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High grade glial tumors (HGGs) including anaplastic astrocytoma (WHO Grade-III) and glioblastoma multiforme (GBM, WHO Grade-IV) are among the most malignant cancers known to man. Due to their defective mitochondria, HGG cells consume glucose via glycolysis even in the presence of oxygen. Overall survival is worse in HGG patients that are hyperglycemic. Unlike normal neural cells, HGG cells cannot efficiently metabolize ketone bodies for energy. Thus, a metabolic treatment based on therapeutic ketosis (reduced glucose with elevated ketone bodies) was proposed to treat GBM and was supoported from preclinical studies. Caprylic (octanoic) acid, a monocarboxylated saturated fatty acid, is among the best producers of ketone bodies and induces necrosis of experimental tumors at high dose. Caprylic acid is enriched in coconut and in goat's milk. It is also a post-translational modifier of the ghrelin hormone and is produced in trace amounts in human tissues. Caprylic acid is a straight-chain isomer of the antiepileptic valproic acid, which is used in treatment of HGG-associated seizures and which may increase survival in GBM patients according to epidemiological observations. Among the valproic acids analogs tested, caprylic acid is the most potent molecule to block C6 astrocytoma cell growth in vitro and accumulates selectively within glial cells as shown by Positron Emission Tomography in vivo. Caprylic acid blocks glycolysis both in healthy liver and in malignant liver cells, which is more prominent in the latter and also lowers blood glucose. Noteworthy, caprylic acid exerts neuroprotective- and mitochondria-protective effects in several models of neurodegenerative diseases. Boost injections of caprylic acid at non-toxic levels during classical ketogenic metabolic therapy may fortify antitumor actions and reduce systemic toxicity by differential programming of mitochondrial and other metabolic pathways.

Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as ‘test-of-cure’ tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.

BackgroundD-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) can form a complex that can regulate the major metabolic pathways, however, the exact mechanism remains unknown. We analyzed a possibility of NADH-channeling from GAPDH-NADH complex to LDH isozymes using enzymes from different cells.ResultsEnzyme-kinetics and NADH-binding studies showed that LDH can use GAPDH-NADH complex as a substrate. LDH activity with GAPDH-NADH complex was challenged with anti-LDH antibodies to show that the channeled and the diffusive reactions always take place in parallel. The channeling path is dominant only in assays with limiting free-NADH concertation that mimic cytosolic conditions. Analytical ultracentrifugation showed that the channeling does not require a high affinity complex. Molecular dynamics calculations and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at subsaturating NADH concentrations. The interaction sites are conserved between LDH isozymes from heart and muscle, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites on LDH and GAPDH tetramers, showed that NAD(H)-channeling within the LDH-GAPDH complex, can be an extension of NAD(H)-channeling between the adjacent subunits in each tetramer.ConclusionsIn the case of a transient (GAPDH-NADH)-LDH complex, the relative contribution from the channeled and the diffusive paths depends on the overlap betweenoff-rates for the transient (GAPDH-NADH)-LDH complex andoff-rates for the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths.Highlights- Substrate channeling molecular mechanism can regulate energy production and aerobic and anaerobic metabolism in cells- LDH and GAPDH can form a channeling complex only at sub-saturating NADH concentration- Channeled and diffusive paths always compete and take place in parallel- NADH channeling does not require a high-affinity complex- NADH channeling within GAPDH-LDH complex is an extension of NAD(H) channeling within each tetramer- Allosteric modulation of NADH binding affinity in GAPDH tetramer can regulate NAD(H) channeling

18 p.-7 fig., Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease., This study was supported by grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (SAF2014-52050-R and SAF2017-83229-R to A.G.A). J.G.E. and D.G.W. were recipients of FPI fellowships from the Ministerio de Economía, Industria y Competitividad. K.B. research is supported by Knut and Alice Wallenberg foundation and the Beijer Institute. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites' life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids' metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosome brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style.

Trypanosomatids sequester large parts of glucose metabolism inside specialised peroxisomes, called glycosomes. Many studies have shown that correct glycosomal compartmentalization of glycolytic enzymes is essential for bloodstream-form Trypanosoma brucel. The recent finding of pore-forming activities in glycosomal membrane preparations and extensions of the trypanosome glycolysis computer model with size-selective pores sparked again an old debate on the extent of (im)permeability of the glycosomal membrane and whether glycosomally located glycolytic enzymes could and should also be present with some activity in the cytosol. This review presents a critical discussion of the experimental and theoretical evidence for and against the different hypotheses. (C) 2014 Elsevier B.V. All rights reserved.

