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Characterization of an Acyl-CoA Thioesterase That Functions as a Major Regulator of Peroxisomal Lipid Metabolism*

  • Mary C. Hunt
    Correspondence
    To whom correspondence should be addressed: Dept. of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry C1-74, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden. Tel.: 46-8-58581293; Fax: 46-8-58581260
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Karianne Solaas
    Footnotes
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • B. Frode Kase
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Stefan E.H. Alexson
    Affiliations
    Department of Medical Laboratory Sciences and Technology, Division of Clinical Chemistry, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Stockholm, Sweden
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  • Author Footnotes
    * This work was supported by the Swedish Society for Medical Research, the Swedish Medical Research Council, the Swedish Natural Science Research Council, Hjärt-Lungfonden, and Amersham Biosciences, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) AF441166.
    § Present address: Inst. for Nutrition Research, University of Oslo, NO-0316 Oslo, Norway.
Open AccessPublished:October 22, 2001DOI:https://doi.org/10.1074/jbc.M106458200
      Peroxisomes function in β-oxidation of very long and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. Here we have cloned and characterized a peroxisomal acyl-CoA thioesterase from mouse, named PTE-2 (peroxisomal acyl-CoA thioesterase 2). PTE-2 is ubiquitously expressed and induced at mRNA level by treatment with the peroxisome proliferator WY-14,643 and fasting. Induction seen by these treatments was dependent on the peroxisome proliferator-activated receptor α. Recombinant PTE-2 showed a broad chain length specificity with acyl-CoAs from short- and medium-, to long-chain acyl-CoAs, and other substrates including trihydroxycoprostanoyl-CoA, hydroxymethylglutaryl-CoA, and branched chain acyl-CoAs, all of which are present in peroxisomes. Highest activities were found with the CoA esters of primary bile acids choloyl-CoA and chenodeoxycholoyl-CoA as substrates. PTE-2 activity is inhibited by free CoASH, suggesting that intraperoxisomal free CoASH levels regulate the activity of this enzyme. The acyl-CoA specificity of recombinant PTE-2 closely resembles that of purified mouse liver peroxisomes, suggesting that PTE-2 is the major acyl-CoA thioesterase in peroxisomes. Addition of recombinant PTE-2 to incubations containing isolated mouse liver peroxisomes strongly inhibited bile acid-CoA:amino acidN-acyltransferase activity, suggesting that this thioesterase can interfere with CoASH-dependent pathways. We propose that PTE-2 functions as a key regulator of peroxisomal lipid metabolism.
      PPARα
      peroxisome proliferator-activated receptor α
      CoA
      coenzyme A
      PTE-2
      peroxisomal acyl-CoA thioesterase-2
      CoASH
      coenzyme A, reduced
      THCA-CoA
      trihydroxycoprostanoyl-CoA
      DHCA
      dihydroxycoprostanoyl-CoA
      CA-CoA
      choloyl-CoA
      CDCA-CoA
      chenodeoxycholoyl-CoA
      DMN-CoA
      4,8-di-methylnonanoyl-CoA
      HMG-CoA
      3-hydroxy-3-methyl-glutaryl-CoA
      BAAT
      bile acid-CoA:amino acidN-acyltransferase
      FXR
      farnesoid-X-receptor
      RXR
      retinoid-X-receptor
      BSA
      bovine serum albumin
      PBS
      phosphate-buffered saline
      HPLC
      high-pressure liquid chromatography
      GFP
      green fluorescent protein
      DTNB
      5,5′-dithiobis(2-nitrobenzoic acid)
      Tritc
      tetramethylrhodamine-5-isothiocyanate
      HIV
      human immunodeficiency virus
      Peroxisomes are cellular organelles present in all eukaryotic cells. They play an indispensable role in the metabolism of a variety of lipids including very long-chain fatty acids, dicarboxylic fatty acids, bile acids, prostaglandins, leukotrienes, thromboxanes, pristanic acid, and xenobiotic fatty acids (for review, see Refs.
      • Van Veldhoven P.P.
      • Mannaerts G.P.
      and
      • Reddy J.K.
      • Hashimoto T.
