Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.     

Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.     

Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation. Inhibition of long chain acyl-CoA synthetase by triacsins caused inhibition of Raji cell proliferation

Triacsins A, B, C, and D are new inhibitors of long chain acyl-CoA synthetase (EC 6.2.1.3) and possess different inhibitory potencies against the enzyme (Tomoda, H., Igarashi, K., and Omura, S. (1987) Biochim. Biophys. Acta 921, 595-598). Acyl-CoA synthetase activity in the membrane fraction of Raji cells was also inhibited by triacsins. The same hierarchy of inhibitory potency as that against the enzyme from other sources, triacsin C greater than triacsin A much greater than triacsin D greater than or equal to triacsin B, was observed. When Raji cells were cultivated in the presence of triacsins, cell proliferation was inhibited in a dose-dependent fashion. The drug concentrations required for 50% inhibition of cell growth at day 2 were calculated to be 1.8 microM for triacsin A, much greater than 20 microM for triacsin B, 1.0 microM for triacsin C, and much greater than 15 microM for triacsin D, demonstrating a hierarchy for inhibitory potency of triacsins similar to that against the acyl-CoA synthetase activity. To understand the role of long chain acyl-CoA synthetase in animal cells, the effect of triacsins on the lipid metabolism of Raji cells was studied. When intact Raji cells were incubated with [14C]oleate in the presence of individual triacsins, the incorporation of [14C]oleate into each of the lipid fractions such as phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol was inhibited to an analogous extent. A common hierarchy, triacsin C greater than triacsin A much greater than triacsin D greater than triacsin B, was shown for the inhibition in each synthesis of the three lipids, which was identical with that for acyl-CoA synthetase. These findings indicate that the inhibition of acyl-CoA synthetase is well correlated with the inhibition of lipid synthesis. Taken together, the data strongly suggest that the inhibition of acyl-CoA synthetase by triacsins leads to the inhibition of lipid synthesis and eventually to the inhibition of proliferation of Raji cells.     

Activation of a thioesterase specific for very-long-chain fatty acids by adrenergic agonists in perfused hearts.

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.     

Identification of PTE2, a human peroxisomal long-chain acyl-CoA thioesterase

Computer-based approaches identified PTE2 as a candidate human peroxisomal acyl-CoA thioesterase gene. The PTE2 gene product is highly similar to the rat cytosolic and mitochondrial thioesterases, CTE1 and MTE1, respectively, and terminates in a tripeptide sequence, serine-lysine-valine(COOH), that resembles the consensus sequence for type-1 peroxisomal targeting signals. PTE2 was targeted to peroxisomes and recombinant PTE2 showed intrinsic acyl-CoA thioesterase activity with a pH optimum of 8.5. A comparison of PTE2 and PTE1 thioesterase activities across multiple acyl-CoA substrates indicated that while PTE1 was most active on medium-chain acyl-CoAs, with little activity on long-chain acyl-CoAs, PTE2 displayed high activity on medium- and long-chain acyl-CoAs. The identification of PTE2 therefore offers an explanation for the observed long-chain acyl-CoA thioesterase activity of mammalian peroxisomes.     

Triacsin C: A differential inhibitor of arachidonoyl-CoA synthetase and nospecific long chain acyl-CoA synthetase,

Triacsins A,B,C, and D are newly discovered compounds isolated from the culture filtrate of streptomyces which are known to inhibit nonspecific long chain acyl-CoA synthetase (EC 6.2.1.3.). These inhibitors have not been previously studied with regard to their effects on arachidonoyl-CoA synthetase, an enzyme which specifically utilizes arachidonate and other icosanoid precursor fatty acids. To explore his question, we used triacsin C, a potent inhibitor of the nonspecific acyl-CoA synthetase. Triacsin C was found to inhibit the action of arachidonoyl-CoA synthetase and the nonspecific enzyme in sonicates of HSDM₁C₁ mouse fibrosarcoma cells. Importantly, however, the triacsin concentration and length of pre-incubation with the enzymes could be adjusted to almost completely inhibit (>80%) the nonspecific long chain acyl CoA-synthetase, with less than 20% inhibition of arachidonoyl-CoA synthetase. Using intact cultured cells exposed to 1 ug/ml traicsin for up to 15 minutes, we unexpectedly observed preferential inhibition of arachidonoyl-CoA synthetase activity. In intact cell studies, arachidonoyl-CoA synthetase was inhibited > 90%, with 55–60% inhibition of the nonspecific acyl-CoA synthetase. As additional evidence of its inhibition of acyl-CoA synthetase enzymes in intact cells, triacsin c inhibited both fatty acid uptake into cells and icosanoid production, metabolic processes which in certain cell types appear to be dependent on acyl-CoA synthetase activity. Thus, triacsin C is a novel inhibitor which can alter the fatty metabolism of intact cells. This compound can be of significant value in determining the specific cellular functions of the two acyl-CoA synthetase enzymes.     

