Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP-metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production.

Pancreatic beta cells secrete insulin in response to plasma glucose. The ATP-sensitive potassium channel (KATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that pyruvate kinase (PK) in close proximity to beta cell KATP locally produces the ATP that inhibits KATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited KATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded KATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of KATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit KATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on KATP channel closure by glucose, further suggesting that PK is not an important regulator of KATP . In contrast, addition of mitochondrial inhibitors robustly increased KATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that oxidative phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial oxidative phosphorylation is the primary controller of KATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit KATP activity in excised patches. PEP solutions only inhibit KATP activity if the pH is unbalanced. Modulating pyruvate kinase has minimal effects on KATP activity. Mitochondrial inhibition, in contrast, robustly potentiates KATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via oxidative phosphorylation to close KATP . Mitochondrial oxidative phosphorylation is therefore the main source of the ATP that inhibits the KATP activity of pancreatic beta cells.

Wheat germ peptide improves glucose metabolism and insulin resistance in HepG2 hepatocytes via regulating SOCS3/IRS1/Akt pathway.

Evidence has demonstrated that oxidative stress plays a crucial role in regulating cellular glucose metabolism. In previous studies, wheat germ peptide (WGP) was found to effectively mitigate oxidative stress induced by high glucose. Based on the information provided, we hypothesized that WGP could exhibit antihyperglycemic and anti-insulin-resistant effects in cells. The insulin-resistant cell model was established by insulin stimulation. The glucose consumption, glycogen content, and the activities of hexokinase and pyruvate kinase following WGP treatment were measured. The protein expression of SOCS3, phosphorylated insulin receptor substrate-1 (p-IRS1), IRS1, phosphorylated protein kinase B (p-Akt), Akt, glucose transporter 2 (GLUT2), phosphorylated GSK 3ß, GSK 3ß, FOXO1, G6P, and phosphoenolpyruvate carboxykinase were assessed by western blot analysis. Our results demonstrated that WGP treatment increased cellular glucose consumption and glycogen synthesis and enhanced hexokinase and pyruvate kinase activities. Additionally, WGP treatment was observed to cause a significant reduction in the expression of SOCS3, FOXO1, G6P, and phosphoenolpyruvate carboxykinase, as well as in the ratio of p-IRS1/IRS1. Conversely, the expression of GLUT2 and the ratios of p-Akt/Akt and p-GSK3ß/GSK3ß were upregulated by WGP. These findings suggested that WGP can activate the SOCS3/IRS1/Akt signaling pathway, thus promoting the phosphorylation of GSK-3ß and increasing the expression of FOXO1 and GLUT2, which contribute to enhancing glycogen synthesis, inhibiting gluconeogenesis, and promoting glucose transport in insulin-resistant HepG2 cells.

Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cells.

IL-17-producing antigen-specific human T cells elicit potent antitumor activity in mice. Yet, refinement of this approach is needed to position it for clinical use. While activation signal strength regulates IL-17 production by CD4+ T cells, the degree to which T cell antigen receptor (TCR) and costimulation signal strength influences Th17 immunity remains unknown. We discovered that decreasing TCR/costimulation signal strength by incremental reduction of αCD3/costimulation beads progressively altered Th17 phenotype. Moreover, Th17 cells stimulated with αCD3/inducible costimulator (ICOS) beads produced more IL-17A, IFNγ, IL-2, and IL-22 than those stimulated with αCD3/CD28 beads. Compared with Th17 cells stimulated with the standard, strong signal strength (three beads per T cell), Th17 cells propagated with 30-fold fewer αCD3/ICOS beads were less reliant on glucose and favored the central carbon pathway for bioenergetics, marked by abundant intracellular phosphoenolpyruvate (PEP). Importantly, Th17 cells stimulated with weak αCD3/ICOS beads and redirected with a chimeric antigen receptor that recognizes mesothelin were more effective at clearing human mesothelioma. Less effective CAR Th17 cells generated with high αCD3/ICOS beads were rescued by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1), a PEP regulator. Thus, Th17 therapy can be improved by using fewer activation beads during manufacturing, a finding that is cost effective and directly translatable to patients.

Mitochondrial phosphoenolpyruvate carboxykinase promotes tumor growth in estrogen receptor-positive breast cancer via regulation of the mTOR pathway.

BACKGROUND: Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer. METHODS: We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer. RESULTS: PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown. CONCLUSION: PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.

Phosphoenolpyruvate reallocation links nitrogen fixation rates to root nodule energy state.