      ). The peroxisomal β-oxidation system contains two sets of enzymes, one of which is involved in the oxidation of branched chain fatty acids and intermediates in the hepatic bile acid biosynthetic pathway and consists of one or two branched-chain acyl-CoA oxidase(s), ad-specific bifunctional protein and the sterol carrier-like protein x (SCPx). The second pathway is involved in the oxidation of very long straight-chain fatty acids, CoA esters of prostaglandins, leukotrienes, and thromboxanes (prostanoids) and dicarboxylic acids. This system is composed of a straight-chain acyl-CoA oxidase, anl-specific bifunctional protein and straight-chain 3-ketoacyl-CoA thiolase. However, these two pathways are not mutually exclusive (
      • Van Veldhoven P.P.
      • Mannaerts G.P.
      ). All enzymes in this latter pathway are induced by peroxisome proliferators, whereas the enzymes in the former β-oxidation pathway are not. Peroxisome proliferators are a group of structurally diverse compounds including hypolipidemic drugs, which induce peroxisome proliferation, hepatomegaly, and cause hepatocarcinogenesis in rodent liver. These peroxisome proliferators, together with free fatty acids, act as ligands for the peroxisome proliferator-activated receptor α (PPARα)1 (
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      ,
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      ). The PPARα is a nuclear receptor that plays a central role in fatty acid metabolism and induces the expression of many enzymes involved in peroxisomal and mitochondrial β-oxidation and ω-oxidation of fatty acids. Targeted disruption of the PPARα gene in mouse has established a key role for this receptor as a mediator of lipid metabolism (
      • Lee S.S.
      • Pineau T.
      • Drago J.
      • Lee E.J.
      • Owens J.W.
      • Kroetz D.L.
      • Fernandez-Salguero P.M.
      • Westphal H.
      • Gonzalez F.J.
      ,
      • Aoyama T.
      • Peters J.M.
      • Iritani N.
      • Nakajima T.
      • Furihata K.
      • Hashimoto T.
      • Gonzalez F.J.
      ,
      • Hunt M.C.
      • Lindquist P.J.G.
      • Peters J.M.
      • Gonzalez F.J.
      • Diczfalusy U.
      • Alexson S.E.H.
      ,
      • Kroetz D.L.
      • Yook P.
      • Costet P.
      • Bianchi P.
      • Pineau Y.
      ) and in the adaptive response to fasting (
      • Hunt M.C.
      • Lindquist P.J.G.
      • Peters J.M.
      • Gonzalez F.J.
      • Diczfalusy U.
      • Alexson S.E.H.
      ,
      • Kroetz D.L.
      • Yook P.
      • Costet P.
      • Bianchi P.
      • Pineau Y.
      ,
      • Kersten S.
      • Seydoux J.
      • Peters J.M.
      • Gonzalez F.J.
      • Desvergne B.
      • Wahli W.
      ,
      • Leone T.C.
      • Weinheimer C.J.
      • Kelly D.P.
      ).
      Prior to transport into peroxisomes and β-oxidation, all substrates must be activated to their CoA ester, which occurs at different cellular locations. Long-chain acyl-CoA synthetases are present in different subcellular membranes (
      • Lewin T.M.
      • Kim J.H.
      • Granger D.A.
      • Vance J.E.
      • Coleman R.A.
      ,
      • Gargiulo C.E.
      • Stuhlsatz-Krouper S.M.
      • Schaffer J.E.
      ) and long-chain acyl-CoA synthetase in the peroxisomal membrane activates very long and long-chain fatty acids and possibly branched-chain fatty acids to their CoA esters. Dicarboxylic fatty acids, prostanoids, and di- and trihydroxycoprostanic acid are activated to their CoA esters in the endoplasmic reticulum. These CoA esters are then transported into peroxisomes, possibly via ABC transporters. Peroxisomal β-oxidation of very long and long-chain fatty acyl-CoAs results in chain-shortening of these esters, and the chain-shortened products can be transported as carnitine esters to mitochondria for further degradation. However, peroxisomes appear to have important roles in β-oxidation of a number of xenobiotic carboxylic acids which may only be partially metabolized in peroxisomes (
      • Yamada J.
      • Itoh S.
      • Horie S.
      • Watanabe T.
      • Suga T.
      ,
      • Yamada J.
      • Ogawa S.
      • Horie S.
      • Watanabe T.
      • Suga T.
      ). The β-oxidation of other CoA esters, such as prostanoids results in chain-shortening in peroxisomes, which are subsequently excreted in urine as the free carboxylic acid (for review, see Ref.
      • Diczfalusy U.
      ). Similarly the β-oxidation of dicarboxylic acids results in a chain-shortening in peroxisomes with the dicarboxylic acid being excreted in urine as the free acid or as glycine conjugates. Several of these carboxylic acids are only slowly β-oxidized, implying that intraperoxisomal CoASH may be sequestered to such an extent that it becomes limiting. To maintain appropriate CoASH levels and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases are likely to play important roles (for review, see Ref.
