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Graphical abstract, Highlights • Novel cell-free protein synthesis reaction buffer improves performance by 400%. • Enhanced performance is maintained across the synthesis of different proteins. • Protein synthesis performance is robust across different cell lysate batches and E. coli strains. • Buffer components affect aspects of reaction kinetics in differing ways., Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

Even with increasing evidence for roles of glycolytic enzymes in controlling cancerous characteristics, the best target of candidate metabolic enzymes for lessening malignancy remains under debate. Pyruvate is a main glycolytic metabolite that could be mainly converted into either lactate by Lactate Dehydrogenase A (LDHA) or acetyl-CoA by Pyruvate Dehydrogenase E1 component α subunit (PDHA1) catalytic complex. In tumor cells, accumulating lactate is produced whereas the conversion of pyruvate into mitochondrial acetyl-CoA is less active compared with their normal counterparts. This reciprocal molecular association makes pyruvate metabolism a potential choice of anti-cancer target. Cellular and molecular changes were herein assayed in Head and Neck Squamous Cell Carcinoma (HNSCC) cells in response to LDHA and PDHA1 loss in vitro, in vivo and in clinic. By using various human cancer databases and clinical samples, LDHA and PDHA1 levels exhibit reversed prognostic roles. In vitro analysis demonstrated that decreased cell growth and motility accompanied by an increased sensitivity to chemotherapeutic agents was found in cells with LDHA loss whereas PDHA1-silencing exhibited opposite phenotypes. At the molecular level, it was found that oncogenic Protein kinase B (PKB/Akt) and Extracellular signal-regulated kinase (ERK) singling pathways contribute to pyruvate metabolism mediated HNSCC cell growth. Furthermore, LDHA/PDHA1 changes in HNSCC cells resulted in a broad metabolic reprogramming while intracellular molecules including polyunsaturated fatty acids and nitrogen metabolism related metabolites underlie the malignant changes. Collectively, our findings reveal the significance of pyruvate metabolic fates in modulating HNSCC tumorigenesis and highlight the impact of metabolic plasticity in HNSCC cells.

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. In this study, we observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties, as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress., P. L. -M. was funded by a PhD fellowship from the ‘‘la Caixa’’ Foundation and received financial support from the Universidad de Zaragoza, Fundación Bancaria Ibercaja, and Fundación CAI (CM 1/16) during his stay at the UW–Madison. We acknowledge Ministerio de Economía, Industria y Competitividad for grant AGL2015-66177 (J. A. C.), the University of Zaragoza for grant UZ-2015-BIO-01 (J. A. C.), and OTRI (University of Zaragoza), Ministerio de Ciencia, Innovación y Universidades for grants CTQ2013-44367-C2-2-P and BFU2016-75633-P (R. H. -G.) and BFU2016-78232-P (A. V. -C.), Gobierno de Aragón for grants E34_R17 and LMP58_18 (R. H. -G.) and E45_17R (J. A. C and A. V. -C.) and FEDER (2014–2020) funds for “Building Europe from Aragón”. R. H. -G. and A. V. -C. thank the ARAID foundation for support. The research leading to these results also received funding from the FP7 (2007–2013) under BioStruct-X (grant agreement N283570 and BIOSTRUCTX_5186). We also thank the ALBA synchrotron radiation source, in particular, the beamline XALOC facility. J. A. and I. D. acknowledge access to UEA Faculty of Science Research Facilities. J. A. acknowledges funding support from the Biotechnology and biological Sciences Research Council (BBSRC; BB/P010660/1) and the Spanish Ministry of Science, Innovation and Universities through grant PID2019-109395GB-100. T. H. acknowledges a BBSRC DTP studentship. Financial support from the Universidad de Sevilla (Acciones Especiales del VI Plan Propio de Investigación y Transferencia) is also acknowledged. This work was also supported by National Institutes of Health (NIH) Grant GM065386 (J. M. D.), NIH National Research Service Award T32 GM007215 (J. B.) and the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP) DGE-1256259 (J. B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

International audience; The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process

