysis via reversed-phase ion-pairing liquid chromatography coupled to a stand alone orbitrap mass spectrometer 
Analytical chemistry  2010;82(8):3212-3221.
We present a liquid chromatography-mass spectrometry (LC-MS) method that capitalizes on the mass-resolving power of the orbitrap to enable sensitive and specific measurement of known and unanticipated metabolites in parallel, with a focus on water soluble species involved in core metabolism. The reversed phase LC method, with a cycle time 25 min, involves a water-methanol gradient on a C18 column with tributylamine as the ion pairing agent. The MS portion involves full scans from 85 – 800 m/z at 1 Hz and 100,000 resolution in negative ion mode on a stand alone orbitrap (“Exactive”). The median limit of detection, across 80 metabolite standards, was 5 ng/mL with linear range typically ≥ 100-fold. For both standards and a cellular extract from Saccharomyces cerevisiae (Baker’s yeast), the median inter-run relative standard deviation in peak intensity was 8%. In yeast exact, we detected 137 known compounds, whose 13C-labeling patterns could also be tracked to probe metabolic flux. In yeast engineered to lack a gene of unknown function (YKL215C), we observed accumulation of an ion of m/z 128.0351, which we subsequently confirmed to be oxoproline, resulting in annotation of YKL215C as an oxoprolinase. These examples demonstrate the suitability of the present method for quantitative metabolomics, fluxomics, and discovery metabolite profiling.
PMCID: PMC2863137  PMID: 20349993
12.  Genetic Basis of Metabolome Variation in Yeast 
PLoS Genetics  2014;10(3):e1004142.
Metabolism, the conversion of nutrients into usable energy and biochemical building blocks, is an essential feature of all cells. The genetic factors responsible for inter-individual metabolic variability remain poorly understood. To investigate genetic causes of metabolome variation, we measured the concentrations of 74 metabolites across 100 segregants from a Saccharomyces cerevisiae cross by liquid chromatography-tandem mass spectrometry. We found 52 quantitative trait loci for 34 metabolites. These included linkages due to overt changes in metabolic genes, e.g., linking pyrimidine intermediates to the deletion of ura3. They also included linkages not directly related to metabolic enzymes, such as those for five central carbon metabolites to ira2, a Ras/PKA pathway regulator, and for the metabolites, S-adenosyl-methionine and S-adenosyl-homocysteine to slt2, a MAP kinase involved in cell wall integrity. The variant of ira2 that elevates metabolite levels also increases glucose uptake and ethanol secretion. These results highlight specific examples of genetic variability, including in genes without prior known metabolic regulatory function, that impact yeast metabolism.
Author Summary
Many traits, from human height to E. coli growth rate, quantitatively vary across members of a species. Among the most medically and agriculturally important traits are levels of cellular metabolites, such as cholesterol levels in humans or starch in food crops. Metabolic variation in yeast also holds practical importance with some Saccharomyces strains better suited to making ethanol for biofuel and others tailored to making flavorful wine. This metabolic heterogeneity can be used to gain insight into general principles of metabolic regulation which effect metabolite abundance in eukaryotes. To this end, we examined inter-strain differences in metabolism in over 100 closely related S. cerevisiae strains. We identified over 50 genetic loci that control the levels of specific metabolites, including not only loci that encode metabolic enzymes, but also those that encode global cellular regulators. For example, differences in the sequence of ira2, an inhibitor of Ras, lead to differences in central carbon metabolite levels, and polymorphisms in slt2, a poorly characterized MAP kinase, alter levels of sulfur-containing metabolites. These findings provide insights into the mechanisms cells use to control metabolite concentrations.
PMCID: PMC3945093  PMID: 24603560
13.  Growth-limiting Intracellular Metabolites in Yeast Growing under Diverse Nutrient Limitations 
Molecular Biology of the Cell  2010;21(1):198-211.
We find that the metabolome of nutrient-limited yeast varies dramatically with the limiting nutrient's identity. Low glutamine is a hallmark of nitrogen limitation, ATP of phosphorus limitation, and pyruvate of carbon limitation. The availability of these metabolites can quantitatively account for the nutrient-limited yeast's growth rate.
Microbes tailor their growth rate to nutrient availability. Here, we measured, using liquid chromatography-mass spectrometry, >100 intracellular metabolites in steady-state cultures of Saccharomyces cerevisiae growing at five different rates and in each of five different limiting nutrients. In contrast to gene transcripts, where ∼25% correlated with growth rate irrespective of the nature of the limiting nutrient, metabolite concentrations were highly sensitive to the limiting nutrient's identity. Nitrogen (ammonium) and carbon (glucose) limitation were characterized by low intracellular amino acid and high nucleotide levels, whereas phosphorus (phosphate) limitation resulted in the converse. Low adenylate energy charge was found selectively in phosphorus limitation, suggesting the energy charge may actually measure phosphorus availability. Particularly strong concentration responses occurred in metabolites closely linked to the limiting nutrient, e.g., glutamine in nitrogen limitation, ATP in phosphorus limitation, and pyruvate in carbon limitation. A simple but physically realistic model involving the availability of these metabolites was adequate to account for cellular growth rate. The complete data can be accessed at the interactive website
PMCID: PMC2801714  PMID: 19889834
14.  Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia coli 
Nature chemical biology  2009;5(8):593-599.
Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use liquid chromatography-tandem mass spectrometry to quantify more than 100 metabolite concentrations in aerobic, exponentially growing E. coli with glucose, glycerol, or acetate as the carbon source. The total observed intracellular metabolite pool is approximately 300 mM. A small number of metabolites dominate the metabolome on a molar basis, with glutamate most abundant. Metabolite concentration exceeds Km for most substrate-enzyme pairs. An exception is lower glycolysis, where concentrations of intermediates are near the Km of their consuming enzymes and all reactions are near equilibrium. This may facilitate efficient flux reversibility given thermodynamic and osmotic constraints. The data and analyses presented here highlight the ability to identify organizing metabolic principles from systems-level absolute metabolite concentration data.
PMCID: PMC2754216  PMID: 19561621
15.  Analytical strategies for LC-MS-based targeted metabolomics 
Recent advances in mass spectrometry are enabling improved analysis of endogenous metabolites. Here we discuss several issues relevant to developing liquid chromatography-electrospray ionization-mass spectrometry methods for targeted metabolomics (i.e., quantitative analysis of dozens to hundreds of specific metabolites). Sample preparation and liquid chromatography approaches are discussed, with an eye towards the challenge of dealing with a diversity of metabolite classes in parallel. Evidence is presented that heated electrospray ionization (ESI) generally gives improved signal compared to the more traditional unheated ESI. Applicability to targeted metabolomics of triple-quadruple mass spectrometry operating in multiple reaction monitoring (MRM) mode and high mass-resolution full scan mass spectrometry (e.g., time-of-flight, Orbitrap) are described. We suggest that both are viable solutions, with MRM preferred when targeting a more limited number of analytes, and full scan preferred for its potential ability to bridge targeted and untargeted metabolomics.
PMCID: PMC2607197  PMID: 18502704
16.  Isotope Ratio-Based Profiling of Microbial Folates 
Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using −75°C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly 13C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.
PMCID: PMC1909916  PMID: 17360194
17.  Identifying Decomposition Products in Extracts of Cellular Metabolites 
Analytical biochemistry  2006;358(2):273-280.
Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, it would accordingly seem that the best method for extracting a particular metabolite is the one giving the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that ≥ 50% water gave increased yield of nucleosides and bases compared to ≤ 20% water, as determined by liquid chromatography-tandem mass spectrometry analysis of the resulting extracts. Spiking of the extracts with isotope-labeled nucleotides revealed, however, that the high yield of nucleosides and bases occurred due to decomposition of nucleotides in the water-rich condition, not good extraction. Spiking combined with isotope labeling provides a general approach to detecting decomposition products in extracts of cellular metabolites. For extraction of E. coli with methanol:water, cold temperature and a high methanol fraction minimizes artifacts due to metabolite decomposition.
PMCID: PMC1868396  PMID: 16962982
Metabolomics; metabolism; extraction; bacteria; sampling; stability; LC-MS/MS; triple quadrupole; small molecule
18.  Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia 
The impact of oncogene activation and hypoxia on energy metabolism is analyzed by integrating quantitative measurements into a redox-balanced metabolic flux model. Glutamine-driven oxidative phosphorylation is found to be a major ATP source even in oncogene-expressing or hypoxic cells.
The integration of oxygen uptake measurements and LC-MS-based isotope tracer analyses in a redox-balanced metabolic flux model enabled quantitative determination of energy generation pathways in cultured cells.In transformed mammalian cells, even in hypoxia (1% oxygen), oxidative phosphorylation produces the majority of ATP.The oncogene Ras simultaneously increases glycolysis and decreases oxidative phosphorylation, thus resulting in no net increase in ATP production.Glutamine is the major source of high-energy electrons for oxidative phosphorylation, especially upon Ras activation.
Mammalian cells can generate ATP via glycolysis or mitochondrial respiration. Oncogene activation and hypoxia promote glycolysis and lactate secretion. The significance of these metabolic changes to ATP production remains however ill defined. Here, we integrate LC-MS-based isotope tracer studies with oxygen uptake measurements in a quantitative redox-balanced metabolic flux model of mammalian cellular metabolism. We then apply this approach to assess the impact of Ras and Akt activation and hypoxia on energy metabolism. Both oncogene activation and hypoxia induce roughly a twofold increase in glycolytic flux. Ras activation and hypoxia also strongly decrease glucose oxidation. Oxidative phosphorylation, powered substantially by glutamine-driven TCA turning, however, persists and accounts for the majority of ATP production. Consistent with this, in all cases, pharmacological inhibition of oxidative phosphorylation markedly reduces energy charge, and glutamine but not glucose removal markedly lowers oxygen uptake. Thus, glutamine-driven oxidative phosphorylation is a major means of ATP production even in hypoxic cancer cells.