      • Hunt M.C.
      • Alexson S.E.H.
      ). The hydrolysis of CoA esters to the free acids requires the presence of an acyl-CoA thioesterase, an enzyme that functions to hydrolyze CoA esters to the free acid and CoASH. To date, however, the specific thioesterase(s) active on CoA esters of prostanoids or dicarboxylic acids have not been identified.
      Another important reaction that occurs in liver peroxisomes is the formation of bile acids. The primary bile acids, cholic acid and chenodeoxycholic acid, are formed by a number of enzymatic modifications of the cholesterol backbone by P-450 enzymes. The di- or trihydroxycoprostanoyl-CoA (DHCA-CoA or THCA-CoA) formed undergoes a final β-oxidative cleavage of the side chain in peroxisomes with the release of propionyl-CoA, to form chenodeoxycholoyl-CoA and choloyl-CoA, respectively (
      • Pedersen J.I.
      • Gustafsson J.
      ,
      • Kase F.
      • Björkhem I.
      • Pedersen J.I.
      ). The peroxisomal bile acid-CoA:amino acid N-acyltransferase (BAAT) then catalyzes the conjugation of the CoA-activated bile acid to taurine or glycine prior to secretion from liver into bile. Recently, a multiorganellar bile acid-CoA thioesterase activity, which hydrolyzes the bile acid-CoA esters to free bile acids and CoASH, has been characterized in human liver peroxisomes (
      • Solaas K.
      • Ulvestad A.
      • Söreide O.
      • Kase B.F.
      ,
      • Solaas K.
      • Sletta R.J.
      • Søreide O.
      • Kase B.F.
      ). This bile acid-CoA thioesterase activity described may compete with the BAAT enzyme for the bile acid-CoA substrate, thereby influencing intracellular levels of free and conjugated bile acids.
      The existence of selective acyl-CoA thioesterases or a “broad range” acyl-CoA thioesterase could provide important control points in the oxidation of many peroxisomal substrates and to regulate intracellular levels of CoA esters and CoASH. This may be especially important during times of high β-oxidation and fatty acid overload, to generate free CoASH necessary for fatty acid β-oxidation to proceed. Acyl-CoA thioesterase activity has indeed been shown to be present in peroxisomes (
      • Osmundsen H.
      • Neat C.E.
      • Borrebaek B.
      ,
      • Berge R.K.
      • Flatmark T.
      • Osmundsen H.
      ,
      • Alexson S.E.H.
      • Osmundsen H.
      • Berge R.K.
      ,
      • Wilcke M.
      • Alexson S.E.H.
      ), and isolated rat brown adipose tissue peroxisomes contain acyl-CoA thioesterase(s) active on short- and medium-chain acyl-CoAs, which are inhibited by CoASH (
      • Alexson S.E.H.
      • Osmundsen H.
      • Berge R.K.
      ). Rat liver peroxisomes showed broad acyl-CoA thioesterase activity on C2–C22 acyl-CoAs, and one enzyme was partially purified, which was shown to be most active on myristoyl-CoA (
      • Wilcke M.
      • Alexson S.E.H.
      ). To date, several peroxisomal acyl-CoA thioesterases have been cloned from yeast, mouse, and human. PTE-Ia and -Ib have been cloned from mouse, which belong to a novel family of Type I acyl-CoA thioesterases, with related enzymes also in cytosol and mitochondria (
      • Hunt M.C.
      • Nousiainen S.E.B.
      • Huttunen M.K.
      • Orii K.
      • Svensson L.T.
      • Alexson S.E.H.
      ). Acyl-CoA thioesterases have been identified in yeast and human peroxisomes, named PTE1 (
      • Jones J.M.
      • Nau K.
      • Geraghty M.T.
      • Erdmann R.
      • Gould S.J.
      ). The human homologue of PTE1 was previously identified as hACTEIII/hTE, a protein that interacted with and activated the HIV-1 Nef protein (
      • Watanabe H.
      • Shiratori T.
      • Shoji H.
      • Miyatake S.
      • Okazaki Y.
      • Ikuta K.
      • Sato T.
      • Saito T.
      ,
      • Liu L.X.
      • Margottin F.
      • Le Gall S.
      • Schwartz O.
      • Selig L.
      • Benarous R.
      • Benichou S.
      ). In the present study we have cloned the mouse homologue of PTE1/hACTEIII/hTE, which we have named mouse peroxisomal acyl-CoA thioesterase 2 (PTE-2). We have characterized this enzyme in detail, to examine its putative function in peroxisomal β-oxidation. Characterization of PTE-2 suggests that it is a major thioesterase with an array of functions in peroxisomal lipid metabolism.