Triacsin C: a differential inhibitor of arachidonoyl-CoA synthetase and nonspecific long chain acyl-CoA synthetase

Triacsins A, B, C, and D are newly discovered compounds isolated from the culture filtrate of streptomyces which are known to inhibit nonspecific long chain acyl-CoA synthetase (EC 6.2.1.3.). These inhibitors have not been previously studied with regard to their effects on arachidonoyl-CoA synthetase, an enzyme which specifically utilizes arachidonate and other icosanoid precursor fatty acids. To explore this question, we used triacsin C, a potent inhibitor of the nonspecific acyl-CoA synthetase. Triacsin C was found to inhibit the action of arachidonoyl-CoA synthetase and the nonspecific enzyme in sonicates of HSDM1C1 mouse fibrosarcoma cells. Importantly, however, the triacsin concentration and length of pre-incubation with the enzymes could be adjusted to almost completely inhibit (greater than 80%) the nonspecific long chain acyl CoA-synthetase, with less than 20% inhibition of arachidonoyl-CoA synthetase. Using intact cultured cells exposed to 1 ug/ml triacsin for up to 15 minutes, we unexpectedly observed preferential inhibition of arachidonoyl-CoA synthetase activity. In intact cell studies, arachidonoyl-CoA synthetase was inhibited greater than 90%, with 55-60% inhibition of the nonspecific acyl-CoA synthetase. As additional evidence of its inhibition of acyl-CoA synthetase enzymes in intact cells, triacsin C inhibited both fatty acid uptake into cells and icosanoid production, metabolic processes which in certain cell types appear to be dependent on acyl-CoA synthetase activity. Thus, triacsin C is a novel inhibitor which can alter the fatty metabolism of intact cells. This compound can be of significant value in determining the specific cellular functions of the two acyl-CoA synthetase enzymes.     

The expression of cytosolic and mitochondrial type II acyl-CoA thioesterases is upregulated in the porcine corpus luteum during pregnancy

Acyl-CoA thioesterases hydrolyze acyl-CoAs to free fatty acids and CoASH, thereby regulating fatty acid metabolism. This activity is catalyzed by numerous structurally related and unrelated enzymes, of which several acyl-CoA thioesterases have been shown to be regulated via the peroxisome proliferator-activated receptor alpha, strongly linking them to fatty acid metabolism. Two protein families have recently been characterized, the type I acyl-CoA thioesterase gene family and the type II protein family, which are expressed in cytosol, mitochondria and peroxisomes. Still, only little is known about regulation of their expression and precise functions in vivo. In the present study, we have investigated the activity and expression of acyl-CoA thioesterase in the porcine ovary during different phases of the estrus cycle. The activity was low in homogenates obtained during the immature and follicular phases, increasing nearly 4-fold during the luteal phase, with the highest activity being found in the pregnant corpus luteum (about 7-fold higher than in immature follicles). The increase in homogenate activity in corpus luteum from pregnant pigs was due to a moderate increase in the cytosolic activity, and an approximately 20-25-fold increase in the mitochondrial fraction. Western blot analysis showed no detectable expression of the type I acyl-CoA thioesterases (CTE-I and MTE-I) and revealed that the increased activity in cytosol and mitochondria is due to increased expression of the type II acyl-CoA thioesterases (CTE-II and MTE-II). This apparent hormonal regulation of expression of the type II acyl-CoA thioesterase may provide new insights into the functions of these enzymes in the mammalian ovary.     

Simultaneous measurement of prostaglandin and arachidonoyl CoA formed from arachidonic acid in rabbit kidney medulla microsomes: the roles of Zn2+ and Cu2+ as modulators of formation of the two products.

