scholarly journals Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors

2015 ◽  
Vol 21 (2) ◽  
pp. 111-116 ◽  
Author(s):  
Philip J. Hopcroft ◽  
David I. Fisher
Keyword(s):  
Fatty Acid ◽  
Mammalian Cells ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Structure Activity ◽  
Malonyl Coa ◽  
Scale Normalization ◽  
Development Structure ◽  
Screening Cascade

The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography–mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure–activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z′ of >0.6, and has high reproducibility of EC50 values.

scholarly journals Identification of a Novel Mycobacterial 3-Hydroxyacyl-Thioester Dehydratase, HtdZ (Rv0130), by Functional Complementation in Yeast

2008 ◽  
Vol 190 (11) ◽  
pp. 4088-4090 ◽  
Author(s):  
Aner Gurvitz ◽  
J. Kalervo Hiltunen ◽  
Alexander J. Kastaniotis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mycobacterium Tuberculosis ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Functional Complementation ◽  
Type Ii ◽  
Mutant Cells ◽  
Dehydratase Activity

ABSTRACT We report on the identification of Mycobacterium tuberculosis HtdZ (Rv0130), representing a novel 3-hydroxyacyl-thioester dehydratase. HtdZ was picked up by the functional complementation of Saccharomyces cerevisiae htd2Δ cells lacking the dehydratase of mitochondrial type II fatty acid synthase. Mutant cells expressing HtdZ contained dehydratase activity, recovered their respiratory ability, and partially restored de novo lipoic acid synthesis.

scholarly journals Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase

1997 ◽  
Vol 327 (1) ◽  
pp. 267-273 ◽  
Author(s):  
P. Grattan ROUGHAN
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Acetyl Coa ◽  
Minimal Processing ◽  
Malonyl Coa ◽  
Substrate Channelling ◽  
Isolated Chloroplasts

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10–20 μM, assuming a stromal volume of 66 μl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 μM. Malonyl-CoA concentrations were also very low (< 0.1–3.0 μM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 μM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA–acyl-carrier-protein transacylase reactions at the concentrations of metabolites measured here are < 0.1–4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.

scholarly journals Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis

1991 ◽  
Vol 273 (3) ◽  
pp. 787-790 ◽  
Author(s):  
J Naggert ◽  
A Witkowski ◽  
B Wessa ◽  
S Smith
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Lambda Repressor ◽  
Thioesterase Ii ◽  
Chain Lengths

Thioesterase I, a constituent domain of the multifunctional fatty acid synthase, and thioesterase II, an independent monofunctional protein, catalyse the chain-terminating reaction in fatty acid synthesis de novo at long and medium chain lengths respectively. The enzymes have been cloned and expressed in Escherichia coli under the control of the temperature-sensitive lambda repressor. The recombinant proteins are full-length catalytically competent thioesterases with specificities indistinguishable from those of the natural enzymes.

scholarly journals Central Resistin Regulates Hypothalamic and Peripheral Lipid Metabolism in a Nutritional-Dependent Fashion

Endocrinology ◽  
2008 ◽  
Vol 149 (9) ◽  
pp. 4534-4543 ◽  
Author(s):  
María J. Vázquez ◽  
C. Ruth González ◽  
Luis Varela ◽  
Ricardo Lage ◽  
Sulay Tovar ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Mrna Expression ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Inhibitory Effect ◽  
De Novo Lipogenesis ◽  
Dependent Manner ◽  
Fed State

Evidence suggests that the adipocyte-derived hormone resistin (RSTN) directly regulates both feeding and peripheral metabolism through, so far, undefined hypothalamic-mediated mechanisms. Here, we demonstrate that the anorectic effect of RSTN is associated with inappropriately decreased mRNA expression of orexigenic (agouti-related protein and neuropeptide Y) and increased mRNA expression of anorexigenic (cocaine and amphetamine-regulated transcript) neuropeptides in the arcuate nucleus of the hypothalamus. Of interest, RSTN also exerts a profound nutrition-dependent inhibitory effect on hypothalamic fatty acid metabolism, as indicated by increased phosphorylation levels of both AMP-activated protein kinase and its downstream target acetyl-coenzyme A carboxylase, associated with decreased expression of fatty acid synthase in the ventromedial nucleus of the hypothalamus. In addition, we also demonstrate that chronic central RSTN infusion results in decreased body weight and major changes in peripheral expression of lipogenic enzymes, in a tissue-specific and nutrition-dependent manner. Thus, in the fed state central RSTN is associated with induced expression of fatty acid synthesis enzymes and proinflammatory cytokines in liver, whereas its administration in the fasted state does so in white adipose tissue. Overall, our results indicate that RSTN controls feeding and peripheral lipid metabolism and suggest that hepatic RSTN-induced insulin resistance may be mediated by central activation of de novo lipogenesis in liver.

