Fatty Acyl Coenzyme A Research Articles

FIT2 is an acyl-coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis.

The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis.

Structural basis for disulphide-CoA inhibition of a butyryl-CoA hexameric thioesterase

Acyl-coenzyme A thioesterases (ACTs) catalyse the hydrolysis of thioester bonds between fatty-acyl chains and coenzyme A (CoA), producing a free fatty-acyl chain and CoA. These enzymes are expressed ubiquitously across prokaryotes and eukaryotes, and play important roles in lipid metabolism. There are 25 thioesterase families, subdivided based on their active site configuration, protein oligomerization, and substrate specificity. Understanding the mechanism of regulation within these families is important due to their roles in controlling the cell concentration of a range of fatty acids and CoA-bound compounds. Here we report a structural basis for a novel mode of inhibition of an ACT from Staphylococcus aureus. The enzyme displays a hotdog fold composed of five β-strands wrapping around a central α-helix, and an additional 30 residue α-helix located at its C-terminus. We show that the enzyme is a hexamer and has specificity towards butyryl-CoA. Structural analysis revealed putative catalytic residues, and we show through site directed mutagenesis that Asn28, Asp43, and Thr60 are critical for activity. Additionally, we show that the Asn28Ala destabilises the enzyme oligomeric state into two distinct populations. Co-crystallization of the enzyme with the substrate butyryl-CoA produced a crystal with three CoA ligands bound in the enzyme active sites: CoA, butyryl-CoA, and disulphide-CoA, the latter of which inhibits enzyme activity. Our study provides new insights into the structure and specificity of hexameric thioesterases, inhibitory feedback mechanisms, and possible biotechnological applications in short-chain fatty acid production such as biofuels, pharmaceuticals, and industrial compounds.

Nifedipine Modulates Renal Lipogenesis via the AMPK-SREBP Transcriptional Pathway.

Lipid accumulation in renal cells has been implicated in the pathogenesis of obesity-related kidney disease, and lipotoxicity in the kidney can be a surrogate marker for renal failure or renal fibrosis. Fatty acid oxidation provides energy to renal tubular cells. Ca2+ is required for mitochondrial ATP production and to decrease reactive oxygen species (ROS). However, how nifedipine (a calcium channel blocker) affects lipogenesis is unknown. We utilized rat NRK52E cells pre-treated with varying concentrations of nifedipine to examine the activity of lipogenesis enzymes and lipotoxicity. A positive control exposed to oleic acid was used for comparison. Nifedipine was found to activate acetyl Coenzyme A (CoA) synthetase, acetyl CoA carboxylase, long chain fatty acyl CoA elongase, ATP-citrate lyase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase, suggesting elevated production of cholesterol and phospholipids. Nifedipine exposure induced a vast accumulation of cytosolic free fatty acids (FFA) and stimulated the production of reactive oxygen species, upregulated CD36 and KIM-1 (kidney injury molecule-1) expression, inhibited p-AMPK activity, and triggered the expression of SREBP-1/2 and lipin-1, underscoring the potential of nifedipine to induce lipotoxicity with renal damage. To our knowledge, this is the first report demonstrating nifedipine-induced lipid accumulation in the kidney.

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)-binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2-like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.

Minireview: malonyl CoA, AMP-activated protein kinase, and adiposity.

An increasing body of evidence has linked AMP-activated protein kinase (AMPK) and malonyl coenzyme A (CoA) to the regulation of energy balance. Thus, factors that activate AMPK and decrease the concentration of malonyl CoA in peripheral tissues, such as exercise, decrease triglyceride accumulation in the adipocyte and other cells. The data reviewed here suggest that this is related to the fact that these factors concurrently increase fatty acid oxidation, decrease the esterification of fatty acids to form glycerolipids, and, by mechanisms still unknown, increase energy expenditure. Malonyl CoA contributes to these events because it is an allosteric inhibitor of carnitine palmitoyltransferase, the enzyme that controls the transfer of long-chain fatty acyl CoA from the cytosol to the mitochondria, where they are oxidized. AMPK activation in turn increases fatty acid oxidation (by effects on enzymes that govern malonyl CoA synthesis and possibly its degradation) and inhibits triglyceride synthesis. It also increases the expression of uncoupling proteins and the transcriptional regulator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha), which could possibly increase energy expenditure. Recent studies suggest that the ability of leptin, adiponectin, 5'-aminoimidazole 4-carboxamide riboside (AICAR), adrenergic agonists, and metformin to diminish adiposity may be mediated, at least in part, by AMPK activation in peripheral tissues. In addition, preliminary studies suggest that malonyl CoA and AMPK take part in fuel-sensing and signaling mechanisms in the hypothalamus that could regulate food intake and energy expenditure.