Under physiological conditions, small amounts of free arachidonic acid (AA) is released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) act competitively on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To date, there is no information about the factors deciding the metabolic fate of free AA into these two pathways. In this study, we tried to establish a method for the simultaneous measurement of PG and AA-CoA synthesis from exogenous AA in microsomes from rabbit kidney medulla. The kidney medulla microsomes were incubated with [14C]-AA in 0.1 M-Tris/HCI buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. When 60 microM AA was used as the substrate, indomethacin (an inhibitor of COX) and triacsin C (an inhibitor of ACS) reduced only PG and AA-CoA formation, respectively. On the other hand, when 5 microM AA was used as the substrate, indomethacin and triacsin C came to increase significantly the AA-CoA and PG formation, respectively. Thus, the experiments utilizing indomethacin and triacsin C revealed that the incubation using 60 microM AA can simultaneously detect the changes in the activities of COX and ACS caused by drugs, while the incubation using 5 microM AA can detect the changes in the product formation elicited by the resulting shunt of AA. Further, using these incubation conditions, the effects of Zn2+ and Cu2+ on the PG and AA-CoA formation were examined. Zn2+ inhibited the AA-CoA synthesis from 60 microM AA without affecting the PG synthesis. In contrast, when 5 microM AA was used as the substrate, a significant increase in the PG formation was observed in the presence of this ion, indicating that drug actions on the PG formation from AA by the kidney medulla microsomes may change depending on the substrate concentration. On the other hand, Cu2+ increased PG synthesis and inhibited AA-CoA synthesis from both 60 and 5 microM AA. These results suggest that the simultaneous measurements of PG and AA-CoA formation by the kidney medulla microsomes under high (60 microM) and low (5 microM) substrate concentrations can investigate the direct and indirect actions of drugs on the COX and ACS activities, and are useful for clarifying the haemostatic control of the metabolic fate of AA into the two enzymatic pathways. Furthermore, this study showed that Zn2+ and Cu2+ can modulate PG and AA-CoA formation by affecting COX activity, ACS activity, and/or the AA flow into the two enzymatic pathways.     

The peroxisome proliferator-induced cytosolic type I acyl-CoA thioesterase (CTE-I) is a serine-histidine-aspartic acid alpha /beta hydrolase.

Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317-34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.     

Inhibition of acyl-CoA synthetase by triacsins

Triacsin A, 1-hydroxy-3-(E,E-2',4'-undecadienylidine) triazene and triacsin C, 1-hydroxy-3-(E,E,E-2',4',7'-undecatrienylidine) triazene are potent inhibitors of acyl-CoA synthetase (EC 6.2.1.3). The concentrations of triacsin A required for 50% inhibition of acyl-CoA synthetase from Pseudomonas aeruginosa and from rat liver are 17 and 18 microM, and those of triacsin C are 3.6 and 8.7 microM, respectively. Kinetic analysis indicates that inhibition of triacsin A is non-competitive with respect to the two substrates ATP and coenzyme A, but is competitive with respect to long-chain fatty acids. The apparent Ki value is 8.97 microM when oleic acid is used as substrate. Acid hydrolysis of triacsins results in corresponding polyenic aldehydes with no activity. This suggests that the N-hydroxytriazene moiety is essential for inhibitory activity against acyl-CoA synthetase.     

Peroxisome proliferator-induced long chain acyl-CoA thioesterases comprise a highly conserved novel multi-gene family involved in lipid metabolism

Long chain acyl-CoA esters are important intermediates in degradation and synthesis of fatty acids, as well as having important functions in regulation of intermediary metabolism and gene expression. Although the physiological functions for most acyl-CoA thioesterases have not yet been elucidated, previous data suggest that these enzymes may be involved in lipid metabolism by modulation of cellular concentrations of acyl-CoAs and fatty acids. In line with this, we have cloned four highly homologous acyl-CoA thioesterase genes from mouse, showing multiple compartmental localizations. The nomenclature for these genes has tentatively been assigned as CTE-I (cytosolic), MTE-I (mitochondrial), and PTE-Ia and Ib (peroxisomal), based on the identification of putative targeting signals. Although the various isoenzymes show between 67% and 94% identity at amino acid level, each individual enzyme shows a specific tissue expression. Our data suggest that all four genes are located within a very narrow cluster on chromosome 12 in mouse, similar to a sequence cluster on human chromosome 14, which identified four genes homologous to the mouse thioesterase genes. Four related genes were also identified in Caenorhabditis elegans, all containing putative PTS1 targeting signals, suggesting that the ancestral type I thioesterase gene(s) is/are of peroxisomal origin. All four thioesterases are differentially expressed in tissues examined, but all are inducible at mRNA level by treatment with the peroxisome proliferator clofibrate, or during the physiological condition of fasting, both of which conditions cause a perturbation in overall lipid homeostasis. These results strongly support the existence of a novel multi-gene family cluster of mouse acyl-CoA thioesterases, each with a distinct function in lipid metabolism.     

