Showing papers on "ATP citrate lyase published in 2013"

DNMT1 is regulated by ATP-citrate lyase and maintains methylation patterns during adipocyte differentiation

Abstract: During adipocyte differentiation, significant epigenomic changes occur in association with the implementation of the adipogenic program. We have previously shown that histone acetylation increases during differentiation in a manner dependent on acetyl coenzyme A (acetyl-CoA) production by the enzyme ATP-citrate lyase (ACL). Whether ACL regulates nuclear targets in addition to histones during differentiation is not clear. In this study, we report that DNA methyltransferase 1 (DNMT1) levels in adipocytes are controlled in part by ACL and that silencing of DNMT1 can accelerate adipocyte differentiation. DNMT1 gene expression is induced early in 3T3-L1 adipocyte differentiation during mitotic clonal expansion and is critical for maintenance of DNA and histone H3K9 methylation patterns during this period. In the absence of DNMT1, adipocyte-specific gene expression and lipid accumulation occur precociously. Later in differentiation, DNMT1 levels decline in an ACL-dependent manner. ACL-mediated suppression of DNMT1 occurs at least in part by promoting expression of microRNA 148a (miR-148a), which represses DNMT1. Ectopic expression of miR-148a accelerates differentiation under standard conditions and can partially rescue a hypermethylation-mediated differentiation block. The data suggest a role for DNMT1 in modulating the timing of differentiation and describe a novel ACL-miR-148a-dependent mechanism for regulating DNMT1 during adipogenesis.

Both Decrease in ACL1 Gene Expression and Increase in ICL1 Gene Expression in Marine-Derived Yeast Yarrowia lipolytica Expressing INU1 Gene Enhance Citric Acid Production from Inulin

Abstract: In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.

Inhibition of ATP Citrate Lyase Induces an Anticancer Effect via Reactive Oxygen Species: AMPK as a Predictive Biomarker for Therapeutic Impact

Abstract: De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase ( p -AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p -AMPK. Mitochondrial-deficient ρ 0 cells retained high levels of ROS and p -AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p -AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p -AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p -AMPK reflects cellular ROS levels in vitro and in vivo . Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p -AMPK could be a predictive biomarker for its therapeutic outcome.

Metabolomic and transcriptomic profiling of human K- ras oncogene transgenic rats with pancreatic ductal adenocarcinomas

Abstract: Pancreatic ductal adenocarcinoma (PDAC) is one of the most debilitating malignancies in humans, and one of the reasons for this is the inability to diagnose this disease early in its development. To search for biomarkers that can be used for early diagnosis of PDAC, we established a rat model of human PDAC in which expression of a human K-ras(G12V) oncogene and induction of PDAC are regulated by the Cre/lox system. In the present study, transgenic rats bearing PDAC and control transgenic rats with normal pancreatic tissues were used for metabolomic analysis of serum and pancreatic tissue by non-targeted and targeted gas chromatography-mass spectrometry and transcriptomic analysis of pancreatic tissue by microarray. Comparison of the metabolic profiles of the serum and pancreatic tissue of PDAC-bearing and control rats identified palmitoleic acid as a metabolite, which was significantly decreased in the serum of PDAC-bearing animals. Transcriptomic analysis indicated that several transcripts involved in anaerobic glycolysis and nucleotide degradation were increased and transcripts involved in the trichloroacetic acid cycle were decreased. Other transcripts that were changed in PDAC-bearing rats were adenosine triphosphate citrate lyase (decreased: fatty acid biosynthesis), fatty acid synthase (increased: fatty acid biosynthesis) and arachidonate 5-lipoxygenase activating protein (increased: arachidonic acid metabolism). Overall, our results suggest that the decreased serum levels of palmitoleic acid in rats with PDAC was likely due to its decrease in pancreatic tissue and that palmitoleic acid should be investigated in human samples to assess its diagnostic significance as a serum biomarker for human PDAC.