Trypanosomatids sequester large parts of glucose metabolism inside specialised peroxisomes, called glycosomes. Many studies have shown that correct glycosomal compartmentalization of glycolytic enzymes is essential for bloodstream-form Trypanosoma brucel. The recent finding of pore-forming activities in glycosomal membrane preparations and extensions of the trypanosome glycolysis computer model with size-selective pores sparked again an old debate on the extent of (im)permeability of the glycosomal membrane and whether glycosomally located glycolytic enzymes could and should also be present with some activity in the cytosol. This review presents a critical discussion of the experimental and theoretical evidence for and against the different hypotheses. (C) 2014 Elsevier B.V. All rights reserved.

RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity. An E. coli strain that requires PNPase for viability has suppressed growth in the presence of increased citrate concentration. Transcriptome analysis reveals a PNPase-mediated response to citrate, and PNPase deletion broadly impacts on the metabolome. In vitro, citrate directly binds and modulates PNPase activity, as predicted by crystallographic data. Binding of metal-chelated citrate in the active site at physiological concentrations appears to inhibit enzyme activity. However, metal-free citrate is bound at a vestigial active site, where it stimulates PNPase activity. Mutagenesis data confirmed a potential role of this vestigial site as an allosteric binding pocket that recognizes metal-free citrate. Collectively, these findings suggest that RNA degradative pathways communicate with central metabolism. This communication appears to be part of a feedback network that may contribute to global regulation of metabolism and cellular energy efficiency.

In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.

One current challenge in metabolic systems biology is to map out the regulation networks that control metabolism. From progress in this area, we conclude that non-transcriptional mechanisms (e.g. metabolite-protein interactions and protein phosphorylation) are highly relevant in actually controlling metabolic function. Furthermore, recent results highlight more functions of enzymes and metabolites than currently appreciated in genome-scale metabolic reconstructions, thereby adding another level of complexity. Combining experimental analyses and modeling efforts we are also beginning to understand how metabolic behavior emerges. Particularly, we recognize that metabolism is not simply a dull workhorse process but rather takes very active control of itself and other cellular processes, rendering true system-level understanding of metabolism possibly more difficult than for other cellular systems.

One current challenge in metabolic systems biology is to map out the regulation networks that control metabolism. From progress in this area, we conclude that non-transcriptional mechanisms (e.g. metabolite-protein interactions and protein phosphorylation) are highly relevant in actually controlling metabolic function. Furthermore, recent results highlight more functions of enzymes and metabolites than currently appreciated in genome-scale metabolic reconstructions, thereby adding another level of complexity. Combining experimental analyses and modeling efforts we are also beginning to understand how metabolic behavior emerges. Particularly, we recognize that metabolism is not simply a dull workhorse process but rather takes very active control of itself and other cellular processes, rendering true system-level understanding of metabolism possibly more difficult than for other cellular systems.

Time-dependent regulation analysis is a new methodology that allows us to unravel, both quantitatively and dynamically, how and when functional changes in the cell are brought about by the interplay of gene expression and metabolism. In this first experimental implementation, we dissect the initial and late response of baker's yeast upon a switch from glucose-limited growth to nitrogen starvation. During nitrogen starvation, unspecific bulk degradation of cytosolic proteins and small organelles (autophagy) occurs. If this is the primary cause of loss of glycolytic capacity, one would expect the cells to regulate their glycolytic capacity through decreasing simultaneously and proportionally the capacities of the enzymes in the first hour of nitrogen starvation. This should lead to regulation of the flux which is initially dominated by changes in the enzyme capacity. However, metabolic regulation is also known to act fast. To analyse the interplay between autophagy and metabolism, we examined the first 4 h of nitrogen starvation in detail using time-dependent regulation analysis. Some enzymes were initially regulated more by a breakdown of enzyme capacity and only later through metabolic regulation. However, other enzymes were regulated metabolically in the first hours and then shifted towards regulation via enzyme capacity. We conclude that even initial regulation is subtle and governed by different molecular levels.

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites' life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids' metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosoma brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style.

