ATP Requirement Research Articles

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The 13C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0g/L isoprene with a yield of 0.267g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.

Accumulation of the components of cyclic electron flow around photosystem I in C4 plants, with respect to the requirements for ATP.

By concentrating CO2, C4 photosynthesis can suppress photorespiration and achieve high photosynthetic efficiency, especially under conditions of high light, high temperature, and drought. To concentrate CO2, extra ATP is required, which would also require a change in photosynthetic electron transport in C4 photosynthesis from that in C3 photosynthesis. Several analyses have shown that the accumulation of the components of cyclic electron flow (CEF) around photosystem I, which generates the proton gradient across thylakoid membranes (ΔpH) and functions in ATP production without producing NADPH, is increased in various NAD-malic enzyme and NADP-malic enzyme C4 plants, suggesting that CEF may be enhanced to satisfy the increased need for ATP in C4 photosynthesis. However, in C4 plants, the accumulation patterns of the components of two partially redundant pathways of CEF, NAD(P)H dehydrogenase-like complex and PROTON GRADIENT REGULATION5-PGR5-like1 complex, are not identical, suggesting that these pathways may play different roles in C4 photosynthesis. Accompanying the increase in the amount of NDH, the expression of some genes which encode proteins involved in the assembly of NDH is also increased at the mRNA level in various C4 plants, suggesting that this increase is needed to increase the accumulation of NDH. To better understand the relation between CEF and C4 photosynthesis, a reverse genetic approach to generate C4 transformants with respect to CEF will be necessary.

Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

The invasive blood-stage malaria parasite – the merozoite – induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142.

To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.

13C metabolic flux analysis at a genome-scale

Metabolic models used in 13C metabolic flux analysis generally include a limited number of reactions primarily from central metabolism. They typically omit degradation pathways, complete cofactor balances, and atom transition contributions for reactions outside central metabolism. This study addresses the impact on prediction fidelity of scaling-up mapping models to a genome-scale. The core mapping model employed in this study accounts for (75 reactions and 65 metabolites) primarily from central metabolism. The genome-scale metabolic mapping model (GSMM) (697 reaction and 595 metabolites) is constructed using as a basis the iAF1260 model upon eliminating reactions guaranteed not to carry flux based on growth and fermentation data for a minimal glucose growth medium. Labeling data for 17 amino acid fragments obtained from cells fed with glucose labeled at the second carbon was used to obtain fluxes and ranges. Metabolic fluxes and confidence intervals are estimated, for both core and genome-scale mapping models, by minimizing the sum of square of differences between predicted and experimentally measured labeling patterns using the EMU decomposition algorithm.Overall, we find that both topology and estimated values of the metabolic fluxes remain largely consistent between core and GSM model. Stepping up to a genome-scale mapping model leads to wider flux inference ranges for 20 key reactions present in the core model. The glycolysis flux range doubles due to the possibility of active gluconeogenesis, the TCA flux range expanded by 80% due to the availability of a bypass through arginine consistent with labeling data, and the transhydrogenase reaction flux was essentially unresolved due to the presence of as many as five routes for the inter-conversion of NADPH to NADH afforded by the genome-scale model. By globally accounting for ATP demands in the GSMM model the unused ATP decreased drastically with the lower bound matching the maintenance ATP requirement. A non-zero flux for the arginine degradation pathway was identified to meet biomass precursor demands as detailed in the iAF1260 model. Inferred ranges for 81% of the reactions in the genome-scale metabolic (GSM) model varied less than one-tenth of the basis glucose uptake rate (95% confidence test). This is because as many as 411 reactions in the GSM are growth coupled meaning that the single measurement of biomass formation rate locks the reaction flux values. This implies that accurate biomass formation rate and composition are critical for resolving metabolic fluxes away from central metabolism and suggests the importance of biomass composition (re)assessment under different genetic and environmental backgrounds. In addition, the loss of information associated with mapping fluxes from MFA on a core model to a GSM model is quantified.

PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H(+)-ATP Synthase.

The mitochondrial H(+)-ATP synthase synthesizes most of cellular ATP requirements by oxidative phosphorylation (OXPHOS). The ATPase Inhibitory Factor 1 (IF1) is known to inhibit the hydrolase activity of the H(+)-ATP synthase insituations that compromise OXPHOS. Herein, we demonstrate that phosphorylation of S39 in IF1 by mitochondrial protein kinase A abolishes its capacity to bind the H(+)-ATP synthase. Only dephosphorylated IF1 binds and inhibits both the hydrolase and synthase activities of the enzyme. The phosphorylation status of IF1 regulates the flux of aerobic glycolysis and ATP production through OXPHOS in hypoxia and during the cell cycle. Dephosphorylated IF1 is present in human carcinomas. Remarkably, mouse heart contains a large fraction of dephosphorylated IF1 that becomes phosphorylated and inactivated upon invivo β-adrenergic stimulation. Overall, we demonstrate the essential function of the phosphorylation of IF1 in regulating energy metabolism and speculate that dephosho-IF1 might play a role in signaling mitohormesis.

The Role of Mitochondrially Derived ATP in Synaptic Vesicle Recycling

Synaptic mitochondria are thought to be critical in supporting neuronal energy requirements at the synapse, and bioenergetic failure at the synapse may impair neural transmission and contribute to neurodegeneration. However, little is known about the energy requirements of synaptic vesicle release or whether these energy requirements go unmet in disease, primarily due to a lack of appropriate tools and sensitive assays. To determine the dependence of synaptic vesicle cycling on mitochondrially derived ATP levels, we developed two complementary assays sensitive to mitochondrially derived ATP in individual, living hippocampal boutons. The first is a functional assay for mitochondrially derived ATP that uses the extent of synaptic vesicle cycling as a surrogate for ATP level. The second uses ATP FRET sensors to directly measure ATP at the synapse. Using these assays, we show that endocytosis has high ATP requirements and that vesicle reacidification and exocytosis require comparatively little energy. We then show that to meet these energy needs, mitochondrially derived ATP is rapidly dispersed in axons, thereby maintaining near normal levels of ATP even in boutons lacking mitochondria. As a result, the capacity for synaptic vesicle cycling is similar in boutons without mitochondria as in those with mitochondria. Finally, we show that loss of a key respiratory subunit implicated in Leigh disease markedly decreases mitochondrially derived ATP levels in axons, thus inhibiting synaptic vesicle cycling. This proves that mitochondria-based energy failure can occur and be detected in individual neurons that have a genetic mitochondrial defect.

Abstract SY42-03: One carbon metabolism and cancer

Abstract Cancer cells adapt their metabolism to allow for growth and proliferation, while maintaining survival under conditions of fluctuating nutrient availability. Recent studies have shown that cancer cells are highly dependent on the non-essential amino acid serine. We have found a key requirement for serine is to maintain the one carbon cycle needed for nucleotide synthesis. Serine depletion impedes nucleotide synthesis and inhibits proliferation, a response that can be rescued by providing glycine and one-carbon units in the form of formate. More recently, we have been examining the fate of serine and the effect of serine depletion on other pathways that require one-carbon metabolism, such as NADPH production and the methionine cycle. We found that the adaptation to serine starvation involves diversion of glycolytic intermediates into the de novo SSP, resulting in a greater dependence on OXPHOS to provide energy. Interestingly, the serine produced during this period of adaptation is channeled into antioxidant defense through generation of both glutathione and NADPH, to help buffer the ROS produced from this increase in OXPHOS. Defects in the ability to make this adaptation and limit ROS, as seen in cells lacking p53, impedes the ability of cells to cope with a loss of exogenous serine. Serine is also required to support the S-adenosyl methionine (SAM) cycle, although surprisingly we have found that this reflected a requirement for ATP rather than one-carbon units. As a consequence, serine depletion caused a decrease in the conversion of methionine to SAM and a rapid remodeling of DNA methylation. We are currently examining the consequences on gene specific methylation and expression. Funded by CR-UK and ERC Citation Format: Oliver Maddocks, Christiaan Labuschagne, Karen H. Vousden. One carbon metabolism and cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY42-03. doi:10.1158/1538-7445.AM2015-SY42-03