      DISCUSSION

      In this study we have cloned and characterized the mouse peroxisomal acyl-CoA thioesterase 2, called PTE-2, which is localized in peroxisomes. This enzyme is the mouse homologue of human PTE1 (
      • Jones J.M.
      • Nau K.
      • Geraghty M.T.
      • Erdmann R.
      • Gould S.J.
      ), which was first identified by the yeast two-hybrid system as hACTEIII and hTE, a HIV-1 Nef-binding protein (
      • Watanabe H.
      • Shiratori T.
      • Shoji H.
      • Miyatake S.
      • Okazaki Y.
      • Ikuta K.
      • Sato T.
      • Saito T.
      ,
      • Liu L.X.
      • Margottin F.
      • Le Gall S.
      • Schwartz O.
      • Selig L.
      • Benarous R.
      • Benichou S.
      ). There is a lot of confusion regarding the nomenclature of acyl-CoA thioesterases, and based on a recent attempt to accomplish a more uniform terminology (
      • Hunt M.C.
      • Alexson S.E.H.
      ), we propose the name PTE-2 for this acyl-CoA thioesterase, as the nomenclature PTE-I has already been assigned to two enzymes that are members of a novel multigene family, with other members in mitochondria and cytosol (
      • Hunt M.C.
      • Nousiainen S.E.B.
      • Huttunen M.K.
      • Orii K.
      • Svensson L.T.
      • Alexson S.E.H.
      ) (Table III). However, the rule applied to the nomenclature of yeast enzymes states that they remain known by the name applied when they were first identified, therefore this enzyme will remain known as PTE1. A human PTE2 cloned as a putative peroxisomal enzyme (
      • Jones J.B.
      • Gould S.J.
      ) (Table III) shows 100% identity to human MTE-I (mitochondrial acyl-CoA thioesterase) without its N-terminal sequence. This MTE-I belongs to a novel family of acyl-CoA thioesterases in human, with localizations also in cytosol and peroxisomes.
      M. C. Hunt, A. Rautanen, T. L. T. Svensson, and S. E. H. Alexson, manuscript in preparation.
      Table IIINomenclature for peroxisomal acyl-CoA thioesterases
      Enzyme (proposed name)SpeciesLocalizationOther nameClonedRef.
      mPTE-IaMousePeroxisomalGene
      • Hunt M.C.
      • Nousiainen S.E.B.
      • Huttunen M.K.
      • Orii K.
      • Svensson L.T.
      • Alexson S.E.H.
      mPTE-IbMousePeroxisomalGene
      • Hunt M.C.
      • Nousiainen S.E.B.
      • Huttunen M.K.
      • Orii K.
      • Svensson L.T.
      • Alexson S.E.H.
      hPTE-IHumanPeroxisomalGeneHuntet al., manuscript in preparation.
      MTE-IHumanPutativePTE2cDNA
      • Jones J.B.
      • Gould S.J.
      Mitochondrial
      PTE1YeastPeroxisomalcDNA
      • Jones J.M.
      • Nau K.
      • Geraghty M.T.
      • Erdmann R.
      • Gould S.J.
      PTE-2HumanPeroxisomalhTEcDNA
      • Liu L.X.
      • Margottin F.
      • Le Gall S.
      • Schwartz O.
      • Selig L.
      • Benarous R.
      • Benichou S.
      PeroxisomalhACTEIIIcDNA
      • Watanabe H.
      • Shiratori T.
      • Shoji H.
      • Miyatake S.
      • Okazaki Y.
      • Ikuta K.
      • Sato T.
      • Saito T.
      PeroxisomalPTE1cDNA
      • Jones J.M.
      • Nau K.
      • Geraghty M.T.
      • Erdmann R.
      • Gould S.J.
      PeroxisomalGeneThis article
      MousePeroxisomalcDNAThis article
      The HIV-1 Nef, which interacts with the thioesterase PTE-2, is a cytosolic 27-kDa myristoylated protein that is required for high viral load and full pathological effect of HIV (
      • Kestler W.H.
      • Ringler D.J.
      • Mori K.
      • Panicali D.L.
      • Shegal P.K.
      • Daniel M.D.
      • Desrosiers R.C.
      ). It is thought that Nef activity triggers endocytosis of CD4 and major histocompatibility complex class-I molecules. Although the interaction of Nef with the acyl-CoA thioesterase is not fully understood, it has been shown that Nef contains sites critical for binding of the human thioesterase, which results in down-regulation of CD4 (
      • Cohen G.B.
      • Rangan V.S.
      • Chen B.K.
      • Smith S.
      • Baltimore D.
      ). The binding of Nef to the thioesterase also targets Nef to peroxisomes in vivo and increases thioesterase activity in vitro (
      • Watanabe H.
      • Shiratori T.
      • Shoji H.
      • Miyatake S.
      • Okazaki Y.
      • Ikuta K.
      • Sato T.
      • Saito T.
      ). It was also shown that abolishment of the interaction of the thioesterase with Nef resulted in impairment of Nef biological functions (
      • Liu L.X.
      • Heveker N.
      • Fackler O.T.
      • Arold S.
      • Le Gall S.
      • Janvier K.P.B.M.
      • Dumas C.
      • Schwartz O.
      • Benichou S.
      • Benarous R.
      ). It then became evident that although this thioesterase may have a putative function in the pathogenesis of HIV, the enzyme may also be involved in fatty acid metabolism. In 1999, Jones et al. (
      • Jones J.M.
      • Nau K.
      • Geraghty M.T.
      • Erdmann R.
      • Gould S.J.
      ) again characterized this enzyme in human, which they named PTE1, together with a yeast homologue, showing these proteins are targeted to the peroxisomal lumen. The yeast PTE1 identified showed regulation at mRNA level by growth on oleate. In yeast, β-oxidation of fatty acids is confined only to peroxisomes, and growth of yeast on oleate as sole carbon source results in increased expression of peroxisomal enzymes and proliferation of peroxisomes. Disruption of PTE1 in yeast resulted in a loss of approximately 80% of total cellular thioesterase activity, demonstrating that PTE1 is the major long-chain acyl-CoA thioesterase in yeast grown on oleate. Furthermore, this deletion impaired growth on oleate, suggesting that efficient β-oxidation in yeast requires the expression of this thioesterase, possibly to closely regulate the intraperoxisomal CoASH levels. Our data now suggest that PTE-2 is in fact a major acyl-CoA thioesterase, which can hydrolyze a wide variety of CoA esters in peroxisomes also in the mouse. The difference in acyl-CoA chain length specificity of PTE-2 in our study compared with previous studies is due to the inclusion of albumin to the thioesterase assay when measuring activity with acyl-CoAs longer than decanoyl-CoA. With addition of albumin, it became evident that PTE-2 hydrolyzes all acyl-CoAs of two carbons up to twenty carbons with very similarVmax. The inhibition of PTE-2 activity by CoASH indicates that this enzyme can “sense” intraperoxisomal CoASH levels, and thus when there is a requirement for CoASH, PTE-2 is active, whereas during times of high free CoASH, PTE-2 can be inhibited. Peroxisomes contain a distinct pool of CoASH (
      • Van Broekhoven A.
      • Peeters M.C.
      • Debeer L.J.
      • Mannaerts G.P.
      ,
      • Van Veldhoven P.P.
      • Mannaerts G.P.
      ), which has been reported to change during fasting and treatment with peroxisome proliferators (
      • Horie S.
      • Isobe M.
      • Suga T.
      ). The extent of CoASH sequestration may therefore depend on the size of the CoASH pool, the amount and type of lipids being trapped in the β-oxidation systems in the peroxisome, as well as activities of acyl-CoA thioesterases and possibly a recently cloned peroxisomal nudix hydrolase, which apparently can hydrolyze CoASH to yield 3′,5′-ADP and the corresponding 4′-phosphopantetheine derivative (
      • Gasmi L.
      • McLennan A.G.
      ).

