Showing papers on "Pyruvate dehydrogenase phosphatase published in 2013"

A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence

Abstract: In response to tenacious stress signals, such as the unscheduled activation of oncogenes, cells can mobilize tumour suppressor networks to avert the hazard of malignant transformation. A large body of evidence indicates that oncogene-induced senescence (OIS) acts as such a break, withdrawing cells from the proliferative pool almost irreversibly, thus crafting a vital pathophysiological mechanism that protects against cancer. Despite the widespread contribution of OIS to the cessation of tumorigenic expansion in animal models and humans, we have only just begun to define the underlying mechanism and identify key players. Although deregulation of metabolism is intimately linked to the proliferative capacity of cells, and senescent cells are thought to remain metabolically active, little has been investigated in detail about the role of cellular metabolism in OIS. Here we show, by metabolic profiling and functional perturbations, that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAF(V600E), an oncogene commonly mutated in melanoma and other cancers. BRAF(V600E)-induced senescence was accompanied by simultaneous suppression of the PDH-inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2). The resulting combined activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress. Abrogation of OIS, a rate-limiting step towards oncogenic transformation, coincided with reversion of these processes. Further supporting a crucial role of PDH in OIS, enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAF(V600E)-driven melanoma development. Finally, depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition, and caused regression of established melanomas. These results reveal a mechanistic relationship between OIS and a key metabolic signalling axis, which may be exploited therapeutically.

M2 isoform of pyruvate kinase is dispensable for tumor maintenance and growth

Abstract: Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.

A novel regulatory defect in the branched-chain α-keto acid dehydrogenase complex due to a mutation in the PPM1K gene causes a mild variant phenotype of maple syrup urine disease

Abstract: This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others.

Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.

Abstract: Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes. Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and α-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts. Exome sequencing identified two heterozygous mutations (c.875C > G and c.535A > G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, α-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and α-KGDH E2 subunits. Accordingly, the production of 14CO2 by patient fibroblasts after incubation with 14Cglucose, 14Cbutyrate or 14C3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and α-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants. We report here a putative case of impaired free or H protein-derived lipoic acid attachment due to LIPT1 mutations as a cause of PDH and α-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies.

Nonmetabolic functions of pyruvate kinase isoform M2 in controlling cell cycle progression and tumorigenesis.

Abstract: Pyruvate kinase catalyzes the rate-limiting final step of glycolysis, generating adenosine triphosphate (ATP) and pyruvate. The M2 tumor-specific isoform of pyruvate kinase (PKM2) promotes glucose uptake and lactate production in the presence of oxygen, known as aerobic glycolysis or the Warburg effect. As recently reported in Nature, PKM2, besides its metabolic function, has a nonmetabolic function in the direct control of cell cycle progression by activating β-catenin and inducing expression of the β-catenin downstream gene CCND1 (encoding for cyclin D1). This nonmetabolic function of PKM2 is essential for epidermal growth factor receptor (EGFR) activation-induced tumorigenesis.

Decarboxylation of pyruvate to acetaldehyde for ethanol production by hyperthermophiles.

Abstract: Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles.

Identification of a Selective Small-Molecule Inhibitor Series Targeting the Eyes Absent 2 (Eya2) Phosphatase Activity

Abstract: Eya proteins are essential coactivators of the Six family of homeobox transcription factors and also contain a unique protein tyrosine phosphatase activity, belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for a subset of Six1-mediated transcription, making this a unique type of transcriptional control. It is also responsible for directing cells to the repair instead of apoptosis pathway upon DNA damage. Furthermore, the phosphatase activity of Eya is critical for transformation, migration, invasion, and metastasis of breast cancer cells. Thus, inhibitors of the Eya phosphatase activity may be antitumorigenic and antimetastatic, as well as sensitize cancer cells to DNA damage-inducing therapies. In this article, we identified a previously unknown chemical series using high-throughput screening that inhibits the Eya2 phosphatase activity with IC(50)s ranging from 1.8 to 79 µM. Compound activity was confirmed using an alternative malachite green assay and H2AX, a known Eya substrate. Importantly, these Eya2 phosphatase inhibitors show specificity and do not significantly inhibit several other cellular phosphatases. Our studies identify the first selective Eya2 phosphatase inhibitors that can potentially be developed into chemical probes for functional studies of Eya phosphatase or into anticancer drugs in the future.

Characterization of pyruvate uptake in Escherichia coli K-12.

Abstract: The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.