PMCID: PMC3882799  PMID: 24301801
cancer bioenergetics; isotope tracing; metabolic flux analysis
19.  Absence of detectable arsenate in DNA from arsenate-grown GFAJ-1 cells 
Science (New York, N.Y.)  2012;337(6093):10.1126/science.1219861.
A strain of Halomonas bacteria, GFAJ-1, has been reported to be able to use arsenate as a nutrient when phosphate is limiting, and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.
PMCID: PMC3845625  PMID: 22773140
20.  Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells 
Nature  2013;497(7451):10.1038/nature12138.
Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and its contents are internalized into cells via large, heterogeneous vesicles known as macropinosomes. Oncogenic Ras proteins have been shown to stimulate macropinocytosis but the functional contribution of this uptake mechanism to the transformed phenotype remains unknown1-3. Here we show that Ras-transformed cells utilize macropinocytosis to transport extracellular protein into the cell. The internalized protein undergoes proteolytic degradation, yielding amino acids including glutamine that can enter central carbon metabolism. Accordingly, the dependence of Ras-transformed cells on free extracellular glutamine for growth can be suppressed by the macropinocytic uptake of protein. Consistent with macropinocytosis representing an important route of tumor nutrient uptake, its pharmacological inhibition compromised the growth of Ras-transformed pancreatic tumor xenografts. These results identify macropinocytosis as a mechanism by which cancer cells support their unique metabolic needs and point to the possible exploitation of this process in the design of anti-cancer therapies.
PMCID: PMC3810415  PMID: 23665962
21.  Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides 
Chemistry & Biology  2013;20(11):1386-1398.
Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.
Graphical Abstract
•Maf proteins represent a family of nucleoside triphosphate pyrophosphatases•Maf proteins hydrolyze the canonical nucleotides dTTP, UTP, and CTP•Maf proteins are also active against m5UTP, m5CTP, pseudo-UTP, and m7GTP•Maf structures reveal the molecular mechanisms of their substrate selectivity
Tchigvintsev et al. show that Maf proteins are a family of nucleotide pyrophosphatases active against both canonical and modified nucleotides. This suggests that Mafs might have a dual role in cell division and in the prevention of the incorporation of modified nucleotides into cellular nucleic acids.
PMCID: PMC3899018  PMID: 24210219
22.  Cancer mistunes methylation 
Nature chemical biology  2013;9(5):293-294.
Metabolic aberrations affecting protein and DNA methylation are a potential source of cancer. A new study shows that the metabolic enzyme nicotinamide N-methyl-transferase, which is overexpressed in several types of tumors, can enhance cancer aggressiveness by draining methyl groups from S-adenosyl-methionine.
PMCID: PMC3733116  PMID: 23594782
23.  Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation 
Molecular cell  2012;48(1):52-62.
Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively-cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19’s substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery.
PMCID: PMC3472130  PMID: 22902555
25.  Saturated Very Long Chain Fatty Acids Are Required for the Production of Infectious Human Cytomegalovirus Progeny 
PLoS Pathogens  2013;9(5):e1003333.
Human cytomegalovirus hijacks host cell metabolism, increasing the flux of carbon from glucose to malonyl-CoA, the committed precursor to fatty acid synthesis and elongation. Inhibition of acetyl-CoA carboxylase blocks the production of progeny virus. To probe further the role of fatty acid metabolism during infection, we performed an siRNA screen to identify host cell metabolic enzymes needed for the production of infectious cytomegalovirus progeny. The screen predicted that multiple long chain acyl-CoA synthetases and fatty acid elongases are needed during infection, and the levels of RNAs encoding several of these enzymes were upregulated by the virus. Roles for acyl-CoA synthetases and elongases during infection were confirmed by using small molecule antagonists. Consistent with a role for these enzymes, mass spectrometry-based fatty acid analysis with13C-labeling revealed that malonyl-CoA is consumed by elongases to produce very long chain fatty acids, generating an approximately 8-fold increase in C26-C34 fatty acid tails in infected cells. The virion envelope was yet further enriched in C26-C34 saturated fatty acids, and elongase inhibitors caused the production of virions with lower levels of these fatty acids and markedly reduced infectivity. These results reveal a dependence of cytomegalovirus on very long chain fatty acid metabolism.
Author Summary
Herpes viruses modulate cellular pathways to generate the building blocks that are necessary for their replication. Human cytomegalovirus alters metabolism of infected cells and causes a dramatic increase in lipid biosynthesis. We have investigated the role of lipid pathways in the viral life cycle and discovered that the virus requires several host enzymes that are responsible for the synthesis of very long chain fatty acids. Interestingly, very long chain fatty acids are substantially increased in the lipids of infected cells and saturated forms of these fatty acids are selectively incorporated into the envelope of the virus. Drugs that inhibit the synthesis of very long chain fatty acids generate virus particles with reduced infectivity. The discovery that human cytomegalovirus depends on the production of particular fatty acids furthers our understanding of virus-host cell interaction and suggests potential novel strategies for antiviral therapies.
PMCID: PMC3656100  PMID: 23696731

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