Pathogenic bacteria sense the host environment and regulate expression of virulence-related genes. Environmental signals like temperature, bicarbonate/CO2 and glucose induce toxin production in Bacillus anthracis, but the mechanisms by which these signals contribute to virulence and overall physiological adaptation remains elusive. An integrated, systems level investigation using transcriptomics and iTRAQ-based proteomics was done to assess the effect of temperature, bicarbonate/CO2 and glucose on B. anthracis. Significant changes observed in amino acid, carbohydrate, energy and nucleotide metabolism indicates events of metabolic readjustments by environmental factors. Directed induction of genes involved in polyamine biosynthesis and iron metabolism revealed the redirection of cellular metabolite pool towards iron uptake. Protein levels of glycolytic enzymes, ptsH and Ldh along with transcripts involved in immune evasion (mprF, bNOS, Phospholipases and asnA), cell surface remodeling (rfbABCD, antABCD, and cls) and utilization of lactate (lutABC) and inositol showed constant repression under environmental perturbations. Discrepancies observed in mRNA/protein level of genes involved in glycolysis, protein synthesis, stress response and nucleotide metabolism hinted at the existence of additional regulatory layers and illustrated the utility of an integrated approach. The above findings might assist in the identification of novel adaptive strategies of B. anthracis during host associated survival and pathogenesis. Biological Significance In this study, the changes observed at both transcript and protein level were quantified and integrated to understand the effect of host environmental factors (host temperature, bicarbonate and glucose) in shaping the physiology and adaptive strategies of a fully virulent strain of B. anthracis for efficient survival and virulence in its host. Perturbations affecting toxin production were found to concordantly affect vital metabolic pathways and several known as well as novel virulence factors. These changes act as a valuable asset for generating testable hypotheses that can be further verified by detailed molecular and mutant studies to identify novel adaptive strategies of B. anthracis during infection. Adaptation of an integrated transcriptomics and proteomics approach also led to the identification of discrepancies between mRNA/protein levels among genes across major functional categories. Few of these discrepancies have been previously reported in literature for model organisms. However their existence in B. anthracis and that too as a result of growth perturbations have not been reported till date. These findings demonstrate a substantial role of regulatory processes post mRNA synthesis via post transcriptional, translational or protein degradation mechanisms. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis). Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies., Author Summary An increasing number of mathematical models are being built and analysed in order to obtain a better understanding of specific biological systems. These quantitative models contain parameters that need to be measured or estimated. Because of experimental errors or lack of data, our knowledge about these parameters is uncertain. Our work explores the effect of including these uncertainties in model analysis. Therefore, we studied a particularly well curated model of the energy metabolism of the parasite Trypanosoma brucei, responsible for African sleeping sickness. We first collected all the information we could find about how the model parameters were defined on a website, the SilicoTryp wiki (http:///silicotryp.ibls.gla.ac.uk/wiki/). From this information, we were able to quantify our uncertainty about each parameter, thus allowing us to analyse the model while explicitly taking these uncertainties into account. We found that, even though the model was well-defined and most of its parameters were experimentally measured, taking into account the remaining uncertainty allows us to gain more insight into model behavior. We were able to identify previously unrecognised fragilities of the model, leading to new hypotheses amenable to experimental testing.

In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.

In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.

The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12] from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-angstrom resolution with an x-ray data set collected by the Laue method. For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec. The structure was solved by using the Bacillus stearothermophilus enzyme model [Skarzynski, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data set which was 37% complete. The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of >60% of the reflections by 6-fold density averaging. After molecular dynamics refinement, the current molecular model has an R factor of 17.6%. Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-angstrom resolution, reveals important structural differences in the NAD binding region. These are of great interest for the design of specific inhibitors of the parasite enzyme.

Biology and medicine have become 'big science', even though we may not always like this: genomics and the subsequent analysis of what the genomes encode has shown that interesting living organisms require manymore than 300 gene products to interact.We once thought that somewhere in this jungle of interacting macromolecules was hidden the molecule that constitutes the secret of Life, and therewith of health and disease. Now we know that, somehow, the secret of Life is the jungle of interactions. Consequently, we need to find the Rosetta Stones, i.e. interpretations of this jungle of systems biology. We need to find, perhaps convoluted, paths of understanding and intervention. Systems biochemistry is a good place to start, as it has the foothold that what goes in must come out. In the present paper, we review two strategies, which look at control and regulation. We discuss the difference between control and regulation and prove a relationship between them. ©The Authors Journal compilation.

