Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.     

Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins

Protein glycation by methylglyoxal is a nonenzymatic post-translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end-products. Methylglyoxal-derived advanced glycation end-products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI-TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolase's activity loss, in a glycation-dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end-products were determined by MALDI-TOF. Yeast cells experienced selective pressure towards efficient use of d-glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important post-translational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through over-expression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Over-expression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.     

The SUMO pathway functions in mouse oocyte maturation

Sumoylation is an important posttranslational modification in which SUMO (small ubiquitin-related modifier) proteins are bonded covalently to their substrates. Studies on the roles of sumoylation in cell cycle regulation have been emerging in both mitosis from yeast to mammals and meiosis in budding yeast, but the functions of sumoylation in mammalian meiosis, especially in oocyte meiotic maturation are not well known. Here, we examined the localization and expression of SUMO-1 and SUMO-2/3, the two basic proteins in the sumoylation pathway and investigated their roles through overexpression of Senp2 during mouse oocyte maturation. Immunofluorescent staining revealed differential patterns of SUMO-1 and SUMO-2/3 localization: SUMO-1 was localized to the spindle poles in prometaphase I, MI and MII stages, around the separating homologues in anaphase I and telophase I stages of first meiosis, while SUMO-2/3 was mainly concentrated near centromeres during mouse oocyte maturation. Immunoblot analysis uncovered the different expression profiles of SUMO-1 and SUMO-2/3 modified proteins during mouse oocyte maturation. Overexpression of Senp2, a SUMO-specific isopeptidase, caused changes of SUMO-modified proteins and led to defects in MII spindle organization in mature eggs. These results suggest that the SUMO pathway may play an indispensable role during mouse oocyte meiotic maturation.Key words: sumoylation, mouse oocyte maturation, overexpression, Senp2, MII spindle     

Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress

Abstract

Cancer cells reprogram metabolism to maintain rapid proliferation under often stressful conditions. Glycolysis and glutaminolysis are two central pathways that fuel cancer metabolism. Allosteric regulation and metabolite driven post-translational modifications of key metabolic enzymes allow cancer cells glycolysis and glutaminolysis to respond to changes in nutrient availability and the tumor microenvironment. While increased aerobic glycolysis (the Warburg effect) has been a noted part of cancer metabolism for over 80 years, recent work has shown that the elevated levels of glycolytic intermediates are critical to cancer growth and metabolism due to their ability to feed into the anabolic pathways branching off glycolysis such as the pentose phosphate pathway and serine biosynthesis pathway. The key glycolytic enzymes phosphofructokinase-1 (PFK1), pyruvate kinase (PKM2) and phosphoglycerate mutase 1 (PGAM1) are regulated by upstream and downstream metabolites to balance glycolytic flux with flux through anabolic pathways. Glutamine regulation is tightly controlled by metabolic intermediates that allosterically inhibit and activate glutamate dehydrogenase, which fuels the tricarboxylic acid cycle by converting glutamine derived glutamate to α-ketoglutarate. The elucidation of these key allosteric regulatory hubs in cancer metabolism will be essential for understanding and predicting how cancer cells will respond to drugs that target metabolism. Additionally, identification of the structures involved in allosteric regulation will inform the design of anti-metabolism drugs which bypass the off-target effects of substrate mimics. Hence, this review aims to provide an overview of allosteric control of glycolysis and glutaminolysis.     

HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.     

A Fresh View of Glycolysis and Glucokinase Regulation: History and Current Status

This minireview looks back at a century of glycolysis research with a focus on the mechanisms of flux regulation. Traditionally, glycolysis is regarded as a feeder pathway that prepares glucose for further catabolism and energy production. However, glycolysis is much more than that, in particular in those tissues that express the low affinity glucose-phosphorylating enzyme glucokinase. This enzyme equips the glycolytic pathway with a special steering function for the regulation of intermediary metabolism. In beta cells, glycolysis acts as a transducer for triggering and amplifying physiological glucose-induced insulin secretion. On the basis of these considerations, I have defined a glycolytic flux regulatory unit composed of the two fructose ester steps of this pathway with various enzymes and metabolites that regulate glycolysis.     

Activity-dependent SUMOylation of the brain-specific scaffolding protein GISP

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.     