A novel nuclear protein phosphatase 2C negatively regulated by ABL1 is involved in abiotic stress and panicle development in rice.

Abstract: Type 2C protein phosphatase plays an important role in the signal transduction of stress response in plants. In this paper, we identified a novel stress-induced type 2C protein phosphatase gene OsSIPP2C1 from rice. OsSIPP2C1 contains a complete open reading frame of 1,074 bp, encoding a protein with 357 amino acids. OsSIPP2C1 expression was up-regulated by high salt, PEG6000 and exogenous ABA, and enhanced in the abl1 mutant under normal, salt, or drought condition. Interestingly, OsSIPP2C1 expression was increased during the early panicle development. Subcellular localization assay using rice protoplast cells indicated that OsSIPP2C1 was predominantly located in the nucleus. Together, it is suggested that a nuclear PP2C protein OsSIPP2C1 negatively regulated by ABL1 is involved in abiotic stress and panicle development in rice.

The serine/threonine phosphatase PPM1B (PP2Cβ) selectively modulates PPARγ activity.

Abstract: Reversible phosphorylation is a widespread molecular mechanism to regulate the function of cellular proteins, including transcription factors. Phosphorylation of the nuclear receptor PPARγ (peroxisome-proliferator-activated receptor γ) at two conserved serine residue (Ser(112) and Ser(273)) results in an altered transcriptional activity of this transcription factor. So far, only a very limited number of cellular enzymatic activities has been described which can dephosphorylate nuclear receptors. In the present study we used immunoprecipitation assays coupled to tandem MS analysis to identify novel PPARγ-regulating proteins. We identified the serine/threonine phosphatase PPM1B [PP (protein phosphatase), Mg(2+)/Mn(2+) dependent, 1B; also known as PP2Cβ] as a novel PPARγ-interacting protein. Endogenous PPM1B protein is localized in the nucleus of mature 3T3-L1 adipocytes where it can bind to PPARγ. Furthermore we show that PPM1B can directly dephosphorylate PPARγ, both in intact cells and in vitro. In addition PPM1B increases PPARγ-mediated transcription via dephosphorylation of Ser(112). Finally, we show that knockdown of PPM1B in 3T3-L1 adipocytes blunts the expression of some PPARγ target genes while leaving others unaltered. These findings qualify the phosphatase PPM1B as a novel selective modulator of PPARγ activity.

The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase.

Abstract: Recent studies proposed a functional coupling between 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3)-dependent testosterone formation and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1)-mediated interconversion of glucocorticoids through competition for the luminal pyridine nucleotide pool. To test this hypothesis, we used human embryonic kidney-293 cells transfected with 17β-HSD3 and/or 11β-HSD1, in the absence or presence of hexose-6-phosphate dehydrogenase that generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the endoplasmic reticulum and determined enzyme activities. As an endogenous cell model, mouse MA-10 Leydig cells were used. 17β-HSD3-dependent reduction of Δ4-androstene-3,17-dione was affected by neither coexpression with 11β-HSD1 nor overexpression or knockdown of hexose-6-phosphate dehydrogenase. In contrast, knockdown of glucose-6-phosphate dehydrogenase decreased 17β-HSD3 activity, indicating dependence on cytoplasmic NADPH. Upon selective permeabilization of the plasma membrane by digitonin, 17β-HSD3 but not 11β-HSD1 was detected by antibodies against C-terminal epitope tags, suggesting a cytoplasmic orientation of 17β-HSD3. The cytoplasmic orientation was confirmed using proteinase K digestion of microsomal preparations and by analysis of glycosylation of wild-type 17β-HSD3 and chimera in which the N-terminal anchor sequences between 17β-HSD3 and 11β-HSD1 were exchanged. In conclusion, the results demonstrate a cytoplasmic orientation of 17β-HSD3 and dependence on glucose-6-phosphate dehydrogenase-generated NADPH, explaining the lack of a direct functional coupling with the luminal 11β-HSD1-mediated glucocorticoid metabolism.

Ptc6 is required for proper rapamycin-induced down-regulation of the genes coding for ribosomal and rRNA processing proteins in S. cerevisiae.

Abstract: Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li+. Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic genetic interactions of the ptc1 mutation with components of the TOR pathway, thus suggesting different cellular roles for both isoforms. We show here that the rapamycin-sensitive phenotype of ptc6 cells is unrelated to the reported role of Pt6 in controlling pyruvate dehydrogenase activity. Lack of Ptc6 results in substantial attenuation of the transcriptional response to rapamycin, particularly in the subset of repressed genes encoding ribosomal proteins or involved in rRNA processing. In contrast, repressed genes involved in translation are Ptc6-independent. These effects cannot be attributed to the regulation of the Sch9 kinase, but they could involve modulation of the binding of the Ifh1 co-activator to specific gene promoters.