Legume-rhizobium symbiosis in root nodules fixes nitrogen to satisfy the plant's nitrogen demands. The nodules' demand for energy is thought to determine nitrogen fixation rates. How this energy state is sensed to modulate nitrogen fixation is unknown. Here, we identified two soybean (Glycine max) cystathionine ß-synthase domain-containing proteins, nodule AMP sensor 1 (GmNAS1) and NAS1-associated protein 1 (GmNAP1). In the high-nodule energy state, GmNAS1 and GmNAP1 form homodimers that interact with the nuclear factor-Y C (NF-YC) subunit (GmNFYC10a) on mitochondria and reduce its nuclear accumulation. Less nuclear GmNFYC10a leads to lower expression of glycolytic genes involved in pyruvate production, which modulates phosphoenolpyruvate allocation to favor nitrogen fixation. Insight into these pathways may help in the design of leguminous crops that have improved carbon use, nitrogen fixation, and growth.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Proteomic analysis reveals molecular mechanism of Cd2+ tolerance in the leaves of halophyte Halogeton glomeratus.

Halogeton glomeratus (H. glomeratus) is categorized as a halophyte, it can potentially endure not only salt but also heavy metals. The aim of this work was to study the molecular mechanisms underlying the Cd2+ tolerance of halophyte H. glomeratus seedlings. For that we used a combination of physiological characteristics and data-independent acquisition-based proteomic approaches. The results revealed that the significant changes of physiological characteristics of H. glomeratus occurred under approximately 0.4 mM Cd2+ condition and that Cd2+ accumulated in Cd2+-treated seedling roots, stems and leaves. At the early stage of Cd2+ stress, numerous differentially abundant proteins related to "phosphoenolpyruvate carboxylase", "transmembrane transporters", and "vacuolar protein sorting-associated protein" took important roles in the response of H. glomeratus to Cd2+ stress. At the later stage of Cd2+ stress, some differentially abundant proteins involved in "alcohol-forming fatty acyl-CoA reductase", "glutathione transferase", and "abscisic acid receptor" were considered to regulate the adaptation of H. glomeratus exposed to Cd2+ stress. Finally, we found various detoxification-related differentially abundant proteins related to Cd2+ stress. These biological processes and regulators synergistically regulated the Cd2+ tolerance of H. glomeratus. SIGNIFICANCE: The halophyte, H.glomeratus, has a strong tolerance to salinity, also survives in the heavy metal stress. At present, there are few reports on the comprehensive characterization and identification of Cd2+ response and adaption related regulators in H.glomeratus. This research focuses on the molecular mechanisms of H. glomeratus tolerance to Cd2+ stress at proteome levels to uncover the novel insight of the Cd2+-related biological processes and potential candidates involved in the response and adaption mechanism. The results will help elucidate the genetic basis of this species' tolerance to Cd2+ stress and develop application prospect of wild genetic resources to heavy metal phytoremediation.

Hepatic overexpression of protein targeting to glycogen attenuates obesity and improves hyperglycemia in db/db mice.

Increased liver glycogen content has been shown to reduce food intake, attenuate obesity, and improve glucose tolerance in a mouse model of high-fat diet (HFD)-induced obesity. Here we studied the contribution of liver glycogen to the regulation of obesity and glucose metabolism in a model of type 2 diabetes and obesity, namely the db/db mouse. To this end, we crossed db/db mice with animals overexpressing protein targeting to glycogen (PTG) in the liver to generate db/db mice with increased liver glycogen content (db/db-PTG). Hepatic PTG overexpression reduced food intake and fat weight and attenuated obesity and hyperglycemia in db/db mice. Db/db-PTG mice showed similar energy expenditure and physical activity to db/db mice. PTG overexpression reduced liver phosphoenolpyruvate carboxykinase (PEPCK) protein levels and repressed hepatic glucose production in db/db mice. Moreover, increased liver glycogen elevated hepatic ATP content in these animals. However, lipid metabolism was not modified by PTG overexpression. In conclusion, increased liver glycogen content ameliorates the diabetic and obesity phenotype in db/db mice.

Dietary iron modulates hepatic glucose homeostasis via regulating gluconeogenesis.

Iron exerts significant influences on glucose metabolism. However, the regulatory mechanisms underlying disordered glucose response remains largely unclear. The aim of this study was to examine the impact of dietary iron on hepatic gluconeogenesis in mice and in rat liver-derived cells. High iron models of C57BL/6J mice were fed with 1.25 g Fe/kg diets for 9 weeks, and high-iron BRL-3A cell models were treated with 250 µmol/L FeSO4 for 12 h and 24 h. Our data showed that higher iron intake resulted in higher hepatic iron without iron toxicity, and reduced body weight gain with no difference of food intakes. High dietary iron significantly increased 61% of hepatic glycogen deposition, but exhibited impairment in glucose responses in mice. Moreover, high dietary iron suppressed hepatic gluconeogenesis by repressing the expression of key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Meanwhile, mice fed with higher iron diets exhibited both decreased AMP-activated protein kinase (AMPK) activity and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) protein levels. Furthermore, in BRL-3A cells, iron treatment increased cellular glucose uptake, and altered gluconeogenesis rhythmically by regulating the activation of AMPK and expression of PGC-1α successively. This study demonstrated that dietary high iron was able to increase hepatic glycogen deposition by enhancement of glucose uptake, and suppress hepatic gluconeogenesis by regulation of AMPK and PGC-1α.