SUMMARYAfrican trypanosomes have emerged as promising unicellular model organisms for the next generation of systems biology. They offer unique advantages, due to their relative simplicity, the availability of all standard genomics techniques and a long history of quantitative research. Reproducible cultivation methods exist for morphologically and physiologically distinct life-cycle stages. The genome has been sequenced, and microarrays, RNA-interference and high-accuracy metabolomics are available. Furthermore, the availability of extensive kinetic data on all glycolytic enzymes has led to the early development of a complete, experiment-based dynamic model of an important biochemical pathway. Here we describe the achievements of trypanosome systems biology so far and outline the necessary steps towards the ambitious aim of creating a ‘Silicon Trypanosome’, a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology. We expect that, in the long run, the quantitative modelling enabled by the Silicon Trypanosome will play a key role in selecting the most suitable targets for developing new anti-parasite drugs.

Previous studies indicated increased levels of protein oxidation in brain from subjects with Alzheimer's disease (AD), raising the question of whether oxidative damage is a late effect of neurodegeneration or precedes and contributes to the pathogenesis of AD. Hence, in the present study we used a parallel proteomic approach to identify oxidatively modified proteins in inferior parietal lobule (IPL) from subjects with mild cognitive impairment (MCI) and early stage-AD (EAD). By comparing to age-matched controls, we reasoned that such analysis could help in understanding potential mechanisms involved in upstream processes in AD pathogenesis. We have identified four proteins that showed elevated levels of protein carbonyls: carbonic anhydrase II (CA II), heat shock protein 70 (Hsp70), mitogen-activated protein kinase I (MAPKI), and syntaxin binding protein I (SBP1) in MCI IPL. In EAD IPL we identified three proteins: phosphoglycerate mutase 1 (PM1), glial fibrillary acidic protein, and fructose bisphospate aldolase C (FBA-C). Our results imply that some of the common targets of protein carbonylation correlated with AD neuropathology and suggest a possible involvement of protein modifications in the AD progression. Antioxid. Redox Signal. 12, 327–336.

Time-dependent regulation analysis is a new methodology that allows us to unravel, both quantitatively and dynamically, how and when functional changes in the cell are brought about by the interplay of gene expression and metabolism. In this first experimental implementation, we dissect the initial and late response of baker's yeast upon a switch from glucose-limited growth to nitrogen starvation. During nitrogen starvation, unspecific bulk degradation of cytosolic proteins and small organelles (autophagy) occurs. If this is the primary cause of loss of glycolytic capacity, one would expect the cells to regulate their glycolytic capacity through decreasing simultaneously and proportionally the capacities of the enzymes in the first hour of nitrogen starvation. This should lead to regulation of the flux which is initially dominated by changes in the enzyme capacity. However, metabolic regulation is also known to act fast. To analyse the interplay between autophagy and metabolism, we examined the first 4 h of nitrogen starvation in detail using time-dependent regulation analysis. Some enzymes were initially regulated more by a breakdown of enzyme capacity and only later through metabolic regulation. However, other enzymes were regulated metabolically in the first hours and then shifted towards regulation via enzyme capacity. We conclude that even initial regulation is subtle and governed by different molecular levels.

A high rate of glycolytic flux, even in the presence of oxygen, is a central metabolic hallmark of neoplastic tumors. Cancer cells preferentially utilize glycolysis in order to satisfy their increased energetic and biosynthetic requirements. This metabolic phenotype has been confirmed in human studies using positron emission tomography (PET) with F-18-2-fluoro-deoxy-glucose which have demonstrated that tumors take up 10-fold more glucose than adjacent normal tissues in vivo. The high glucose metabolism of cancer cells is caused by a combination of hypoxia-responsive transcription factors, activation of oncogenic proteins and the loss of tumor suppressor function. Over-expression of HIF-1 alpha and myc, activation of ras and loss of p53 function each have been found to stimulate glycolysis in part by activating a family of regulatory bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB). The PFKFB enzymes synthesize fructose-2,6-bisphosphate (F2,6BP) which allosterically activates 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. PFK-1 is inhibited by ATP when energy stores are abundant and F2,6BP can override this inhibition and enhance glucose uptake and glycolytic flux. It is therefore not surprising that F2,6BP synthesis is stimulated by several oncogenic alterations which simultaneously cause both enhanced consumption of glucose and growth. Importantly, these studies suggest that selective depletion of intracellular F2,6BP in cancer cells may suppress glycolytic flux and decrease their survival, growth and invasiveness. This review will summarize the requirement of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases for the regulation of glycolysis in tumor cells and their potential utility as targets for the development of antineoplastic agents. United States Department of Health & Human Services National Institutes of Health (NIH) - USA (1P20RR018733) United States Department of Health & Human Services National Institutes of Health (NIH) - USA (1R01CA116428) Leukemia and Lymphoma Society United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) ( R01CA116428) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Center for Research Resources (NCRR) ( P20RR018733)

The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5 - 10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on K(m) and V(max). The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.