The effects of nitric oxide and peroxynitrite on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To clarify factors deciding the metabolic fate of free AA into these two pathways, we investigated the effects of a nitric oxide (NO) donor 1-hydroxyl-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), and peroxynitrite (ONOO(-)) on the formation of PG and AA-CoA from high and low concentrations of AA (60 and 5 micro M) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 micro M [14C]-AA in 0.1M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced GSH and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. When 60 micro M AA was used as the substrate concentration, NOC7 stimulated the PG formation at 0.5 micro M, and inhibited it at 50 and 100 micro M, without affecting the AA-CoA formation. When 5 micro M AA was used as the substrate concentration, NOC7 showed no effect on the PG and AA-CoA formation up to 10 micro M or below, but enhanced the AA-CoA formation with a coincident decrease in the PG formation at 50 micro M or over. Experiments utilizing a NO antidote, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, revealed that the observed effects of NOC7 using 60 and 5 micro M AA are caused by NO. On the other hand, ONOO(-) stimulated the PG formation from 60 micro M AA, with no alteration in the AA-CoA formation at a concentration of 100 micro M, but when 5 micro M AA was used as the substrate concentration, it was without effect on the PG and AA-CoA formation. These findings indicate that actions of NO and ONOO(-) on the PG and AA-CoA formation by the kidney medulla microsomes may change depending on the substrate concentration. The effects of NO using 5 micro M AA were reversed by the addition of the superoxide generating system (xanthine-xanthine oxidase plus catalase), indicating that superoxide is a vital modulator of the action of NO. These results suggest that NO, but not ONOO(-), can be a regulator of the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that superoxide may play an important role in the action of NO.     

Identification of peroxisomal acyl-CoA thioesterases in yeast and humans

A computer-based screen of the Saccharomyces cerevisiae genome identified YJR019C as a candidate oleate-induced gene. YJR019C mRNA levels were increased significantly during growth on fatty acids, suggesting that it may play a role in fatty acid metabolism. The YJR019C product is highly similar to tesB, a bacterial acyl-CoA thioesterase, and carries a tripeptide sequence, alanine-lysine-phenylalanineCOOH, that closely resembles the consensus sequence for type-1 peroxisomal targeting signals. YJR019C directed green fluorescence protein to peroxisomes, and biochemical studies revealed that YJR019C is an abundant component of purified yeast peroxisomes. Disruption of the YJR019C gene caused a significant decrease in total cellular thioesterase activity, and recombinant YJR019C was found to exhibit intrinsic acyl-CoA thioesterase activity of 6 units/mg. YJR019C also shared significant sequence similarity with hTE, a human thioesterase that was previously identified because of its interaction with human immunodeficiency virus-Nef in the yeast two-hybrid assay. We report here that hTE is also a peroxisomal protein, demonstrating that thioesterase activity is a conserved feature of peroxisomes. We propose that YJR019C and hTE be renamed as yeast and human PTE1 to reflect the fact that they encode peroxisomal thioesterases. The physical segregation of yeast and human PTE1 from the cytosolic fatty acid synthase suggests that these enzymes are unlikely to play a role in formation of fatty acids. Instead, the observation that PTE1 contributes to growth on fatty acids implicates this thioesterase in fatty acid oxidation.     

Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis

A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.     

Molecular cloning and characterization of MT-ACT48, a novel mitochondrial acyl-CoA thioesterase.

While characterizing Eps15 partners, we identified a 48-kDa polypeptide (p48) which was precipitated by Eps15-derived glutathione S-transferase fusion proteins. A search in a murine expressed sequence tag data base with N-terminal microsequences of p48 led to the identification of two complete cDNA clones encoding two isoforms of a 439-amino acid protein sharing 95% nucleic and amino acid identity. Northern blot and immunoblotting studies showed that p48 was ubiquitously expressed. A significant homology (19% identity and 40% similarity) between p48 and rat brain cytosolic acyl-CoA thioesterase was observed in an 80-amino acid C-terminal domain, retrieved from proteins from human, nematode, and plants. The thioesterase function of p48 was further demonstrated against long chain acyl-CoAs in a spectrophotometric assay. Furthermore, data obtained from sequence analysis showed that p48 contained a mitochondrial targeting signal, cleaved in mature protein as assessed by microsequencing. The mitochondrial localization of both endogenous and transfected p48 was confirmed by confocal microscopy. These results indicate that p48, called MT-ACT48 (mitochondrial acyl-CoA thioesterase of 48 kDa), defines a novel family of mitochondrial long chain acyl-CoA thioesterases.     