BnaPPT1 is essential for chloroplast development and seed oil accumulation in Brassica napus.

INTRODUCTION: Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis. OBJECTIVES: This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus. METHODS: We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function. RESULTS: The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%. CONCLUSION: Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.

Adenylate Kinase Isozyme 3 Regulates Mitochondrial Energy Metabolism and Knockout Alters HeLa Cell Metabolism.

The balance between oxidative phosphorylation and glycolysis is important for cancer cell growth and survival, and changes in energy metabolism are an emerging therapeutic target. Adenylate kinase (AK) regulates adenine nucleotide metabolism, maintaining intracellular nucleotide metabolic homeostasis. In this study, we focused on AK3, the isozyme localized in the mitochondrial matrix that reversibly mediates the following reaction: Mg2+ GTP + AMP ⇌ Mg2+ GDP + ADP. Additionally, we analyzed AK3-knockout (KO) HeLa cells, which showed reduced proliferation and were detected at an increased number in the G1 phase. A metabolomic analysis showed decreased ATP; increased glycolytic metabolites such as glucose 6 phosphate (G6P), fructose 6 phosphate (F6P), and phosphoenolpyruvate (PEP); and decreased levels of tricarboxylic acid (TCA) cycle metabolites in AK3KO cells. An intracellular ATP evaluation of AK3KO HeLa cells transfected with ATeam plasmid, an ATP sensor, showed decreased whole cell levels. Levels of mitochondrial DNA (mtDNA), a complementary response to mitochondrial failure, were increased in AK3KO HeLa cells. Oxidative stress levels increased with changes in gene expression, evidenced as an increase in related enzymes such as superoxide dismutase 2 (SOD2) and SOD3. Phosphoenolpyruvate carboxykinase 2 (PCK2) expression and PEP levels increased, whereas PCK2 inhibition affected AK3KO HeLa cells more than wild-type (WT) cells. Therefore, we concluded that increased PCK2 expression may be complementary to increased GDP, which was found to be deficient through AK3KO. This study demonstrated the importance of AK3 in mitochondrial matrix energy metabolism.

Propofol ameliorates acute postoperative fatigue and promotes glucagon-regulated hepatic gluconeogenesis by activating CREB/PGC-1α and accelerating fatty acids beta-oxidation.

Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation.

Active coacervate droplets are protocells that grow and resist Ostwald ripening.

Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over their components, keeping the droplets out of equilibrium. This turnover can be used to drive active processes such as growth, and provide an insight into the chemical requirements underlying (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active droplets, and report three key findings that make these droplets into evolvable protocells. First, we show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute to the attractive electrostatic interactions and translational entropy within complex coacervates, active or passive. Finally, we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme and protein concentration, and that a different droplet composition (addition of RNA) leads to altered growth rates and droplet fitness. These findings together make active coacervate droplets a powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a population level.

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation.

When amino acids vary during evolution, the outcome can be functionally neutral or biologically-important. We previously found that substituting a subset of nonconserved positions, "rheostat" positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics. Here, we explored whether "phylogenetic" patterns of change in multiple sequence alignments (such as positions with subfamily specific conservation) predict the locations of functional rheostat positions. To that end, we experimentally tested eight phylogenetic positions in human liver pyruvate kinase (hLPYK), using 10-15 substitutions per position and biochemical assays that yielded five functional parameters. Five positions were strongly rheostatic and three were non-neutral. To test the corollary that positions with low phylogenetic scores were not rheostat positions, we combined these phylogenetic positions with previously-identified hLPYK rheostat, "toggle" (most substitution abolished function), and "neutral" (all substitutions were like wild-type) positions. Despite representing 428 variants, this set of 33 positions was poorly statistically powered. Thus, we turned to the in vivo phenotypic dataset for E. coli lactose repressor protein (LacI), which comprised 12-13 substitutions at 329 positions and could be used to identify rheostat, toggle, and neutral positions. Combined hLPYK and LacI results show that positions with strong phylogenetic patterns of change are more likely to exhibit rheostat substitution outcomes than neutral or toggle outcomes. Furthermore, phylogenetic patterns were more successful at identifying rheostat positions than were co-evolutionary or eigenvector centrality measures of evolutionary change.