Toxic heavy metals such as cadmium (Cd) and copper (Cu) are global problems that are a growing threat to the environment. Despite some heavy metals are required for plant growth and development, others are considered toxic elements and do not play any known physiological role in plant cells. Elevated doses of Cd or Cu cause toxicity in plants and generate damages due to the stress condition and eventually cause a significant reduction in quantity and quality of crop plants. The nitric oxide (NO) donor sodium nitroprusside (SNP) is reported to alleviate the toxicity of some heavy metals like Cd and Cu. In the current study, the role of NO in alleviating stresses of Cd and Cu was investigated in in vitro-grown tobacco (Nicotiana tabacum) Based on plant growth, total chlorophyll contents, contents and activities of rubisco and rubisco activase. According to the results of this study, the growth and total chlorophyll contents of Cd/Cu stressed plants were hugely decreased in the absence of SNP, while the supplementation of SNP resulted in a significant increase of both fresh weight and total chlorophyll contents. Remarkable reductions of Rubisco and rubisco activase contents and activities were observed in Cd and Cu-induced plants. SNP supplementation showed the highest contents and activities of rubisco and rubisco activase compared to the control and Cu/Cd-stressed plants. Taken together, our findings suggest that SNP could play a protective role in regulation of plant responses to abiotic stresses such as Cd and Cu by enhancing Rubisco and Rubisco activase., Graphical abstract, Highlights • Heavy metal ions are believed to act as growth inhibitors and environmental disruptors. • Nitric oxide (NO) plays a functional role in regulation of plant responses to abiotic stresses. • Rubisco involved in the process of atmospheric carbon fixation in photosynthesis. • Rubisco Catalyzes 2 types of reactions (carboxylation and oxygenation). • Rubisco activase removes bound RuBP from inactive, decarboxylated Rubisco in an ATP-dependent reaction.

Histidine phosphorylation is a posttranslational modification that alters protein function and also serves as an intermediate of phosphoryl transfer. Although phosphohistidine is relatively unstable, enzymatic dephosphorylation of this residue is apparently needed in some contexts, since both prokaryotic and eukaryotic phosphohistidine phosphatases have been reported. Here we identify the mechanism by which a bacterial phosphohistidine phosphatase dephosphorylates the nitrogen-related phosphotransferase system, a broadly conserved bacterial pathway that controls diverse metabolic processes. We show that the phosphatase SixA dephosphorylates the phosphocarrier protein NPr and that the reaction proceeds through phosphoryl transfer from a histidine on NPr to a histidine on SixA. In addition, we show that Escherichia coli lacking SixA are outcompeted by wild-type E. coli in the context of commensal colonization of the mouse intestine. Notably, this colonization defect requires NPr and is distinct from a previously identified in vitro growth defect associated with dysregulation of the nitrogen-related phosphotransferase system. The widespread conservation of SixA, and its coincidence with the phosphotransferase system studied here, suggests that this dephosphorylation mechanism may be conserved in other bacteria.

Graphical abstract, Highlights • New metabolic routes and membrane proteins were identified in the organelles. • Exponential and stationary growth-phase cells vary little in glycosomal proteins. • A variety of proteins bearing a typical PTS1 but with unknown function were found., In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as β-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite’s glycosomes.

There is currently a strong interest to derive the biological precursor cis,cis-muconic acid from shikimate pathway-branches to develop a biological replacement for adipic acid. Pioneered by the Frost laboratory this concept has regained interest: Recent approaches (Boles, Alper, Yan) however suffer from low product titres. Here an in silico comparison of all strain construction strategies was conducted to highlight stoichiometric optimizations. Using elementary mode analysis new knock-out strategies were determined in Saccharomyces cerevisiae and Escherichia coli. The strain construction strategies are unique to each pathway-branch and organism, allowing significantly different maximum and minimum yields. The maximum theoretical product carbon yields on glucose ranged from 86% (dehydroshikimate-branch) to 69% (anthranilate-branch). In most cases a coupling of product formation to growth was possible. Especially in S. cerevisiae chorismate-routes a minimum yield constraint of 46.9% could be reached. The knock-out targets are non-obvious, and not-transferable, highlighting the importance of tailored strain construction strategies., Highlights • Maximum theoretical product carbon yields for biological production of cis,cis-muconic acid from glucose were calculated. • Existing strain construction strategies were compared in silico and weaknesses were identified. • New strain construction strategies were determined to allow coupling of product formation to growth for E. coli and S. cerevisiae. • In particular abolishing one target reaction introduces a minimum yield constraint of 28% in S. cerevisiae. • Determined knock-out targets are unique to each pathway and organism, highlighting the importance of tailored strain construction strategies.

Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism., Graphical abstract Image 1, Highlights • The rhodamine-based sensor RSL+ permeates efficiently eukaryotic plasma membrane and accumulates in mitochondria. • RSL+ detects mitochondrial ATP levels in human and mouse skin fibroblasts. • RSL+ detects real-time variations of mitochondrial ATP levels under non-physiological conditions.

Shc proteins play a role in energy metabolism through interaction with the insulin receptor. The aim of this study was to determine whether Shc proteins influence liver glycolysis and gluconeogenesis under both fed and fasted states. Decreased glycolytic and increased gluconeogenic and transamination enzyme activities were observed in ShcKO versus WT mice. Levels of key regulatory metabolites, such as fructose-2,6-bisphosphate, matched the activity of metabolic pathways. Protein levels of glycolytic and gluconeogenic enzymes were not different. pAMPK protein levels increased with fasting and were higher in ShcKO versus WT mice. Therefore, Shc proteins play a role in shifting the metabolism from glucose oxidation to gluconeogenesis and lipid catabolism and should be considered as regulators of fuel selection. Fuel selection and utilization could play a critical role in healthy aging. Characterization of metabolic events in ShcKO mice would help to elucidate how metabolism is influenced by these proteins., Graphical abstract fx1, Highlights • Decreased glycolysis and increased gluconeogenesis in ShcKO mice. • Changes in metabolite levels reflect changes in enzyme activities. • Protein levels of key enzymes remained unchanged. • Shc proteins play a role in metabolic shift and act as regulators of fuel selection.