SUMO-2/3 modification and binding regulate the association of CENP-E with kinetochores and progression through mitosis

SUMOylation is essential for cell-cycle regulation in invertebrates; however, its functions during the mammalian cell cycle are largely uncharacterized. Mammals express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are 96% identical and referred to as SUMO-2/3). We found that SUMO-2/3 localize to centromeres and condensed chromosomes, whereas SUMO-1 localizes to the mitotic spindle and spindle midzone, indicating that SUMO paralogs regulate distinct mitotic processes in mammalian cells. Consistent with this, global inhibition of SUMOylation caused a prometaphase arrest due to defects in targeting the microtubule motor protein CENP-E to kinetochores. CENP-E was found to be modified specifically by SUMO-2/3 and to possess SUMO-2/3 polymeric chain-binding activity essential for kinetochore localization. Our findings indicate that SUMOylation is a key regulator of the mammalian cell cycle, with SUMO-1 and SUMO-2/3 modification of different proteins regulating distinct processes.     

Roles of hemoglobin Allostery in hypoxia-induced metabolic alterations in erythrocytes: simulation and its verification by metabolome analysis

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.     

Energy metabolism transition in multi-cellular human tumor spheroids

It is thought that glycolysis is the predominant energy pathway in cancer, particularly in solid and poorly vascularized tumors where hypoxic regions develop. To evaluate whether glycolysis does effectively predominate for ATP supply and to identify the underlying biochemical mechanisms, the glycolytic and oxidative phosphorylation (OxPhos) fluxes, ATP/ADP ratio, phosphorylation potential, and expression and activity of relevant energy metabolism enzymes were determined in multi-cellular tumor spheroids, as a model of human solid tumors. In HeLa and Hek293 young-spheroids, the OxPhos flux and cytochrome c oxidase protein content and activity were similar to those observed in monolayer cultured cells, whereas the glycolytic flux increased two- to fourfold; the contribution of OxPhos to ATP supply was 60%. In contrast, in old-spheroids, OxPhos, ATP content, ATP/ADP ratio, and phosphorylation potential diminished 50-70%, as well as the activity (88%) and content (3 times) of cytochrome c oxidase. Glycolysis and hexokinase increased significantly (both, 4 times); consequently glycolysis was the predominant pathway for ATP supply (80%). These changes were associated with an increase (3.3 times) in the HIF-1alpha content. After chronic exposure, both oxidative and glycolytic inhibitors blocked spheroid growth, although the glycolytic inhibitors, 2-deoxyglucose and gossypol (IC(50) of 15-17 nM), were more potent than the mitochondrial inhibitors, casiopeina II-gly, laherradurin, and rhodamine 123 (IC(50) > 100 nM). These results suggest that glycolysis and OxPhos might be considered as metabolic targets to diminish cellular proliferation in poorly vascularized, hypoxic solid tumors.     

Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae.

The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions. Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level. The Pasteur effect was thus abolished in these cells. The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation. The concentrations of the enzyme substrates showed only small or insignificant changes. These data imply that the enzyme has a low flux control coefficient for glycolysis. However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration. Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.     

A lack of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast

Yeast SUMO (Smt3) and its mammalian ortholog SUMO-1 are ubiquitin-like proteins that can reversibly be conjugated to other proteins. Among the substrates for SUMO modification in vertebrates are RanGAP1 and RanBP2/Nup358, two proteins previously implicated in nucleocytoplasmic transport. Sumoylated RanGAP1 binds to the nuclear pore complex via RanBP2/Nup358, a giant nucleoporin, which was recently reported to act as a SUMO E3 ligase on some nuclear substrates. However, no direct evidence for a role of the SUMO system in nuclear transport has been obtained so far. By the use of conditional yeast mutants, we examined nuclear protein import in vivo. We show here that cNLS-dependent protein import is impaired in mutants with defective Ulp1 and Uba2, two enzymes involved in the SUMO conjugation reaction. In contrast, other transport pathways such as rgNLS-mediated protein import and mRNA export are not affected. Furthermore, we find that the yeast importin-alpha subunit Srp1 accumulates in the nucleus of ulp1 and uba2 strains but not the importin-beta subunit Kap95, indicating that a lack of Srp1 export might impair cNLS import. In summary, our results provide evidence that SUMO modification in yeast, as has been suspected for vertebrates, plays an important role in nucleocytoplasmic trafficking.     