Contributions of citrate in redox potential maintenance and ATP production: metabolic pathways and their regulation in Lactobacillus panis PM1

Abstract: Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli and can utilize various NADH-reoxidizing routes (e.g., citrate, glycerol, and oxygen) according to environmental conditions. In this study, we investigated the ability of L. panis PM1 to produce succinate, acetate, and lactate via citrate utilization. Possible pathways, as well as regulation, for citrate metabolism were examined on the basis of the genome sequence data and metabolic profiles of L. panis PM1. The presence of citrate led to the up-regulation, at the transcriptional level, of the genes encoding for citrate lyase, malate dehydrogenase, and malic enzyme of the citrate pathways by 10- to 120-fold. The transcriptional regulator of the dha operon coding for glycerol dehydratase of L. panis PM1 repressed the expression of the citrate lyase gene (10-fold). Metabolite analyses indicated that the transcriptional enhancement by citrate stimulated succinate yield. Citrate metabolism contributed to energy production by providing a major alternate pathway for NAD(+) regeneration and allowed acetyl phosphate to yield acetate/ATP instead of ethanol/NAD(+). Additionally, a branching pathway from oxaloacetate to pyruvate increased the pool of lactate, which was then used to produce ATP during stationary phase. However, the redirection of NADH-to-citrate utilization resulted in stress caused by end-products (i.e., succinate and acetate). This stress reduced succinate production by up to 50 % but did not cause significant changes at transcriptional level. Overall, citrate utilization was beneficial for the growth of L. panis PM1 by providing a NAD(+) regeneration route and producing extra ATP.

Ca2+-citrate uptake and metabolism in Lactobacillus casei ATCC 334.

Abstract: The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca(2+) and not as free citrate or the Mg(2+)-citrate complex, thereby identifying Ca(2+)-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca(2+) and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca(2+)-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca(2+)-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca(2+)-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca(2+)-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages.

Correlation of ATP Citrate Lyase and Acetyl CoA Levels with Trichothecene Production in Fusarium graminearum

Abstract: The correlation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride.

Citric Acid Mediates the Iron Absorption from Low Molecular Weight Human Milk Fractions

Abstract: Previously, we have demonstrated increased iron absorption from low molecular weight (LMW) human milk whey fractions. In the present study, we investigated the effect of heat denaturation, zinc (a competitor of iron), duodenal cytochrome b (DcytB) antibody neutralization and citrate lyase treatment on LMW human milk fraction (>5 kDa referred as 5kF) induced ferric iron reduction, solubilization, and uptake in Caco-2 cells. Heat denaturation and zinc inhibited the 5kF fraction induced ferric iron reduction. In contrast, zinc but not heat denaturation abrogated the ferric iron solubilization activity. Despite inhibition of ferric iron reduction, iron uptake in Caco-2 cells was similar from both native and heat denatured 5kF fractions. However, iron uptake was higher from native compared to heat denatured 5kF fractions in the cells preincubated with the DcytB antibody. Citrate lyase treatment inhibited the ferric iron reduction, solubilization, and uptake in Caco-2 cells. These findings demonstrate that citric acid present in human milk solubilizes the ferric iron which could be reduced by other heat labile components leading to increased uptake in intestinal cells.

BUTYRATE SUPPLEMENTATION AFFECTS mRNA ABUNDANCE OF GENES INVOLVED IN GLYCOLYSIS, OXIDATIVE PHOSPHORYLATION AND LIPOGENESIS IN THE RUMEN EPITHELIUM OF HOLSTEIN DAIRY COWS

Abstract: Energy availability in epithelial cells is a crucia l link for maintaining epithelial barrier integrity ; energy depletion is linked to impaired barrier function in several epithelia. This study aimed to elucidate t he effects of exogenous butyrate on mRNA abundance of genes indirectly involved in rumen epithelial barrier integrit y. Sixteen mid-lactation Holstein cows fed a total mix ed ration received a concentrate mix to induce Suba cute Ruminal Acidosis (SARA). For 7 days, while being fed the concentrate mix, cows were assigned either a control treatment or a butyrate treatment, in which cows were fed butyrate at 2.5% daily dry matter in take in the form of a calcium salt. On days 6 and 7, rumen pH was measured continuously and on day 7, rumen biopsies took place. Rumen pH fell below 5.6 for more than 3 hours per day in both treatments, con-fir ming the occurrence of SARA. Microarray and pathway analysis, confirmed by real time PCR, showed that exogenous butyrate significantly increased the mRNA abundance of hexokinase 2 (fold change: 2.07), pyruvate kinase (1.19), cytochrome B-complex 3 (1.18) and ATP Synthase, F0 subunit (1.66), which en-code important glycolytic enzymes. Meanwhile, butyrate decreased mRNA abundance of pyruvate dehydrogenase kinase 2(-2.38), ATP citrate lyase (-2.00) and mito chondrial CoA transporter (-2.27), which en-code enzymes involved in lipogenesis. These data suggest exogeno us butyrate induces a shift towards energy mobiliza tion in the rumen epithelium, which may aid barrier functio n in the rumen epithelium during SARA.