      PTE-2 Can Have a Multitude of Functions in Regulating Peroxisomal Lipid Metabolism

      In the present study we have cloned and characterized the mouse PTE-2 with respect to regulation of expression and kinetic activity. Recombinant PTE-2 hydrolyzed all tested CoA esters, showing an almost complete lack of substrate specificity with respect to the carboxylic acid moiety. The enzyme catalyzes the hydrolysis of straight-chain, saturated and unsaturated acyl-CoAs of 2–20 carbons in chain length with roughly similarVmax. The enzyme also hydrolyzed other CoA esters such as acetoacetyl-CoA, malonyl-CoA, HMG-CoA, clofibroyl-CoA, THCA-CoA and CoA esters of bile acids, and β-oxidation intermediates. This apparent lack of substrate specificity suggests that the binding site of the enzyme is rather nonspecific with respect to the acyl moiety and that the enzyme may recognize the CoASH moiety for binding. This is in line with the observed inhibitory effect of free CoASH, the kinetics of which suggested a competitive mode of action, although the data were not fully conclusive. Notably, all the CoA esters tested as substrates for PTE-2 can be expected to be present in peroxisomes as substrates, intermediates, or end products in lipid metabolism. In combination with the strong regulation of expression via PPARα, the PTE-2 thioesterase may have a multitude of functions in peroxisomes as outlined in Fig. 8. Fatty acids and other substrates for β-oxidation in peroxisomes must first be esterified to their CoA ester. CoASH that is appended to poorly oxidizable substrates may cause a trapping of CoASH and thereby prevent the β-oxidation cycle to continue. Thus, PTE-2 may temporarily hydrolyze substrates for the β-oxidation to release CoASH. We also tested CoA esters of β-oxidation intermediates, 2-trans-decenoyl-CoA and 3-hydroxypalmitoyl-CoA. Both these CoA esters were poorer substrates compared with the corresponding straight-chain acyl-CoA: 2-trans-decenoyl-CoA was hydrolyzed at a rate of about 22% of that observed with decanoyl-CoA with the Km being increased 6-fold and Vmax being decreased, and 3-hydroxypalmitoyl-CoA, which was hydrolyzed at a rate of about 18% of the rate of hydrolysis of palmitoyl-CoA, with the Kmbeing similar but Vmax being much lower. The lower activities of PTE-2 with β-oxidation intermediates indicates that the thioesterase preferentially removes the CoA esters of substrates and end products, while β-oxidation intermediates are allowed to be further oxidized.
      Figure thumbnail gr8
      Figure 8Model for putative functions of PTE-2 in peroxisomes. PTE-2 may function as an auxillary enzyme in β-oxidation of a number of substrates in peroxisomes. The abbreviations used are: VLCFA, very long-chain fatty acids:CAT, carnitine acetyltransferase; COT, carnitine octanoyltransferase; PG, prostaglandins: LT, leukotrienes; Tb, thromboxanes; FFA, free fatty acid; Cn, carnitine.
      However, a number of carboxylic acids, such as prostaglandins, leukotrienes, and thromboxanes are chain-shortened in peroxisomes and excreted in the urine as the free acids, thus requiring an acyl-CoA thioesterase for these processes. The finding in the present study that PTE-2 readily hydrolyzes the CoA ester of prostaglandin F is the first demonstration of an acyl-CoA thioesterase that can have this function.
      In the β-oxidation of pristanic acid, the intermediate 4,8-dimethylnonanoyl-CoA (DMN) is formed and is transported from the peroxisome to the mitochondria as a carnitine ester, for further oxidation. The high activity of recombinant PTE-2 toward DMN-CoA indicates that PTE-2 may also be able to hydrolyze DMN-CoA to DMN and release CoASH for other β-oxidation reactions in peroxisomes if required. The DMN could then be re-esterified to CoASH by the very long-chain acyl-CoA synthetase in peroxisomes (
      • Steinberg S.J.
      • Wang S.J.
      • Kim D.G.
      • Mihalik S.J.
      • Watkins P.A.
      ).

      PTE-2 Is a PPARα Target Gene That May Regulate Bile Acid Formation