Altered cellular metabolism is a fundamental adaptation of cancer during rapid proliferation as a result of growth factor overstimulation. We review different pathways involving metabolic alterations in cancers including aerobic glycolysis, pentose phosphate pathway, de novo fatty acid synthesis, and serine and glycine metabolism. Although oncoproteins, c-MYC, HIF1α and p53 are the major drivers of this metabolic reprogramming, post-transcriptional regulation by microRNAs (miR) also plays an important role in finely adjusting the requirement of the key metabolic enzymes underlying this metabolic reprogramming. We also combine the literature data on the miRNAs that potentially regulate 40 metabolic enzymes responsible for metabolic reprogramming in cancers, with additional miRs from computational prediction. Our analyses show that: (1) a metabolic enzyme is frequently regulated by multiple miRs, (2) confidence scores from prediction algorithms might be useful to help narrow down functional miR-mRNA interaction, which might be worth further experimental validation. By combining known and predicted interactions of oncogenic transcription factors (TFs) (c-MYC, HIF1α and p53), sterol regulatory element binding protein 1 (SREBP1), 40 metabolic enzymes, and regulatory miRs we have established one of the first reference maps for miRs and oncogenic TFs that regulate metabolic reprogramming in cancers. The combined network shows that glycolytic enzymes are linked to miRs via p53, c-MYC, HIF1α, whereas the genes in serine, glycine and one carbon metabolism are regulated via the c-MYC, as well as other regulatory organization that cannot be observed by investigating individual miRs, TFs, and target genes.

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.

PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional Pi deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from −Pi (Pi-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to −Pi growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 ( Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) In vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following Pi resupply). Approx. 1.3 mg of PEPC was purified 660-fold from −Pi suspension cells to apparent homogeneity with a specific activity of 22.3 units · mg−1 of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1 . Pi-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its K m(PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and In vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of −Pi Arabidopsis . Abbreviations: Ab, antibody; anti-RcPEPC IgG, anti-(Ricinus communis PEPC) IgG; AtPPC1, Arabidopsis thaliana PEPC1; CAM, crassulacean acid metabolism; DTT, dithiothreitol; Glc-6-P, glucose 6-phosphate; MALDI, matrix-assisted laser-desorption ionization; MS medium, Murashige and Skoog medium; MS/MS, tandem MS; p107, 107-kDa PEPC polypeptide; oMALDI 2, orthogonal MALDI 2; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; +Pi, Pi-sufficient; −Pi, Pi-deficient; PP2A, protein phosphatase type-2A; PPCK, PEPC kinase; Q-TOF, quadrupole time-of-flight; QqTOF, quadrupole/quadrupole TOF; RT, reverse transcription

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding Km values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent Km (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM)., Highlights • Human colorectal cancer is not a pure hypoxic tumor of the Warburg phenotype. • The total hexokinase activity of CRC cells is close to that in nearby tissues. • In the tumor there is overexpression of VEGFs (A, B, and C) and their receptors. • CRC has higher rates of OXPHOS as compared with healthy tissue cells. • Tumor-surrounding cells cannot fuel via a lactate shunt the growth of CRC cells.

Computational analysis of ADP-glucose pyrophosphorylases predicts a fold with two domains. Co-expression of two polypeptides comprising residues 1-323 and 328-431 from the Escherichia coli ADP-glucose pyrophosphorylase yielded an enzyme form as active as the wild type. The only difference from the wild type was a slightly modified affinity for allosteric effectors. The two polypeptides could not be separated by chromatographic procedures. Separate expression of these polypeptides produced inactive unstable forms. All these results indicated that the ADP-glucose pyrophosphorylase comprises two domains with a strong interaction between them. That interaction is important for allosteric properties and structural stability. Fil: Bejar, Clarisa M.. Michigan State University. Department of Biochemistry and Molecular Biology; Estados Unidos Fil: Ballicora, Miguel. Michigan State University. Department of Biochemistry and Molecular Biology; Estados Unidos Fil: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Centro de Estudios Fotosintéticos y Bioquímicos. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos; Argentina Fil: Iglesias, Alberto Alvaro. Michigan State University. Department of Biochemistry and Molecular Biology; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Preiss, Jack. Michigan State University. Department of Biochemistry and Molecular Biology; Estados Unidos