scholarly journals Reducing FASN expression sensitizes acute myeloid leukemia cells to differentiation therapy

2021 ◽  
Author(s):  
Magali Humbert ◽  
Kristina Seiler ◽  
Severin Mosimann ◽  
Vreni Rentsch ◽  
Katyayani Sharma ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Fatty Acid ◽  
Myeloid Leukemia ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acid Synthesis ◽  
Mrna Levels ◽  
Differentiation Therapy ◽  
Granulocytic Differentiation ◽  
Acute Myeloid

AbstractFatty acid synthase (FASN) is the only human lipogenic enzyme available for de novo fatty acid synthesis and is often highly expressed in cancer cells. We found that FASN mRNA levels were significantly higher in acute myeloid leukemia (AML) patients than in healthy granulocytes or CD34+ hematopoietic progenitors. Accordingly, FASN levels decreased during all-trans retinoic acid (ATRA)-mediated granulocytic differentiation of acute promyelocytic leukemia (APL) cells, partially via autophagic degradation. Furthermore, our data suggest that inhibition of FASN expression levels using RNAi or (-)-epigallocatechin-3-gallate (EGCG) accelerated the differentiation of APL cell lines and significantly re-sensitized ATRA refractory non-APL AML cells. FASN reduction promoted translocation of transcription factor EB (TFEB) to the nucleus, paralleled by activation of CLEAR network genes and lysosomal biogenesis. Together, our data demonstrate that inhibition of FASN expression in combination with ATRA treatment facilitates granulocytic differentiation of APL cells and may extend differentiation therapy to non-APL AML cells.

scholarly journals The effect of dietary docosahexaenoic acid on the expression of lipogenic genes in broilers

2007 ◽  
Vol 58 (2) ◽  
pp. 153 ◽  
Author(s):  
H. J. Chin ◽  
Y. H. Ko ◽  
T. F. Shen ◽  
S. T. Ding
Keyword(s):  
Fatty Acid ◽  
Docosahexaenoic Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Regulatory Element ◽  
Acid Synthesis ◽  
Daily Weight Gain ◽  
Feed Conversion ◽  
Lipogenic Genes ◽  
Triacylglycerol Synthesis

The objectives of this work were to determine the effects of dietary fungal docosahexaenoic acid (DHA) on tissue DHA concentration and lipogenic gene expression in broilers. A fungal (SR-21) meal product containing 31.5% total fat and 32.7% DHA (% of total fatty acids) was fed to chicken broilers at 0, 1, or 3% for 3 weeks. A diet with 1% DHA oil (containing 40% DHA) was also fed to chicken broilers as a positive control. Dietary fungal meal supplementation (3%) improved daily weight gain, food intake, and feed conversion ratio. The fungal meal supplementation increased dietary DHA content and consequently increased the DHA content in plasma, breast muscle (Pectoralis major), and livers in the broilers. The plasma triacylglycerol concentration was decreased by the supplementation of dietary DHA. The data indicate that the dietary DHA treatment modified certain aspects of the lipid metabolism, especially pathways related to triacylglycerol synthesis. Indeed, both the 1% DHA oil and 3% fungal meal treatments decreased the hepatic lipogenic transcription factor sterol regulatory element binding protein 1 (SREBP1) mRNA relative abundance, suggesting that dietary DHA supplementation decreases SREBP1 gene functions. The relative mRNA abundance of the de novo fatty acid synthesis genes, fatty acid synthase and acetyl coenzyme A carboxylase, was reduced by 1% DHA oil and 3% fungal meal treatments, suggesting that dietary DHA supplementation decreases lipogenesis in the livers of the broilers. Taken together, the fungal meal is a suitable dietary supplement to increase tissue DHA content and reduce the expression of hepatic lipogenic genes in broilers.