MALDI-TOF and electrospray tandem mass spectrometric analysis of fatty acyl-CoA esters

Coenzyme A (CoA) thioesters play a central role in many biochemical reactions, and methods for sensitive identification of these compounds are important for studies of cellular processes. The direct analysis of intact long-chain fatty acyl CoA esters was investigated using matrix assisted laser desorption time-of-flight and electrospray ionization mass spectrometry. Both techniques resulted in the formation of abundant positive and negative ions indicative of the molecular weight of the fatty acyl CoA molecular species. Several structurally significant fragment ions, in particular positive ions indicative of the fatty acyl portion of the molecule, were formed during ionization processes. Metastable (MALDI-TOF) and collision-induced decomposition (quadrupole tandem mass spectrometry) of these molecular ion species resulted in the formation of similar product ions independent of the mode of ionization. Abundant product ions, corresponding to the fatty acyl portion as well as the pantetheine-ADP portion of the molecule, were observed with both ionization techniques. Decomposition of the [M + H] + ion of hexadecyl coenzyme A (C16:0 CoA) at m / z 1006 yielded a prominent product ion at m / z 499 which was characteristic of the fatty acyl portion of the molecule. Similar fatty acyl product ion fragments corresponding to a neutral loss of 609 were found for the [M + H] + species of all the fatty acyl CoA esters investigated. This prominent and diagnostic fragment could be used to detect molecular species of CoA esters present in complex biological mixtures.

Induction of peroxisomes by treatment with perfluorooctanoate does not increase rates of H 2O 2 production in intact liver

Increases in acyl coenzyme A (CoA) oxidase activity due to peroxisome proliferation are postulated to cause oxidative stress via elevated production of H 2O 2, leading to DNA damage. These changes are suspected to be responsible for tumor formation caused by non-genotoxic carcinogens which do not bind to DNA but cause proliferation of peroxisomes. However, the activity of the peroxisomal enzyme acyl CoA oxidase assayed in vitro in the presence of excess fatty acyl CoA substrate may not reflect rates of H 2O 2 generation in intact liver where fatty acid supply is carefully controlled in part by delivery of substrate. The purpose of this work was to determine if rates of hepatic H 2O 2 generation were altered in perfused liver and in vivo following induction of H 2O 2-generating acyl CoA oxidase activity. Injection of the potent peroxisome proliferating agent perfluorooctanoate into rats 5 days prior to sacrifice caused an expected 4-fold increase of H 2O 2-generating acyl CoA oxidase activity measured in hepatic homogenates. In contrast, rates of H 2O 2 generation in perfused liver measured spectrophotometrically (660−640 nm) through a lobe of the liver were not altered by perfluorooctanoate treatment (7.3 ± 1.5 vs. 7.8 ± 0.5 μmol gh in livers from untreated control rats). Similar treatment with perfluorooctanoate also increased in vitro acyl CoA oxidase activity 9-fold in livers from deermice; however, rates of elimination of methanoi, a selective substrate for catalase in rodents whose oxidation is limited by the supply of H 2O 2, were not altered significantly in vivo (control, 110 ± 11 μmol gh vs. perfluorooctanoate, 112 ± 32 μmol gh ). Taken together, these data demonstrate that elevation of H 2O 2 formation by acyl CoA oxidase activity measured in vitro is not necessarily associated with increases in rates of H 2O 2 generation in intact perfused liver or in vivo, most likely due to rate-limitation in intact cells by fatty acid supply. These data do not support the hypothesis that the induction of peroxisomes leads to excessive H 2O 2 production and oxidative stress. It follows that alternative hypotheses to explain carcinogenesis caused by peroxisome-proliferating agents need to be considered.