Glutamine‐driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia

Mammalian cells can generate ATP via glycolysis or mitochondrial respiration. Oncogene activation and hypoxia promote glycolysis and lactate secretion. The significance of these metabolic changes to ATP production remains however ill defined. Here, we integrate LC‐MS‐based isotope tracer studies with oxygen uptake measurements in a quantitative redox‐balanced metabolic flux model of mammalian cellular metabolism. We then apply this approach to assess the impact of Ras and Akt activation and hypoxia on energy metabolism. Both oncogene activation and hypoxia induce roughly a twofold increase in glycolytic flux. Ras activation and hypoxia also strongly decrease glucose oxidation. Oxidative phosphorylation, powered substantially by glutamine‐driven TCA turning, however, persists and accounts for the majority of ATP production. Consistent with this, in all cases, pharmacological inhibition of oxidative phosphorylation markedly reduces energy charge, and glutamine but not glucose removal markedly lowers oxygen uptake. Thus, glutamine‐driven oxidative phosphorylation is a major means of ATP production even in hypoxic cancer cells.     

Chronic hypoxia-induced alterations of key enzymes of glucose oxidative metabolism in developing mouse liver are mTOR dependent

Hypoxia is a potent regulator of gene expression and cellular energy metabolism and known to interfere with post-natal growth and development. Although hypoxia can induce adaptive changes in the developing liver, the mechanisms underlying these changes are poorly understood. To elucidate some of the adaptive changes chronic hypoxia induces in the developing liver, we studied the expression of the genes of mammalian target of rapamycin (mTOR) signaling and glucose metabolism, undertook proteomic examination with 2D gel-MS/MS of electron transport chain, and determined activities and protein expression of several key regulatory enzymes of glucose oxidative metabolism. To gain insight into the molecular mechanism underlying hypoxia-induced liver metabolic adaptation, we treated a subset of mice with rapamycin (0.5 mg/kg/day) to inhibit mTOR postnatally. Rapamycin-treated mice showed lower birth weight, lower body weight, and liver growth retardation in a pattern similar to that observed in the hypoxic mice at P30. Rapamycin treatment led to differential impact on the cytoplasmic and mitochondrial pathways of glucose metabolism. Our results suggest a decrease in mTOR activity as part of the mechanisms underlying hypoxia-induced changes in the activities of glycolytic and TCA cycle enzymes in liver. Chronic postnatal hypoxia induces mTOR-dependent differential effects on liver glycolytic and TCA cycle enzymes and as such should be studied further as they have pathophysiological implications in hepatic diseases and conditions in which hypoxia plays a role.     

Solution structure of human SUMO-3 C47S and its binding surface for Ubc9

Small ubiquitin-related modifier SUMO-3 is a member of a growing family of ubiquitin-like proteins (Ubls). So far, four isoforms of SUMO have been identified in humans. It is generally known that SUMO modification regulates protein localization and activity. Previous structure and function studies have been mainly focused on SUMO-1. The sequence of SUMO-3 is 46% identical with that of SUMO-1; nevertheless, functional heterogeneity has been found between the two homologues. Here we report the solution structure of SUMO-3 C47S (residues 14-92) featuring the beta-beta-alpha-beta-beta-alpha-beta ubiquitin fold. Structural comparison shows that SUMO-3 C47S resembles ubiquitin more than SUMO-1. On the helix-sheet interface, a strong hydrophobic interaction contributes to formation of the globular and compact fold. A Gly-Gly motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. In vivo, SUMO modification proceeds via a multistep pathway, and Ubc9 plays an indispensable role as the SUMO conjugating enzyme (E2) in this process. To develop a better understanding of SUMO-3 conjugation, the Ubc9 binding surface on SUMO-3 C47S has been detected by chemical shift perturbation using NMR spectroscopy. The binding site mainly resides on the hydrophilic side of the beta-sheet. Negatively charged and hydrophobic residues of this region are highly or moderately conserved among SUMO family members. Notably, the negatively charged surface of SUMO-3 C47S is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. This work indicates dissimilarities between SUMO-3 and SUMO-1 in tertiary structure and provides insight into the specific interactions of SUMO-3 with its modifying enzyme.