The Structures and Bioactivities of Fatty Acid Synthase Inhibitors

2019 ◽  
Vol 26 (39) ◽  
pp. 7081-7101 ◽  
Author(s):  
Hezhong Jiang ◽  
Tian Gan ◽  
Jiasui Zhang ◽  
Qingyun Ma ◽  
Yan Liang ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Web Of Science ◽  
De Novo ◽  
Research Literature ◽  
Long Chain Fatty Acids ◽  
Research Directions ◽  
The Past ◽  
Structure Activity ◽  
Anti Cancer

Background: Fatty Acid Synthase (FAS or FASN) is a vital enzyme which catalyzes the de novo synthesis of long chain fatty acids. A number of studies have recently been reported that FAS was combined targets for the discovery of anti-obesity and anti-cancer drugs. Great interest has been developed in finding novel FAS inhibitors, and result in more than 200 inhibitors being reported. Methods: The reported research literature about the FAS inhibitors was collected and analyzedsised through major databases including Web of Science, and PubMed. Then the chemical stractures, FAS inhibitory activities, and Structure-Activity Relationships (SAR) were summarized focused on all these reported FAS inhibitors. Results: The 248 FAS inhibitors, which were reported during the past 20 years, could be divided into thiolactone, butyrolactone and butyrolactam, polyphenols, alkaloids, terpenoids, and other structures, in view of their structure characteristics. And the SAR of high inhibitory structures of each type was proposed in this paper. Conclusion: A series of synthetic quinolinone derivatives show strongest inhibitory activity in the reported FAS inhibitors. Natural polyphenols, existing in food and herbs, show more adaptive in medicine exploration because of their safety and efficiency. Moreover, screening the FAS inhibitors from microorganism and marine natural products could be the hot research directions in the future.

scholarly journals Function of Heterologous Mycobacterium tuberculosis InhA, a Type 2 Fatty Acid Synthase Enzyme Involved in Extending C20 Fatty Acids to C60-to-C90 Mycolic Acids, during De Novo Lipoic Acid Synthesis in Saccharomyces cerevisiae

2008 ◽  
Vol 74 (16) ◽  
pp. 5078-5085 ◽  
Author(s):  
Aner Gurvitz ◽  
J. Kalervo Hiltunen ◽  
Alexander J. Kastaniotis
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Lipoic Acid ◽  
Fatty Acid Synthase ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acid Synthesis ◽  
Acid Biosynthesis ◽  
Mycolic Acids

ABSTRACT We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C20 fatty acids to form C60-to-C90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C4 to C8) than was previously thought (>C12). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.

scholarly journals Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

2021 ◽  
Author(s):  
Satyam Khanal ◽  
Roberto Javier Brea Fernandez ◽  
Michael Burkart ◽  
Neal Devaraj
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Type I ◽  
Phospholipid Membrane ◽  
Acetyl Coa ◽  
Chemical Ligation ◽  
Malonyl Coa ◽  
Synthetic Cell ◽  
Membrane Bound

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

Inhibitions of FASN suppress triglyceride synthesis via the control of malonyl-CoA in goat mammary epithelial cells

2017 ◽  
Vol 57 (8) ◽  
pp. 1624 ◽  
Author(s):  
J. Luo ◽  
J. J. Zhu ◽  
Y. T. Sun ◽  
H. B. Shi ◽  
J. Li
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Fatty Acid Synthesis ◽  
Fatty Acid Synthase ◽  
Mammary Epithelial Cells ◽  
Acid Synthesis ◽  
Mammary Epithelial ◽  
Triglyceride Synthesis ◽  
Malonyl Coa ◽  
Goat Mammary Epithelial Cells

Fatty acid synthase (FASN) is the key enzyme for de novo fatty acid synthesis from acetyl-CoA and malonyl-CoA. All the steps involved in fatty acid synthesis by FASN have been clearly defined in monogastrics and ruminants. However, there are no data on the mechanism of how FASN affects triglyceride synthesis. Inhibition of FASN in goat mammary epithelial cells by C75, a synthetic inhibitor of FASN activity, and shRNA markedly suppressed the accumulation of triglyceride in goat mammary epithelial cells. Meanwhile, C75 treatment significantly reduced the relative content of monounsaturated fatty acids (C16:1 and C18:1). Corresponding to the suppression of lipid accumulation, both of C75 and shRNA also decreased the mRNA expression of GPAM, AGPAT6 and DGAT2, all of which are related to triglyceride synthesis. The fact that treatment of malonyl-CoA decreased the expression of these genes is consistent with the results of shRNA treatment. Furthermore, the supplement of malonyl-CoA enhanced the suppression on GPAM, AGPAT6, LPIN1, DGAT1 and DGAT2. The results underscore the role of malonyl-CoA in inhibition of FASN in regulating triglyceride synthesis in goat mammary epithelial cells.

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