Regulation of eicosanoid synthesis in microvessel endothelium: Glucocorticoids do not affect arachidonyl CoA synthetase activity

The properties of acyl coenzyme A (CoA) synthetase activity were characterized in cultured rabbit coronary microvessel endothelial cells. We report here that microvessel endothelial cells contain two long-chain acyl CoA synthetases. One shows activity with a variety of fatty acids, similar to long-chain non-selective fatty acyl CoA synthetases described previously. The other activity was selective for arachidonic acid and other structurally related substrates. Both activities required ATP, Mg 2+ and CoA for optimal activity. The arachidonyl CoA and the non-selective acyl CoA synthetases showed different thermolabilities. Arachidonyl CoA formation was inhibited by greater than 50% after 1 min at 45°C, whereas a 15 min heating treatment was necessary to produce the same relative inhibition of oleoyl CoA synthesis. Glucocorticoid pretreatment (10 −7 M dexamethasone) of the RCME cells did not affect the apparent K m or V max , nor the fatty acid selectivity for either acyl CoA synthetase. Therefore, although fatty acyl CoA synthetases may be involved in limiting eicosanoid formation, these activities do not appear to be glucocorticoid-responsive.

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [(14)C]trioleoylglycerol, releasing [(14)C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [(14)C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [(14)C]oleic acid produced from the lipase reaction to [(14)C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.

Purification and characterization of fatty acid-binding proteins from human fetal lung.

Fatty acid-binding protein (FABP) was isolated, purified, and characterized from developing human fetal lung cytosol by gel filtration and ion-exchange chromatography. FABP exists in three immunochemically identical forms, DE-I, DE-II, and DE-III, having Mr 15,200 +/- 200 each and isoelectric pH 7.8, 6.9, and 5.4, respectively. DE-I is almost lipid-free, DE-II binds mainly long-chain unsaturated fatty acids, and DE-III is an arachidonic acid carrier. One mole of DE-II and DE-III each binds 1 mol of fatty acids noncovalently. Concentrations of all these FABPs increase gradually from early gestation to term. Defatted lung FABP reverses the inhibitory effect of palmitoyl coenzyme A (CoA) (PAL-CoA) on lung glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the hexose monophosphate (HMP) shunt pathway. This protein when added alone activates the enzyme, suggesting that the original submaximal activity is probably due to the presence of endogenous long-chain fatty acyl CoA esters in the cytosols. As FABP is present in relatively high concentration in most mammalian cells, the potent inhibitory effects of long-chain acyl CoA esters on the HMP shunt pathway in vitro are not seen in intact cells.

Altered coenzyme A and carnitine metabolism in pressure-overload hypertrophied hearts.

Changes in the total content and acylation state of coenzyme A (CoA) and carnitine in the heart result in alterations in fatty acid metabolism that may be associated with ventricular dysfunction. The present study was undertaken to determine whether abnormal myocardial CoA and carnitine metabolism occur in pressure-overload hypertrophy and congestive heart failure. Right ventricular hypertrophy was induced in cats by pulmonary artery (PA) banding. Total tissue CoA was reduced by 50% in the hypertrophied right ventricle and 25% in the nonhypertrophied left ventricle of PA-banded cats in the presence or absence of heart failure. No alterations were observed in long-chain fatty acyl CoA levels in either ventricle of PA-banded cats. In addition, a 20% reduction in total CoA was also observed in hypertrophied hearts of rats subjected to aortic banding. Although the CoA content was reduced in hypertrophied cat hearts, myocardial tissue levels of its precursor, pantothenic acid, were unaltered. Total tissue carnitine and long-chain fatty acyl-carnitine levels were unchanged in both ventricles of PA-banded cats; however, total carnitine was reduced by 25% in hypertrophied rat hearts. This study establishes a marked reduction in myocardial CoA content in pressure-overload hypertrophy.