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae.

Abstract: Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 μM, respectively, with V(max) values of 0.294 and 0.173 μmol mg⁻¹ min⁻¹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 μM and for pyruvate from 2.3 to 4.6 mM, while the V(max) changed from 0.806 to 0.673 μmol mg⁻¹ min⁻¹. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate.

Phenylbutyrate increases activity of pyruvate dehydrogenase complex.

Abstract: Altered metabolism is an important hallmark of cancer cells and confers them a selective advantage for survival and proliferation. Metabolism in cancer cells is shifted towards glycolysis and decreased dependence on mitochondrial glucose oxidation, a phenomenon known as ‘Warburg effect’ (aerobic glycolysis). Lactate, the end product of glycolysis, is produced in large excess by cancer cells that used it as metabolic fuel, and has been directly involved in cancer progression. Growth of cancer cells often occurs in a hypoxic microenvironment and thus, these cells rely on anaerobic glycolysis as a primary energy source. Nevertheless, conversion of glucose to lactate persists in cancer cells despite the presence of oxygen (aerobic glycolysis). This adaptation is initiated, in part, by activation of hypoxia-inducible factor 1α (HIF-1) that regulates expression of several glycolytic enzymes, glucose transporters, and mitochondrial enzymes, including pyruvate dehydrogenase kinases (PDKs) which inactivate the pyruvate dehydrogenase complex (PDHC) [1]. The PDHC is an important enzyme in metabolism linking glycolysis to the tricarboxylic acid (TCA) cycle and lipogenic pathway. PDHC catalyzes in mitochondria the irreversible conversion of pyruvate into acetyl-CoA that initiates the TCA cycle. PDHC is formed by three different enzymes: thiamine diphosphate-dependent heterotetrameric (α 2 β 2 ) E1, dihydrolipoamide acetyltransferase (E2), and FAD containing dihydrolipoamide dehydrogenase (E3), that is integrated into the complex by an E3-binding protein (E3BP). Phosphorylation of specific E1α serine residues by PDKs (PDK1, PDK2, PDK3, and PDK4) results in inactivation of PDHC, whereas dephosphorylation by pyruvate dehydrogenase phosphatases (PDP1 and PDP2) restores PDHC activity (Fig. 1). Inhibition of PDHC activity results in conversion of pyruvate into lactate by lactate dehydrogenase (LDH) in the cytoplasm. Deficiency of PDHC is one of the most common inborn errors of energy metabolism and affected patients present with progressive neurological degeneration and lactic acidosis [2]. Dichloroacetate (DCA) increases PDHC activity by inhibition of PDK and is effective in reducing blood and tissue lactate [3]. However, DCA has been associated with hepatocellular and peripheral nerve toxicity. We have recently found that phenylbutyrate, a drug used in patients with urea cycle defects and cancer, results in reduction of phosphorylated E1α and increased

ML285 affects reactive oxygen species’ inhibition of pyruvate kinase M2

Abstract: The ability of all cells to regulate levels of reactive oxygen species (ROS) is vital for controlling many aspects of proliferation and survival and we have discovered that pyruvate kinase M2 (PKM2) is important for cancer cell biology. PKM2 is directly oxidized on Cys358 to inhibit its catalytic activity, which allows for diversion of glucose-6-phosphate into the pentose phosphate pathway. This, in turn, allows the synthesis of NADPH, which is critical for generating reduced glutathione, necessary for ROS detoxification. In a cellular context, our PKM2 activator, ML285 protects the enzyme from oxidation by ROS and results in sensitization to oxidative stress and increased apoptosis.

Genetically engineered yeast

Abstract: A genetically modified Saccharomyces cerevisiae comprising an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from Bacillus cereus AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate semialdehyde. The yeast may also express a 3-hydroxyisobutyrate dehydrogenase (HIBADH) and a 3-hydroxypropanoate dehydrogenase (3-HPDH) and aspartate 1-decarboxylase. Additionally the yeast may express pyruvate carboxylase and aspartate aminotransferase.