15-Hydroperoxyeicosapentaenoic acid, but not eicosapentaenoic acid, shifts arachidonic acid away from cyclooxygenase pathway into acyl-CoA synthetase pathway in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of eicosapentaenoic acid (EPA) and 15-hydroperoxyeicosapentaenoic acid (15-HPEPE) on the PG and AA-CoA formations from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [(14)C]-AA in 0.1M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. EPA reduced the PG and AA-CoA formations from both 60 and 5 microM AA. In contrast, 15-HPEPE decreased the PG formation without affecting the AA-CoA formation from 60 microM AA, and increased the AA-CoA formation at the expense of PG formation when 5 microM AA was used as substrate concentration. The experiments utilizing Fe(2+) and an electron spin resonance (ESR) revealed that 15-HPEPE elicits these effects in the form of hydroperoxy adduct. These results suggest that 15-HPEPE, but not EPA, has the potential to shift AA away from COX pathway into ACS pathway at low substrate concentration (close to the physiological concentration of AA).     

Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism

Previously we demonstrated that supplementation with the polyunsaturated fatty acids (PUFA) arachidonic acid (AA) or docosahexaenoic acid (DHA) increased neurite outgrowth of PC12 cells during differentiation, and that overexpression of rat acyl-CoA synthetase long-chain family member 6 (Acsl6, formerly ACS2) further increased PUFA-enhanced neurite outgrowth. However, whether Acsl6 overexpression enhanced the amount of PUFA accumulated in the cells or altered the partitioning of any fatty acids into phospholipids (PLs) or triacylglycerides (TAGs) was unknown. Here we show that Acsl6 overexpression specifically promotes DHA internalization, activation to DHA-CoA, and accumulation in differentiating PC12 cells. In contrast, oleic acid (OA) and AA internalization and activation to OA-CoA and AA-CoA were increased only marginally by Acsl6 overexpression. Additionally, the level of total cellular PLs was increased in Acsl6 overexpressing cells when the medium was supplemented with AA and DHA, but not with OA. Acsl6 overexpression increased the incorporation of [(14)C]-labeled OA, AA, or DHA into PLs and TAGs. These results do not support a role for Acsl6 in the specific targeting of fatty acids into PLs or TAGs. Rather, our data support the hypothesis that Acsl6 functions primarily in DHA metabolism, and that its overexpression increases DHA and AA internalization primarily during the first 24 h of neuronal differentiation to stimulate PL synthesis and enhance neurite outgrowth.     

Tyrosine phosphatase SHP2 regulates the expression of acyl-CoA synthetase ACSL4

Acyl-CoA synthetase 4 (ACSL4) is implicated in fatty acid metabolism with marked preference for arachidonic acid (AA). ACSL4 plays crucial roles in physiological functions such as steroid synthesis and in pathological processes such as tumorigenesis. However, factors regulating ACSL4 mRNA and/or protein levels are not fully described. Because ACSL4 protein expression requires tyrosine phosphatase activity, in this study we aimed to identify the tyrosine phosphatase involved in ACSL4 expression. NSC87877, a specific inhibitor of the tyrosine phosphatase SHP2, reduced ACSL4 protein levels in ACSL4-rich breast cancer cells and steroidogenic cells. Indeed, overexpression of an active form of SHP2 increased ACSL4 protein levels in MA-10 Leydig steroidogenic cells. SHP2 has to be activated through a cAMP-dependent pathway to exert its effect on ACSL4. The effects could be specifically attributed to SHP2 because knockdown of the phosphatase reduced ACSL4 mRNA and protein levels. Through the action on ACSL4 protein levels, SHP2 affected AA-CoA production and metabolism and, finally, the steroidogenic capacity of MA-10 cells: overexpression (or knockdown) of SHP2 led to increased (or decreased) steroid production. We describe for the first time the involvement of SHP2 activity in the regulation of the expression of the fatty acid-metabolizing enzyme ACSL4.