Citrate synthase from the liver fluke Fasciola hepatica.

Abstract: Citrate synthase catalyses the first step of the Krebs’ tricarboxylic acid cycle. A sequence encoding citrate synthase from the common liver fluke, Fasciola hepatica, has been cloned. The encoded protein sequence is predicted to fold into a largely α-helical protein with high structural similarity to mammalian citrate synthases. Although a hexahistidine-tagged version of the protein could be expressed in Escherichia coli, it was not possible to purify it by nickel-affinity chromatography. Similar results were obtained with a version of the protein which lacks the putative mitochondrial targeting sequence (residues 1 to 29). However, extracts from bacterial cells expressing this version had additional citrate synthase activity after correcting for the endogenous, bacterial activity. The apparent K m for oxaloacetate was found to be 0.22 mM, which is higher than that observed in mammalian citrate synthases. Overall, the sequence and structure of F. hepatica citrate synthase are similar to ones from other eukaryotes, but there are enzymological differences which merit further investigation.

Structural Insights into the Regulation of ACC2 by Citrate

Abstract: Acetyl-CoA carboxylases (ACCs) play critical roles in fatty acid synthesis and oxidation by the catalytic activity of the carboxylation of acetyl-CoA to malonyl-CoA. It is known that ACCs are inactivated through reversible phosphorylation by AMP-activated protein kinase (AMPK) and allosterically activated by citrate. Here, we determined the crystal structures of biotin carboxylase (BC) domain of human ACC2 phosphorylated by AMPK in the presence of citrate in order to elucidate the activation mechanism by citrate. This structure shows that phosphorylated Ser222 is released from the dimer interface, and thereby facilitating the dimerization or oligomerization of the BC domain allosterically. This structural explanation is coincident with the experimental result that the phosphorylated Ser222 was dephosphorylated more easily by protein phosphatase 2A (PP2A) as the citrate concentration increases.

ACLY (ATP citrate lyase)

Abstract: Review on ACLY (ATP citrate lyase), with data on DNA, on the protein encoded, and where the gene is implicated.

Nature inspired platforms for production of acetyl-CoA derived biofuels in S. cerevisiae