Nuclear Magnetic Resonance Approaches in the Study of 2-Oxo Acid Dehydrogenase Multienzyme Complexes—A Literature Review

Abstract: The 2-oxoacid dehydrogenase complexes (ODHc) consist of multiple copies of three enzyme components: E1, a 2-oxoacid decarboxylase; E2, dihydrolipoyl acyl-transferase; and E3, dihydrolipoyl dehydrogenase, that together catalyze the oxidative decarboxylation of 2-oxoacids, in the presence of thiamin diphosphate (ThDP), coenzyme A (CoA), Mg²⁺ and NAD⁺, to generate CO₂, NADH and the corresponding acyl-CoA. The structural scaffold of the complex is provided by E2, with E1 and E3 bound around the periphery. The three principal members of the family are pyruvate dehydrogenase (PDHc), 2-oxoglutarate dehydrogenase (OGDHc) and branched-chain 2-oxo acid dehydrogenase (BCKDHc). In this review, we report application of NMR-based approaches to both mechanistic and structural issues concerning these complexes. These studies revealed the nature and reactivity of transient intermediates on the enzymatic pathway and provided site-specific information on the architecture and binding specificity of the domain interfaces using solubilized truncated domain constructs of the multi-domain E2 component in its interactions with the E1 and E3 components. Where studied, NMR has also provided information about mobile loops and the possible relationship of mobility and catalysis.

Effect of leaves of Artemisia vulgaris L. on growth of rats.

Abstract: To know the effect of leaves of Artemisia vulgaris Linn. on growth of rats, male albino rats were divided into three groups. First group of animals took normal diet while the second and third groups of animals took a mixture of normal diet and powdered leaves of Artemisia vulgaris Linn in the doses of 500 mg/kg and 1 g/kg respectively. In all the three groups growth of rats was checked up to 40 days. On 20th and 40th days of experiment blood pyruvate level was measured. Intestines of rats were also assayed for pyruvate dehydrogenase and transketolase activity. It was found out that growth of rats which took powdered leaves of Artemisia vulgaris Linn. with normal diet was stunted. Growth inhibition was dose dependant. Rats developed symptom of anorexia and became weak. Blood pyruvate level was elevated and intestinal pyrvate dehydrogenase activity was lowered. Pyruvate thus could not be converted to acetyl coenzyme A needed for generation of energy. Lack of energy caused growth inhibition in rats. Further, intestinal transketolase activity was found decreased. Due to this in pentose phosphate pathway there was insufficient production of NADPH and pentose sugars needed for synthesis of nucleic acid. This also caused growth inhibition in rats. Keyword: Artemisia vulgaris Linn., Pyruvate Dehydrogenase, Transketolase, Pentose Phosphate Pathway.

MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE lpdA1 GENE IN S. meliloti

Abstract: The alfalfa-Sinorhizobium symbiosis is one of the best studied for plant-microbe associations. The pyruvate dehydrogenase complex catalyzes the key metabolic step connecting the glycolysis to the Tricarboxylic acid (TCA) cycle. The PDH also participates in anaplerotic synthesis of acetyl-CoA in bacteroids, or when grown in the presence of dicarboxylic acids as the sole carbon sources. The complex consists of multiple copies of the three subunits, pyruvate dehydrogenase (EC 1.2.4.1), that acts as the E1 component of the complex, dihydrolipoamide transacetylase (EC 2.3.1.12), the E2 component, and dihydrolipoamide dehydrogenase (EC 1.8.1.4), that participates as the E3 subunit. The lpdA1 gene was mutated through the site-directed, single crossover recombination, using modified pVIK112 suicide plasmids. The lpdA1 mutant failed to grow on pyruvate, and was significantly delayed on other tested carbon sources. The activity of the pyruvate dehydrogenase complex was not detected in this mutant, and the regulation of the lpdA1 was not dependent on the presence of pyruvate. This mutant was Nod+, but appeared Fix-.

Carbohydrate-Responsive Element Binding Protein

Abstract: The liver is the principal organ responsible for conversion of excess dietary carbohydrate to storage fat. Glucose, a major source of energy in mammals, provides pyruvate, which is either oxidized to generate ATP or converted into triglycerides for storage. The coordinated control of the metabolic pathways that direct the fate of glucose allows for the efficient use of dietary carbohydrate. Key enzymes in these pathways, including pyruvate kinase, acetyl-CoA carboxylase, and fatty acid synthase complex, are regulated by allosteric, post-translational mechanisms that are triggered by increased insulin secretion or nutrient levels. Insulin-induced expression of the steroid response element binding protein (SREBP)-1c, which mediates insulin-dependent activation of lipogenic gene transcription, contributes up to 40% of the triglycerides in liver. The other half of fat synthesis in liver is regulated by the carbohydrate response element binding protein (ChREBP), which operates independently of insulin. ChREBP induces the expression of liver pyruvate kinase (LPK) gene as well as all the lipogenic enzyme-encoding genes.