Detection of Escherichia coli using luminometer with pyruvate kinase.

Portable and quantitative detection of Escherichia coli (E. coli) has the potential to reform clinical diagnostics, food safety, and environmental monitoring. At present, most commercial devices used for pathogen detection have disadvantages such as expensive, highly complex operations, or limited detection specificity. Using the common luminometer and the properties of pyruvate kinase utilizing phosphoenolpyruvate to generate adenosine triphosphate (ATP), we have developed a method that could specifically quantify E. coli. The system is based on a sandwich hybridization procedure wherein both oligonucleotide probes recognize each end of the target of pathogenic 16S rRNAs segment. The detection probe DNA-conjugated pyruvate kinase can link ATP production to the detection of pathogenic nucleic acid in the samples. The luminometer-based system is capable of detecting E. coli with single bacteria resolution. The platform should be easily used to the detection of many other toxic analytes through the application of suitable functional-DNA recognition elements.

The Phosphoenolpyruvate Carboxykinase Is a Key Metabolic Enzyme and Critical Virulence Factor of Leishmania major.

There is currently no effective vaccine against leishmaniasis because of the lack of sufficient knowledge about the Ags that stimulate host-protective and long-lasting T cell-mediated immunity. We previously identified Leishmania phosphoenolpyruvate carboxykinase (PEPCK, a gluconeogenic enzyme) as an immunodominant Ag that is expressed by both the insect (promastigote) and mammalian (amastigote) stages of the parasite. In this study, we investigated the role of PEPCK in metabolism, virulence, and immunopathogenicity of Leishmania major We show that targeted loss of PEPCK results in impaired proliferation of L. major in axenic culture and bone marrow-derived macrophages. Furthermore, the deficiency of PEPCK results in highly attenuated pathology in vivo. BALB/c mice infected with PEPCK-deficient parasites failed to develop any cutaneous lesions despite harboring parasites at the cutaneous site of infection. This was associated with a dramatic reduction in the frequency of cytokine (IFN-γ, IL-4, and IL-10)-producing CD4+ T cells in spleens and lymph nodes draining the infection site. Cells from mice infected with PEPCK-deficient parasites also produced significantly low levels of these cytokines into the culture supernatant following in vitro restimulation with soluble Leishmania Ag. PEPCK-deficient parasites exhibited significantly greater extracellular acidification rate, increased proton leak, and decreased ATP-coupling efficiency and oxygen consumption rates in comparison with their wild-type and addback counterparts. Taken together, these results show that PEPCK is a critical metabolic enzyme for Leishmania, and its deletion results in altered metabolic activity and attenuation of virulence.

Association of phosphoenolpyruvate carboxykinase 1 protein kinase activity-dependent sterol regulatory element-binding protein 1 activation with prognosis of oesophageal carcinoma.

BACKGROUND: Metabolic enzymes have non-canonical functions and play vital roles in the regulation of various cellular activities. Phosphoenolpyruvate carboxykinase 1 (PCK1), a gluconeogenic enzyme, was recently identified as an AKT-dependent protein kinase and promoted sterol regulatory element-binding protein 1 (SREBP1)-dependent lipogenesis. However, association of this protein kinase activity of PCK1 with progression of oesophageal squamous cell carcinoma (ESCC) is unclear. METHODS: We examined 200 ESCC patient samples and prognosis using immunohistochemistry, multivariate Cox regression and Kaplan-Meier Plot analyses. RESULTS: We show that the expression levels of AKT pS473, AKT-regulated PCK1 pS90, PCK1-mediated INSIG1 pS207/INSIG2 pS151 and nuclear SREBP1 were higher in analysed 200 human ESCC specimens than in their adjacent non-tumour tissues; the expression levels of these proteins were significantly and positively correlated with each other in tumour specimens. In addition, the expression levels of PCK1 pS90, INSIG1 pS207/INSIG2 pS151 and SREBP1 were associated with the tumour, node and metastasis stage and progression in ESCC. Importantly, levels of PCK1 pS90 or INSIG1 pS207/INSIG2 pS151 or nuclear SREBP1 were positively correlated with poor prognosis in patients with ESCC, and the combined expression values of PCK1 pS90, INSIG1 pS207/INSIG2 pS151 and nuclear SREBP1 had a better prognostic value than that of each individual protein expression value and was an independent prognostic marker for ESCC. CONCLUSION: These findings reveal the role of PCK1 protein kinase activity-dependent SREBP1 activation in ESCC progression. The regulation of SREBP1 by AKT activation-dependent PCK1 protein kinase activity may provide the potential for the diagnosis and treatment of human ESCC.

Hysteresis of pyruvate phosphate dikinase from Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.