CO2-driven changes in energy and fermentative metabolism in harvested strawberries

Short postharvest exposure of strawberries to high CO2 levels provides significant benefits in reducing decay and controlling physiological disorders during storage at 0°C. To define the different strategies employed by strawberries to tolerate high CO2 concentrations, the impact of different CO2 concentrations on energy and fermentative metabolism was studied under the same conditions of O2 availability. Our data indicate that metabolic depression represents a strategy to effectively adapt to beneficial high CO2 concentrations, with a decrease in ATP levels and in the energy charge, along with moderate ethanolic fermentation. Moreover, the induction of fermentative genes does not appear to be essential for the accumulation of fermentative metabolites. By contrast, when fruit is stored in air without added CO2, the metabolism is not directed towards fermentation and is accompanied by a high ATP/ADP ratio and energy charge. However, when exposed to 40kPa CO2, the excessively low energy charge and excessive decrease in ATP could not match the ATP requirements, in a process that ultimately causes significant perturbations including a high lipid peroxidation.

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni2+-dependent enzyme and Saccharomyces cerevisiae does not express native Ni2+-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01µmolmin−1mg protein−1 and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni2+ concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni2+-dependent enzymes can be functionally expressed in S. cerevisiae.

Knockdown of Cardiolipin Synthase in Human Brain Microvessel Endothelial Cells Modulates Blood Brain Barrier Transport Properties

The blood brain barrier (BBB) is formed by microvessel endothelial cells and is a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin (CL) a mitochondrial (MITO) lipid is required for activity/integrity of the respiratory chain. We examined the role of CL in maintaining barrier properties of brain microvessel endothelial cells. We show that human brain capillary endothelial cells (hCMEC/D3) produce a significant proportion of their basal ATP requirement through MITO oxidative phosphorylation. siRNA knockdown (KD) of the CL biosynthetic enzyme CL synthase (CLS) significantly reduced these cells ability to increase MITO function under conditions of increased metabolic demand compared to controls. In addition, KD of CLS acted as a metabolic switch causing hCMEC/D3 cells to become more dependent on glycolysis. KD of CLS increased [3H]‐2‐deoxyglucose ([3H]‐2DG) uptake compared to controls, and this may thus serve as a mechanism to compensate for the cells reduced MITO energy production efficiency. Finally, KD of CLS did not affect movement of the permeability marker fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell®plates, an in vitro model for the human BBB, but caused in an increase in [3H]‐2DG transport across the monolayer compared to controls. The results suggest that brain endothelial MITO energy status play a role in regulating glucose transport across the human BBB. These observations may, in part, explain why brain capillary endothelial cells possess higher MITO content than endothelial cells derived from non‐BBB regions of the body. (Supported by NSERC and the CRC Program)

Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

Analysis of Mitochondrial DNA in Induced Pluripotent and Embryonic Stem Cells.

The mitochondrial genome has a major role to play in establishing and maintaining pluripotency. Furthermore, mitochondrial DNA (mtDNA) copy is strictly regulated during differentiation. Undifferentiated, pluripotent cells possess fewer than 300 copies of mtDNA, which establishes the mtDNA set point and promotes cell proliferation and, as a result, these cells rely on glycolysis with some support from oxidative phosphorylation (OXPHOS) for the generation of ATP. The mtDNA set point provides the starting point from which cells increase their mtDNA copy number as they differentiate into mature functional cells. Dependent on cell types, mtDNA copy number ranges from ~10 copies in sperm to several thousand in cardiomyocytes. Consequently, differentiating cell types can acquire the appropriate numbers of mtDNA copy to meet their specific requirements for ATP generated through OXPHOS. However, as reprogrammed somatic cells do not always achieve this, it is essential to analyze them for their OXPHOS potential and ability to regulate mtDNA copy number. Here, we describe how to assess mtDNA copy number in pluripotent and differentiating cells using real-time PCR protocols; assess expression of the mtDNA specific replication factors through real-time RT-PCR; identify mtDNA variants in embryonic and induced pluripotent stem cells; determine DNA methylation patterns of the mtDNA-specific replication factors; and assess mitochondrial OXPHOS capacity.