Effects of medicinal herb water extracts on expression of hepatic glucokinase, pyruvate dehydrogenase and acetyl-CoA carboxylase mRNA

Abstract: We studied the anti-diabetic effects of medicinal herb water extracts on expression of hepatic glucokinase (GCK), pyruvate dehydrogenase (PDH), and acetyl-CoA carboxylase (ACC) mRNA. The medicinal herbs used for experiments were Cornus officinalis (CO), Paeonia suffruticosa Andrews (PSA), Discorea japonica Thunb. (DJ), Rehmannia glutinosa (RG), Lycium chinense (LC), and Pyrus pyrifolia (PP). For GCK mRNA expression, CO, RG, and LC water extracts exhibited a more effective activity than other extracts. Cells treated with RG and LC water extracts showed an increase in expression of PDH mRNA to 191% and 124%, respectively, compared to control. Expression of ACC mRNA was significantly higher in LC water extract. These data indicate that CO, RG, and LC water extracts stimulates expression of he patic GCK, PDH, and ACC mRNA. (Korean J Nutr 2013; 46(2): 119 ~ 125)

The Proteome Variation of Rat Skeletal Muscle Induced by Repeated Eccentric Exercise

Abstract: Objective:To research proteome variety on skeletal muscle damage repair process induced by one and repeated eccentric exercise and proteomics mechanism of skeletal muscle repairing made by repeated eccentric exercise;Methods:72 Wistar rats were randomly divided into normal control group,one eccentric exercise group and repeat exercise group a week later.Control group was not done exercise,the rest groups were exercised with 18m/min of speed and-16 degrees of slope.Rats moved for 30min,rest for 5min and then moved for 30min.Repetitive motion was done after a week.Proteome extraction and dielectrophoresis were taken after 0h,24h,48h,72h,168h of one and repeated eccentric exercise,specific proteins were identified by MALDI-TOF MS and sorted according to its function.Results:Proteins expression about energy metabolism and cell damage were reduced especially from 0h to 72h after repeated motion.The proteins number of decreased and increased expression were not different obviously from 0h to 24h and the decreased proteins number was more than number of increased proteins obviously at 72h after repeated motion.During process of damage repair,energy metabolism related proteins included isocitrate dehydrogenase,phosphate glucose mutase,nicotinamide adenine dinucleotide dehydrogenase,hemoglobin binding protein,pyruvate dehydrogenase,triose phosphate isomerase;cell damage repair related proteins were consisted of ubiquitin carboxyl terminal hydrolytic enzyme,immune globulin lambda light chain,intracellular chlorine ion channel protein,protein phosphatase 2c protein,glutathione peroxidase,double function peroxidase and express active;skeletal muscle cell structure protein have heat shock protein,endoplasmic reticulum stations protein and keratin,myosin,mitochondrial membrane protein,etc.Conclusion:Repeated eccentric exercise may promote key enzyme expression of energy metabolism and energy supply for damage cell,accelerate skeletal muscle contraction protein degradation and cell swallow,and scavenge free radicals and slow inflammatory reaction during 24~48h to speed up the skeletal muscle damage repair.

Interaction of Non-Phosphorylated Liver Pyruvate Kinase with Fructose 1,6-Bisphosphate and Peptides that Mimic the Phosphorylatable N-terminus of the Enzyme

Abstract: The interaction of non-phosphorylated L-type pyruvate kinase (L-PK) with fructose 1,6-bisphosphate (FBP), which is an allosteric activator of the phosphorylated enzyme, and peptides that mimic the phosphorylatable N-terminal regulatory domain of the enzyme, was studied. It was found that the catalytic activity of the enzyme was not enhanced in the presence of FBP, and this ligand acted as a relatively weak reversible inhibitor of the enzyme activity in the micromolar concentration range. The phosphorylation site analogue peptides RRASVA and RRAAVA had no effect on the activity of the enzyme, while the phosphorylated peptide RRAS(Pi)VA reversibly inhibited the enzyme and this process was characterised by the Ki value 47 μM. As the phosphorylated form of L-PK is a subject of significant allosteric regulation by FBP, it was concluded that phosphorylation should function as a molecular switch of the allosteric properties of this enzyme.