      Bile acids are formed in liver peroxisomes by β-oxidative cleavage of the side chain of DHCA-CoA or THCA-CoA to chenodeoxycholoyl-CoA and choloyl-CoA, respectively, with the concomitant production of propionyl-CoA. In the last step, bile acid-CoAs are conjugated to taurine or glycine, a reaction catalyzed by the BAAT enzyme that acts as an acyltransferase. In fact the best substrates for PTE-2 were found to be THCA-CoA, CA-CoA, and CDCA-CoA. We have now established that PTE-2 is the bile acid-CoA thioesterase identified previously (
      • Solaas K.
      • Ulvestad A.
      • Söreide O.
      • Kase B.F.
      ,
      • Solaas K.
      • Sletta R.J.
      • Søreide O.
      • Kase B.F.
      ), suggesting that a major function of the PTE-2 in liver may be in regulation of bile acid formation and excretion. This conclusion is further supported by the competition experiment carried out, which showed that addition of a small amount of recombinant PTE-2 to peroxisomes severely suppresses the activity of BAAT, presumably due to consumption of the substrate CDCA-CoA. PPARα has been established as a key regulator of lipid metabolism, but was also shown to be involved in regulation of bile acid metabolism (
      • Hunt M.C.
      • Yang Y.Z.
      • Eggertsen G.
      • Carneheim C.M.
      • Gåfvels M.
      • Einarsson C.
      • Alexson S.E.H.
      ), thus connecting the pathways of bile acid and fatty acid metabolism. Up-regulation of PTE-2 by WY-14,643, together with the PTE-2-mediated suppression of bile acid conjugation with taurine in vitro, could imply that PPARα is also involved in regulating bile acid amidation. Indeed, preliminary data show a reduction in conjugated bile acids in mouse liver following treatment with WY-14,643.
      K. Solaas, M. C. Hunt, V. Pham, G. Alvelius, K. Hultenby, B. F. Kase, and S. E. H. Alexson, manuscript in preparation.
      This functional aspect may be very important in view of the recent interest in the farnesoid-X-receptor (FXR), a nuclear receptor that acts as a biological sensor for the regulation of bile acid biosynthesis, and that is activated by free and conjugated chenodeoxycholic acid (
      • Makishima M.
      • Okamoto A.Y.
      • Repa J.J.
      • Tu H.
      • Learned R.M.
      • Luk A.
      • Hull M.V.
      • Lustig K.D.
      • Mangelsdorf D.J.
      • Shan B.
      ,
      • Parks D.J.
      • Blanchard S.G.
      • Bledsoe R.K.
      • Chandra G.
      • Consler T.G.
      • Kliewer S.A.
      • Stimmel J.B.
      • Willson T.M.
      • Zavacki A.M.
      • Moore D.D.
      • Lehmann J.M.
      ) and cholic acid (
      • Wang H.
      • Chen J.
      • Holoister K.
      • Sowers L.C.
      • Forman B.M.
      ). The FXR/RXR regulates expression of genes involved in bile acid metabolism, such as the human ileal bile acid-binding protein (
      • Grober J.
      • Zaghini I.
      • Fujii H.
      • Jones S.A.
      • Kliewer S.A.
      • Willson T.M.
      • Ono T.
      • Besnard P.
      ) and cholesterol 7α-hydroxylase (
      • del Castillo-Olivares A.
      • Gil G.
      ,
      • Chiang J.Y.
      • Kimmel R.
      • Weinberger C.
      • Stroup D.
      ). Increased PTE-2 activity would therefore result in an increase in free bile acids, which may subsequently be transported to the nucleus to activate the FXR/RXR heterodimer complex. In this way, PTE-2 may mediate cross-talk between the PPARα and the FXR signaling pathways.
      With the identification of the gene for the human PTE-2, a putative peroxisome proliferator-response element was identified at 438 bp upstream of the ATG start site. This site conforms well to the consensus direct repeat 1 (AGGTCAnAGGTCA), which has been shown to bind both PPAR/RXR heterodimers and hepatocyte nuclear factor 4α. It will be of interest to identify if this site is functional in the human promoter in binding these transcription factors, thus leading to possible activation of human PTE-2 by either peroxisome proliferators or fatty acids (the ligands for PPARs (
      • Forman B.M.
      • Chen J.
      • Evans R.M.
      ,
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      )), or acyl-CoAs and CoA esters of peroxisome proliferators (the agonists/antagonists for hepatocyte nuclear factor 4α (
      • Hertz R.
      • Magenheim J.
      • Berman I.
      • Bar-Tana J.
      ,
      • Hertz R.
      • Sheena V.
      • Kalderon B.
      • Berman I.
      • Bar-Tana J.
      )).