Role of Glucose Metabolism and ATP in Maintaining PINK1 Levels during Parkin-mediated Mitochondrial Damage Responses

Mutations in several genes, including PINK1 and Parkin, are known to cause autosomal recessive cases of Parkinson disease in humans. These genes operate in the same pathway and play a crucial role in mitochondrial dynamics and maintenance. PINK1 is required to recruit Parkin to mitochondria and initiate mitophagy upon mitochondrial depolarization. In this study, we show that PINK1-dependent Parkin mitochondrial recruitment in response to global mitochondrial damage by carbonyl cyanide m-chlorophenylhydrazine (CCCP) requires active glucose metabolism. Parkin accumulation on mitochondria and subsequent Parkin-dependent mitophagy is abrogated in glucose-free medium or in the presence of 2-deoxy-D-glucose upon CCCP treatment. The defects in Parkin recruitment correlate with intracellular ATP levels and can be attributed to suppression of PINK1 up-regulation in response to mitochondria depolarization. Low levels of ATP appear to prevent PINK1 translation instead of affecting PINK1 mRNA expression or reducing its stability. Consistent with a requirement of ATP for elevated PINK1 levels and Parkin mitochondrial recruitment, local or individual mitochondrial damage via photoirradiation does not affect Parkin recruitment to damaged mitochondria as long as a pool of functional mitochondria is present in the photoirradiated cells even in glucose-free or 2-deoxy-D-glucose-treated conditions. Thus, our data identify ATP as a key regulator for Parkin mitochondrial translocation and sustaining elevated PINK1 levels during mitophagy. PINK1 functions as an AND gate and a metabolic sensor coupling biogenetics of cells and stress signals to mitochondria dynamics.

Increasing ATP conservation in maltose consuming yeast, a challenge for industrial organic acid production in non-aerated reactors

Anaerobic fermentation processes are economically attractive for industry, as costs for aeration and stirring are greatly reduced. An example of such a process is (bio)ethanol production by Saccharomyces cerevisiae, in which a low but positive ATP gain leads to elevated product yields. However, for other fermentative pathways, such as in an engineered homolactic strain of S. cerevisiae, the net ATP gain for the fermentation of glucose to lactate is null due to the requirement of ATP for product export. Therefore, increasing the conservation of ATP is of major importance for such 'zero-ATP pathways'. One opportunity to increase ATP conservation arises when disaccharides are used as a substrate. In many industrial microorganisms, disaccharides are cleaved by hydrolysis, which results in the dissipation of energy that is available in this cleaving reaction. However, phosphorolytic cleavage could be used to replace the hydrolysis of disaccharides, thereby increasing the ATP yield. In this study, growth of S. cerevisiae on maltose was used as a model. All known native maltose metabolism genes were removed and replaced by a maltose phosphorylase (Lactobacillus sanfranciscensis) and a single overexpressed copy of the native MAL11 maltose transporter. Because maltose phosphorylase cleaves maltose into glucose and β-glucose-1-phosphate, additionally a β-phosphoglucomutase (Lactococcus lactis) was co-expressed in this strain. Anaerobic maltose-limited chemostat cultures showed that replacement of maltose hydrolysis by phosphorolysis increased the biomass yield of the mutant strain by 26% over the wild type strain, demonstrating the potential of phosphorolysis to improve ATP conservation of disaccharide metabolism in industrial microorganisms [1].