Abstract: Author(s): Rodriguez, Sarah | Advisor(s): Keasling, Jay D | Abstract: The work presented within this thesis was motivated by the need for a robust S. cerevisiae system that has the capacity to drive the production of an acetyl-CoA derived industrial product. Systems that generated acetyl-CoA derived products have been engineered in E. coli have demonstrated the utility of a robust host engineered with strong acetyl-CoA production. We focus on the production of acetyl-CoA derived advanced biofuels. Chapter one describes the metabolic engineering endeavors of microbial pathways for advanced biofuels. These include short-chain alcohols from fermentative pathways, fuels from isoprenoid pathways, and fuels from fatty acid pathways. We describe the key advantages to metabolic pathway engineering in S. cerevisiae. We then review unique features of native S. cerevisiae central metabolism and its acetyl-CoA generation. Because we focus on engineering an advanced biofuel derived from the isoprenoid pathway, we describe the advantages of using amorphadiene as a proxy for isoprenoid pathway flux. This dissertation describes the use of a heterologous enzyme, ATP: citrate lyase (ACL), by which acetyl-CoA is formed in the cytosol, whose use was inspired by native mechanisms of lipid accumulating yeast in Chapter two. We aimed to increase production of the sesquiterpene, amorphadiene, by increasing availability of its primary precursor, cytosolic acetyl-CoA. The importance of this aim is underscored by the stoichiometry that dictates that production of one mole amorphadiene biosynthesis requires nine molar equivalents of acetyl-CoA. In S. cerevisiae, acetyl-CoA metabolism takes place in at least four subcellular compartments: nucleus, mitochondria, cytosol and peroxisomes. A challenge lies in increasing precursor flux of cytosolic acetyl-CoA and decreasing acetyl-CoA flux towards other subcellular compartments. Oleaginous, or lipid accumulating yeast, have evolved mechanisms to export units of mitochondrial acetyl-CoA into the cytosol required for lipid biosynthesis. Key to this mechanism is the activity of the ACL enzyme. In these studies, we implemented an ACL from the oleaginous yeast, Aspergillus nidulans, encoded by genes aclA and aclB. In conclusion, we surmised that the expression of ACL genes did indeed alter isoprenoid metabolism. However, it was unable to increase total amorphadiene production. This result is most likely due to poor catalytic activity of the amorphadiene synthase (ADS), the final enzyme synthase leading to the production of amorphadiene, and native mechanisms that work to control increase of acetyl-CoA levels, thus preventing accumulation.In Chapter three, we increased substrate supply and simultaneously increased carbon flux through the committed isoprenoid biosynthetic steps. We demonstrate the utility of expression of E. faecalis homologs of the upper mevalonate pathway as it sequesters carbon from the central metabolism. In particular, here we sought to determine if 1) expression of native and heterologous enzymes of first committed steps of the mevalonate pathway, E. faecalis genes mvaE and mvaS, are required to sequester acetyl-CoA units toward the committed path of isoprenoid biosynthesis or 2) flux through the mevalonate pathway remained unchanged due do inactivation of the native acetyl-CoA synthase, Acs1p. We conclude that indeed, the native Acs1p demonstrates feedback inhibition, and thus accumulates more acetate than heterologous homologues insensitive to feedback inhibition. Also, indeed we find that overexpression of heterologous E. faecalis genes mvaE and mvaS increase both mevalonate and amorphadiene production. However, the expression of genes mvaE and mvaS in combination with the acetyl-CoA synthase does not further increase mevalonate or amorphadiene production. This result is most likely due to a decrease in total enzyme levels of the first committed step of the mevalonate pathway. In Chapter four, we perform targeted metabolic characterization of expression of the ATP: citrate lyase in a citrate generating S. cerevisiae host, BY4742 with its isocitrate dehydrogenase gene, IDH1, deleted. In these studies, we profiled changes in central metabolism arising from the expression of Aspergillus nidulans ACL genes, aclA and aclB, in the host wild type S. cerevisiae strain and in the BY4742 ∆IDH1 strain. We have chosen to utilize IDH1 deleted cells, which have previously been shown to generate high levels of citrate, in order to provide increased substrate for ACL utilization. Targeted metabolic profiling demonstrated increased citrate levels in the ∆IDH1 strain. Furthermore, citrate levels vary with nitrogen availability in the medium. We find that expression of ACL decreased total citrate levels and as previously seen in chapter one, acetyl-CoA levels remain unchanged. However, we find that expression of ACL causes large accumulation of two metabolites. The first metabolite has been positively identified as 2-isopropylmalate and is an acetyl-CoA derived intermediate of leucine biosynthesis. The second metabolite has yet to be positively identified. We also demonstrate that combined expression of ACL with E. faecalis genes mvaE and mvaS improve mevalonate production by 48%, thus demonstrating the use of ACL as an acetyl-CoA generating enzyme for production of acetyl-CoA derived products. In Chapter five, we offer perspectives on future development of S. cerevisiae as cellular host for production of acetyl-CoA derived biofuels. The work presented in this thesis is a first step in re-purposing nature's metabolic mechanisms which lead to lipid accumulation for metabolic engineering acetyl-CoA derived products within S. cerevisiae. Future work aims to improve engineering efforts to mimic oleaginous metabolism and maximize the utilization of cytosolic carbon for the production of a biofuel. Cellular metabolism will be monitored through metabolomic studies and C13 metabolic flux analysis.