      Can PTE-2 Be Considered as an Auxilliary β-Oxidation Enzyme?

      In addition to bile acid intermediates, PTE-2 efficiently hydrolyzes methyl-branched fatty acids (e.g.4,8-dimethylnonanoyl-CoA and 2-methylstearoyl-CoA), which are substrates of the “branched-chain” β-oxidation pathway in peroxisomes. Branched-chain CoA esters appear to be excellent substrates for PTE-2, but are generally slowly metabolized via β-oxidation in peroxisomes. At first sight, there appears to be a paradoxical situation that an acyl-CoA thioesterase should facilitate degradation of lipids. However, as outlined before, it is probably very important that appropriate CoASH levels are maintained in peroxisomes. Such a function is supported by the findings in yeast that deletetion of the gene encoding the yeast homologue PTE1 impairs growth of the PTE1 knock-out strain on oleate. Therefore, a possible function of PTE-2 may be to act as an auxilliary enzyme in β-oxidation of branched-chain fatty acids/bile acid formation. This β-oxidation pathway is not PPARα-regulated, and therefore the PPARα-mediated up-regulation of PTE-2 may function in salvaging CoASH for β-oxidation of fatty acids or alternatively function in (temporarily) decreasing β-oxidation of branched-chain lipids. This could serve to mediate a metabolic cross-talk between PPARα and degradation of this class of lipids in the liver. It should be stressed that it is very likely that PTE-2 can play different functions in different organs which could be related to organ-specific metabolic function of peroxisomes in different tissues.

      Is PTE-2 Involved in Regulation of Cholesterol Synthesis in Peroxisomes?

      As outlined above, the PPARα regulation of PTE-2 may confer a metabolic crosstalk between fatty acid degradation and cholesterol metabolism. A similar metabolic cross-talk may occur by PPARα regulation of PTE-2 activity, which may interefere with peroxisomal cholesterol biosynthesis. The initial enzymes involved in cholesterol synthesis have been demonstrated to be present in peroxisomes. These enzymes include acetoacetyl-CoA thiolase (
      • Thompson S.L.
      • Krisans S.K.
      ), HMG-CoA synthase (
      • Olivier L.M.
      • Kovacs W.
      • Masuda K.
      • Keller G.A.
      • Krisans S.K.
      ), and HMG-CoA reductase (
      • Keller G.A.
      • Barton M.C.
      • Shapiro D.J.
      • Singer S.J.
      ,
      • Keller G.A.
      • Pazirandeh M.
      • Krisans S.
      ). PTE-2 hydrolyzes acetyl-CoA (substrate for acetoacetyl-CoA thiolase), acetoacetyl-CoA (substrate for HMG-CoA synthase), and HMG-CoA (substrate for the HMG-CoA reductase). In addition, while the peroxisomal HMG-CoA reductase is up-regulated 2 h into the light cycle (
      • Rusnak N.
      • Krisans S.K.
      ), PTE-2 is most highly expressed at the end of the light cycle/beginning of the dark cycle, indicating a functionally coordinated regulation of expression, i.e. high expression of PTE-2 when HMG-CoA reductase is expressed at low levels.