Rapid Glucose Depletion Immobilizes Active Myosin V on Stabilized Actin Cables

Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles. This is mediated by myosin Vs transporting cargos along F-actin bundles known as actin cables. Actin cables are dynamic structures regulated by assembly, stabilization, and disassembly. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage. Thus, upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase, translation, and phosphoinositide metabolism. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and that the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton, resulting in disassembly of actin patches with concomitant inhibition of endocytosis and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin V in a newly described state on selectively stabilized actin cables.

Abstract 4319: Inability of putative ovarian cancer stem cells to engage OXPHOS confer 2-deoxyglucose-mediated prevention of in vivo recurrence

Abstract Background: Mortality in ovarian cancer does not usually occur at primary diagnosis but rather in the recurrent setting where patients present with chemoresistance and carcinomatosis. Prevention of recurrence can therefore have an impact on patient survival. Due to its inherent chemoresistance, cancer stem cells that are left after surgery are thought to drive recurrent disease owing to its tumor-initiating properties and enhanced capacity for repair. We demonstrate that the putative epithelial ovarian cancer (EOC) stem cells have a unique bioenergetic profile that can be targeted with the goal of delaying and/or preventing recurrent disease. Methods: In vitro: Pure clones of CD44+/MyD88+ and CD44-/MyD88- EOC cells were treated with 20 mM 2-DG, 5 μM Oligomycin, or 1 mM dinitrophenol (DNP). ATP levels were quantified using Celltiter Glo. Mitochondrial content and membrane potential (MMP) were measured using Mitotracker Green and Red, respectively. In vivo: CD44+/MyD88+/mCherry+ EOC stem cells were injected i.p. in nude mice. Mice received four doses of 12 mg/kg Paclitaxel q3d and then randomized to maintenance with 2-DG (500 mg/kg every MWF) or maintenance with saline. All treatments were given i.p. Tumors were monitored by live in vivo imaging using In Vivo FX System and quantified using the region of interest (ROI) tool based on mCherry fluorescent area. Results: Treatment with Oligomycin or DNP did not affect viability nor ATP content in CD44+/MyD88+ EOC stem cells but 2-DG induced rapid decline in ATP and death, which was not rescued with Succinate co-treatment. In contrast, in CD44-/MyD88- EOC cells, treatment with Oligomycin, DNP, or 2-DG only slightly decreased ATP without inducing cell death. Despite having more mitochondria, EOC stem cells have: (1) lower levels of Cox- I, -II, and -IV; (2) undetectable levels of pyruvate dehydrogenase; (3) and higher levels of UCP2 compared to CD44-/MyD88- EOC cells. Administration of 2-DG, as maintenance treatment post-chemotherapy, significantly delayed progression of recurrent disease and decreased i.p. tumor burden in the xenograft model. All mice maintained in saline recurred with average time-to-recurrence of 11 days. In contrast, only 25% of the mice maintained in 2-DG demonstrated recurrence with average time-to-recurrence of 19 days. Finally, analysis of tumors post-mortem show high levels of the autophagic markers p-AMPK and cleaved LC3 in the 2-DG maintained group compared to control. Conclusion: We demonstrate that the exclusive reliance of EOC stem cells to glycolysis to meet its ATP requirement and its inability to engage OXPHOS even under stress conditions can be exploited to target these otherwise chemoresistant cancer cells for the prevention of recurrence. Our results show the potential of maintenance treatment with glycolysis inhibitors, administered following standard chemotherapy, in improving survival in patients with ovarian cancer. Citation Format: Ayesha B. Alvero, Michele Montagna, Natalia J. Sumi, Won Duk Joo, Vinicius Craveiro, Emma Graham, Gil Mor. Inability of putative ovarian cancer stem cells to engage OXPHOS confer 2-deoxyglucose-mediated prevention of in vivo recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2014-4319

Bottom-up Metabolic Reconstruction of Arabidopsis and Its Application to Determining the Metabolic Costs of Enzyme Production.