FATTY ACID OXIDATION IN SOLUBLE SYSTEMS OF ANIMAL TISSUES

Summary1. The half‐century of investigations directed towards an understanding of the mechanism of β‐oxidation of fatty acids may be divided into three periods: (a) from 1904 to 1939 when the oxidation could be studied at the level of the intact animal or isolated organ or tissue slice; (b) from 1939 to 1952 when it could be studied at the mitochondrial level; and (c) from 1952 onwards when it could be reconstructed in non‐mitochondrial and soluble enzyme systems.2. The Knoop‐Dakin theory of β‐oxidation could not be directly confirmed owing to the non‐accumulation of any intermediates. The theory was based on deductions from the nature of the end‐products of the metabolism of phenyl fatty acids.3. The study of fatty acid oxidation at the mitochondrial level led to the recognition that the fatty acids are not oxidized as such but only in the form of some derivative whose formation is tied up with oxidative phosphorylation and the production of adenosine triphosphate (ATP).4. The transition from the mitochondrial system to soluble enzymes was facilitated first by the discovery that coenzyme A (CoA) was concerned in acyl transfer reactions and later by the recognition that the active fatty acids are indeed the fatty acyl derivatives of CoA.5. There are four known enzymatic processes by which fatty acyl CoA's are formed: (a) oxidation of pyruvate to acetyl CoA; (b) conversion of fatty acids to fatty acyl CoA's by ATP; (c) replacement of the succinyl group of succinyl CoA by short‐chain fatty acids; and (d) cleavage of β‐ketoacyl CoA's by CoA with formation of a fatty acyl CoA and acetyl CoA.6. Two separate enzymes are known to catalyse the oxidation of fatty acyl CoA's to their corresponding transα, β‐unsaturated derivatives. The first is a green dehydrogenase containing copper and flavin as prosthetic groups which is active upon acyl CoA's from C3 to C8. The metal is essential for the interaction of this dehydrogenase with cytochrome c. The second is a yellow flavoprotein which is active upon acyl CoA's from C4 to C18.7. Unsaturated fatty acyl CoA hydrase catalyses the hydration of transα, β‐ or β, γ‐unsaturated CoA's to their corresponding l(+)‐β‐hydroxyacyl CoA derivatives. The enzyme acts upon all unsaturated derivatives from C4 to at least C12. At equilibrium (pH 9, 250) the ratio β‐hydroxyacyl CoA:total unsaturated acyl CoA is 1. 4:1.8. The β‐hydroxyacyl CoA dehydrogenase catalyses the oxidation of l(+)‐β‐hydroxyacyl CoA by DPN+. The product of oxidation is the corresponding β‐ketoacyl CoA. The enzyme is active over the entire range of fatty acid chain length. The E0of the reaction couple at pH 7.0 and 220 is – 0.224 V. The equilibrium point of the oxidation is strongly pH dependent.9. The β‐ketoacyl CoA cleavage enzyme catalyses the reversible cleavage of β‐ketoacyl CoA's by another molecule of CoA to form acetyl CoA and a new acyl CoA with two carbon atoms less than the parent β‐ketoacyl CoA.10. The new fatty acyl CoA generated in the cleavage reaction undergoes a repeat cycle of β‐oxidation while the C2 unit (acetyl CoA) undergoes condensation with oxalacetate to form citrate.11. Each of the component reactions in the β‐oxidation cycle has been shown to be reversible.12. The asymmetric labelling of acetoacetate formed during oxidation of labelled fatty acids by liver homogenates or mitochondrial suspensions is a phenomenon which can readily be explained in terms of the mechanism of the β‐ketoacyl CoA cleavage enzyme.13. The factors which militate against the accumulation of intermediates during fatty acid oxidation are discussed.14. The accumulation of acetoacetate in any tissue requires a combination of two essential conditions: (a) presence of acyl CoA deacylase; and (b) absence of a β‐ketoacid activation enzyme.15. Assuming that in the diabetic the operation of the citric acid cycle is subnormal by virtue of reduced conversion of glucose to pyruvate, it is possible to explain the accumulation of ketone bodies and its abolition by insulin in terms of the known enzyme reactions of the β‐oxidation cycle.16. The predominance of C16 and C18 fatty acids in lipids may be due to the fact that only the acyl CoA's of these particular acids dissociate to a sufficient degree from combination with the enzymes of the fatty acid oxidizing system as to become available for ester synthesis.It is a great pleasure to acknowledge my indebtedness to Dr Helmut Beinert for his advice and assistance in the preparation of the manuscript and to Drs H. R. Mahler, D. R. Sanadi and S. J. Wakil for their suggestions.