      Role of PTE-2 in Regulation of Short-chain Acyl-CoAs Generated from Peroxisomal β-Oxidation

      β-Oxidation of straight-chain and branched-chain fatty acids produces chain-shortened acyl-CoAs, which may be transferred to mitochondria (as carnitine esters) for further metabolism or be excreted in urine. However, for each cycle in the β-oxidation, acetyl-CoA and propionyl-CoA are also produced. Accumulation of propionyl-CoA can be associated with impaired metabolism (
      • Brass E.P.
      ). Propionyl-CoA is formed from the β-oxidation of pristanoyl-CoA and other branched-chain acyl-CoAs and bile acid intermediates in peroxisomes. This propionyl-CoA may be transferred to carnitine by peroxisomal carnitine acetyltransferase for further metabolism in mitochondria or hydrolyzed to propionic acid. Propionyl-CoA thioesterase activity has previously been identified in peroxisomes (
      • Alexson S.E.H.
      • Osmundsen H.
      • Berge R.K.
      ,
      • Wilcke M.
      • Alexson S.E.H.
      ). Our present data show that PTE-2 can efficiently hydrolyze propionyl-CoA with a Km of <10 μm and a Vmax of about 3.45 μmol/min/mg of protein. Therefore PTE-2 could prevent accumulation of propionyl-CoA and thus release free CoASH for other reactions. In a similar manner, acetyl-CoA units are released during β-oxidation of both very long- and long-chain acyl-CoAs and dicarboxylic acids and are then transferred to carnitine by carnitine acetyltransferase for further metabolism in mitochondria or hydrolyzed to acetate and excreted to cytosol. Free acetate can account for total acetyl-CoA produced in peroxisomes from dicarboxylic and monocarboxylic acids, and acetate production is increased by peroxisome proliferator treatment (
      • Leighton F.
      • Bergseth S.
      • Rørtveit T.
      • Christiansen E.N.
      • Bremer J.
      ). The production of free acetate from acetyl-CoA requires the hydrolysis by an acyl-CoA thioesterase present in peroxisomes (
      • Alexson S.E.H.
      • Osmundsen H.
      • Berge R.K.
      ,
      • Wilcke M.
      • Alexson S.E.H.
      ). Again, recombinant PTE-2 showed high activity toward acetyl-CoA, which constitutes a mechanism to prevent trapping of CoASH in the acetyl-CoA unit.
      In summary, we have shown that PTE-2 acts as a general acyl-CoA thioesterase that is responsible for most of the thioesterase activity detected in isolated peroxisomes. Furthermore, PTE-2 is highly regulated by WY-14,643 and fasting, which identifies PTE-2 as a novel PPARα target gene. Based on the regulation of expression and regulation of enzymatic activity by CoASH levels, it is likely that PTE-2 has important functions in regulating peroxisomal lipid metabolism. We therefore propose that PTE-2 is a major thioesterase found in peroxisomes that may regulate intracellular peroxisomal CoASH levels, controlling β-oxidation of a broad range of acyl-CoA metabolites and the levels of free and conjugated bile acids in liver. PTE-2 could also be a candidate gene for some of the hitherto unidentified disorders of peroxisomal fatty acid metabolism. Targeted disruption of the gene for PTE-2 should provide an interesting model to examine the role of this enzyme in many of the diverse metabolic pathways in peroxisomes.

      Acknowledgments

      We thank Frank Gonzalez and Jeffrey Peters for PPARα-null mice, Jacob Jones for PTE1 antibody, Jan Pedersen for THCA-CoA and 2-methylstearoyl-CoA, Ronald Wanders for 4,8-dimethylnonanoyl-CoA, Kilervo Hiltunen for 2-trans-decenoyl-CoA, Nikolaos Venizelos for 3-hydroxypalmitoyl-CoA, Manfred Held for synthesis of the prostaglandin F-CoA ester, and Kjell Hultenby for GFP antibody. We also gratefully acknowledge the advice and assistance of Cecilia Rustum and Einar Hallberg regarding GFP experiments and for the GFP secondary antibody.

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