The three-dimensional crystal structure of the enzyme glyceraldehyde phosphate dehydrogenase from the kinetoplastid Trypanosoma brucei brucei has been determined at 3.2 Angstrom resolution from a 37% complete data set collected using the Laue method. The crystals used in the structure determination contain one and a half tetrameric enzyme molecules in the asymmetric unit, i.e. six identical subunits. Initial phasing was carried out by the method of molecular replacement using the refined coordinates of hole glyceraldehyde phosphate dehydrogenase from Bacillus stearothermophilus as a search model. The initial electron-density distribution, obtained from the molecular-replacement solution, was greatly improved by a procedure consisting of 36 cycles of iterative non-crystallographic density averaging. During the averaging procedure, the missing reflections (63% of the data) were gradually introduced as map-inversion structure factors. At completion of the procedure, the R factor between averaged map-inversion amplitudes and observed structure-factor amplitudes was 19.0% for all data between 7.0 and 3.2 Angstrom resolution, and that between the map-inversion amplitudes and later recorded structure-factor amplitudes was 41.9%. After model building into the resulting averaged electron-density map, refinement by molecular-dynamics procedures with X-PLOR provided the current model, which has an R factor of 17.6% for 34 835 reflections between 7.0 and 3.2 Angstrom resolution. The refined model, comprising 2735 protein atoms plus one NAD(+) molecule and two sulfate ions per subunit, has r.ms. deviations from ideality of 0.02 Angstrom for bond lengths and 3.6 degrees for bond angles. All subunits, located either within the tetrameric molecule or within the half tetramer present in the asymmetric unit, are related to each other by almost exact twofold symmetry. The overall structure of the glycosomal glyceraldehyde phosphate dehydrogenase subunit and its quaternary arrangement in the tetrameric molecule are similar to that of the enzyme of lobster and Bacillus stearothermophilus (with r.m.s. differences between equivalent C alpha positions of 0.71 and 0.64 Angstrom, respectively). The main differences between the structures is the presence of three insertions, plus the substitution of a beta-strand by a short alpha-helix, both occurring at the surface of the glycosomal enzyme subunit.

The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5 - 10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on K(m) and V(max). The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.

Shigella flexneri, a Gram‐negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. During infection of epithelial cells, Shigella escape from the phagosome to the cytosol, where they reroute host cell glycolysis to obtain nutrients for proliferation. Septins, a poorly understood component of the cytoskeleton, can entrap cytosolic Shigella targeted to autophagy in cage‐like structures to restrict bacterial proliferation. Although bacterial entrapment by septin caging has been the subject of intense investigation, the role of septins and the autophagy machinery in the proliferation of noncaged Shigella is mostly unknown. Here, we found that intracellular Shigella fail to efficiently proliferate in SEPT2‐, SEPT7‐, or p62/SQSTM1‐depleted cells. Consistent with a failure to proliferate, single cell analysis of bacteria not entrapped in septin cages showed that the number of metabolically active Shigella in septin‐ or p62‐depleted cells is reduced. Targeted metabolomic analysis revealed that host cell glycolysis is dysregulated in septin‐depleted cells, suggesting a key role for septins in modulation of glycolysis. Together, these results suggest that septins and the autophagy machinery may regulate metabolic pathways that promote the proliferation of intracellular Shigella not entrapped in septin cages.

Protein-protein interactions (PPIs) are an effective means to orchestrate intricate biological processes required to sustain life. Approximately 650 000 PPIs underlie the human interactome; thus underscoring its complexity and the manifold signaling outputs altered in response to changes in specific PPIs. This minireview illustrates the growing arsenal of PPI assemblies and offers insights into how these varied PPI regulatory modalities are relevant to customized drug discovery, with a focus on cancer. First, known and emerging PPIs and PPI-targeted drugs of both natural and synthetic origin are categorized. Building on these discussions, the merits of PPI-guided therapeutics over traditional drug design are discussed. Finally, a compare-and-contrast section for different PPI blockers, with gain-of-function PPI interventions, such as PROTACS, is provided.