Large-scale modeling of plant metabolism provides the possibility to compare and contrast different cellular and environmental scenarios with the ultimate aim of identifying the components underlying the respective plant behavior. The existing models of Arabidopsis (Arabidopsis thaliana) are top-down assembled, whereby the starting point is the annotated genome, in particular, the metabolic genes. Hence, dead-end metabolites and blocked reactions can arise that are subsequently addressed by using gap-filling algorithms in combination with species-unspecific genes. Here, we present a bottom-up-assembled, large-scale model that relies solely on Arabidopsis-specific annotations and results in the inclusion of only manually curated reactions. While the existing models are largely condition unspecific by employing a single biomass reaction, we provide three biomass compositions that pertain to realistic and frequently examined scenarios: carbon-limiting, nitrogen-limiting, and optimal growth conditions. The comparative analysis indicates that the proposed Arabidopsis core model exhibits comparable efficiency in carbon utilization and flexibility to the existing network alternatives. Moreover, the model is utilized to quantify the energy demand of amino acid and enzyme de novo synthesis in photoautotrophic growth conditions. Illustrated by the case of the most abundant protein in the world, Rubisco, we determine its synthesis cost in terms of ATP requirements. This, in turn, allows us to explore the tradeoff between protein synthesis and growth in Arabidopsis. Altogether, the model provides a solid basis for completely species-specific integration of high-throughput data, such as gene expression levels, and for condition-specific investigations of in silico metabolic engineering strategies.

On-line modeling intracellular carbon and energy metabolism of Nannochloropsis sp. in nitrogen-repletion and nitrogen-limitation cultures

In this study, a photobioreactor cultivation system and a calculation method for on-line monitoring of carbon and energy metabolism of microalgae were developed using Nannochloropsis sp. in nitrogen-repletion and nitrogen-limitation cultures. Only 30-60% of carbon fixed in Calvin cycle was used for biomass and the rest was lost in light respiration. The net fixed carbon was assumed to be incorporated into protein, lipids, carbohydrates, and nucleic acids, whose contents calculated on-line fitted well with the experimental measurements. Intracellular ATPs were quantitatively divided for biomass production and cell maintenance, and the result is in accordance with known reports. Due to light limitation induced by high cell concentration in batch cultures, the proportion of CO2 loss in light respiration and the proportion of energy for maintenance rapidly increased in culturing process. Nitrogen starvation reduced the light respiration, thus decreasing CO2 loss and maintenance energy, but no effect on ATP requirement for cell growth.

Effects of long-acting somatostatin analogues on redox systems in rat lens in experimental diabetes

The effects of long-acting somatostatin analogues, angiopeptin (AGP) and Sandostatin (SMS), on the early decline in the lens content of glutathione (GSH), ATP and NADPH and increase in sorbitol were studied in STZ diabetic rats, and comparison was made with the effect of insulin. Three factors prompted this study: (i) the known increase in IGF-1 in ocular tissue in diabetes and antagonistic effect of somatostatins, (ii) the known effect of IGF-1 in increasing lens aldose reductase and (iii) the lack of effect of somatostatins on diabetic hyperglycaemia, the latter enabling a differentiation to be made between effects of hyperglycaemia per se and site(s) of IGF-1/somatostatins. All four metabolites studied showed a significant restoration towards the normal control level after 7days of treatment with AGP and SMS, and AGP was more effective on levels of GSH and ATP. A significant correlation was found between GSH and ATP across all groups at 7days treatment. The redox state changes in diabetes include both NADP+/NADPH and NAD+/NADH in the conversion of glucose to sorbitol and via sorbitol dehydrogenase to fructose with a linked decrease in ATP formation via NAD+/NADH regulation of the glycolytic pathway. The interlinked network of change includes the requirement for ATP in the synthesis of GSH. The present study points to possible loci of action of somatostatins in improving metabolic parameters in the diabetic rat lens via effects on aldose reductase and/or glucose transport at GLUT 3.