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• W1991882913 abstract "Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo β-oxidation directly but instead must first undergo α-oxidation to pristanic acid, which then enters β-oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor α (PPARα) and is up-regulated in mouse liver in a PPARα-dependent manner. Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo β-oxidation directly but instead must first undergo α-oxidation to pristanic acid, which then enters β-oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor α (PPARα) and is up-regulated in mouse liver in a PPARα-dependent manner. Methyl-branched fatty acids such as phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) and pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) are found in ruminant fats, such as dairy products and beef, but are also found in chlorophyll-containing plants and are ingested in the diet. The body cannot metabolize chlorophyll itself, but instead intestinal flora, mainly present in ruminant animals, can cleave off the side chain of chlorophyll, producing phytol. Phytol is then converted to phytanic acid or phytanoyl-CoA depending on if the phytol is metabolized further directly in the intestine or after uptake into cells, respectively (for review, see Ref. 1Jansen G.A. Biochim. Biophys. Acta. 2006; 1763: 1403-1412Crossref PubMed Scopus (73) Google Scholar). Phytanic acid containing a methyl group in the third position is first converted to a 2-methyl-branched fatty acid, pristanic acid, via α-oxidation (2Avigan J. Steinberg D. Gutman A. Mize C.E. Milne G.W.A. Biochem. Biophys. Res. Commun. 1966; 24: 838-844Crossref PubMed Scopus (51) Google Scholar), which can then undergo β-oxidation as outlined in Fig. 1. Before α-oxidation can take place, the phytanic acid is activated to the CoA ester, which can occur either outside the peroxisome by the long-chain acyl-CoA synthetase or inside the peroxisome by the very long-chain acyl-CoA synthetase (3Watkins P.A. Howard A.E. Gould S.J. Avigan J. Mihalik S.J. J. Lipid Res. 1996; 37: 2288-2295Abstract Full Text PDF PubMed Google Scholar, 4Wanders R.J.A. Denis S. van Roermund C.W.T. Jakobs C. ten Brink H.J. Biochim. Biophys. Acta. 1992; 1125: 274-279Crossref PubMed Scopus (34) Google Scholar). α-Oxidation consists of three steps where the phytanoyl-CoA is hydroxylated into 2-hydroxyphytanoyl-CoA by phytanoyl-CoA 2-hydroxylase (PHYH) 2The abbreviations used are:PHYHphytanoyl-CoA 2-hydroxylaseDMN-CoA4,8-dimethylnonanoyl-CoAQ-PCRreal-time PCRAMACR2-methylacyl-CoA racemasePBSphosphate-buffered salineMES4-morpholineethanesulfonic acidGFPgreen fluorescent proteinPPARαperoxisome proliferator-activated receptor αTRITCtetramethylrhodamine isothiocyanate 2The abbreviations used are:PHYHphytanoyl-CoA 2-hydroxylaseDMN-CoA4,8-dimethylnonanoyl-CoAQ-PCRreal-time PCRAMACR2-methylacyl-CoA racemasePBSphosphate-buffered salineMES4-morpholineethanesulfonic acidGFPgreen fluorescent proteinPPARαperoxisome proliferator-activated receptor αTRITCtetramethylrhodamine isothiocyanate in the first step. In the second step formyl-CoA is cleaved off by the 2-hydroxyphytanoyl-CoA lyase, forming pristanal. The formyl-CoA produced is hydrolyzed into formate and oxidized to CO2 in the cytosol. In the third and last step of α-oxidation pristanal is converted into pristanic acid by an aldehyde dehydrogenase (5Verhoeven N.M. Jacobs C. Carney G. Somers M.P. Wanders R.J.A. Rizzo W.B. FEBS Lett. 1998; 429: 225-228Crossref PubMed Scopus (57) Google Scholar). phytanoyl-CoA 2-hydroxylase 4,8-dimethylnonanoyl-CoA real-time PCR 2-methylacyl-CoA racemase phosphate-buffered saline 4-morpholineethanesulfonic acid green fluorescent protein peroxisome proliferator-activated receptor α tetramethylrhodamine isothiocyanate phytanoyl-CoA 2-hydroxylase 4,8-dimethylnonanoyl-CoA real-time PCR 2-methylacyl-CoA racemase phosphate-buffered saline 4-morpholineethanesulfonic acid green fluorescent protein peroxisome proliferator-activated receptor α tetramethylrhodamine isothiocyanate The 2-methyl-branched pristanoyl-CoA formed by α-oxidation, or indeed pristanic acid directly ingested via the diet, is naturally present in two stereoisomers, 2R and 2S, where the peroxisomal pristanoyl-CoA oxidase (ACOX3) is specific for the S stereoisomer. Therefore, the 2R-pristanic acid must first undergo isomerization into the 2S form, which then undergoes β-oxidation. This racemization step is carried out by the 2-methylacyl-CoA racemase (AMACR) (6Schmitz W. Fingerhut R. Conzelmann E. Eur. J. Biochem. 1994; 38: 2991-2999Google Scholar, 7Ferdinandusse S. Denis S. Ijlst L. Dacremont G. Waterham H.R. Wanders R.J.A. J. Lipid Res. 2000; 41: 1890-1896Abstract Full Text Full Text PDF PubMed Google Scholar). The first step in β-oxidation of pristanic acid is the oxidation by ACOX3, and the two subsequent steps, hydration and dehydrogenation, are catalyzed by multifunctional protein-2, with the last step, the thiolytic cleavage, carried out by sterol carrier protein x, with the release of propionyl-CoA in the first and third cycle of β-oxidation (for review, see Ref. 8Verhoeven N.M. Jacobs C. Prog. Lipid Res. 2001; 40: 453-466Crossref PubMed Scopus (76) Google Scholar). After 3 rounds of β-oxidation 4,8-dimethylnonanoyl-CoA (DMN-CoA) is produced, which can then either be hydrolyzed to the free acid or esterified to carnitine for transport to mitochondria for further β-oxidation (9Farrell S.O. Bieber L.L. Arch. Biochem. Biophys. 1983; 222: 123-132Crossref PubMed Scopus (40) Google Scholar, 10Ofman R. el Mrabet L. Dacremont G. Spijer D. Wanders R.J.A. Biochem. Biophys. Res. Commun. 2002; 290: 629-634Crossref PubMed Scopus (21) Google Scholar). These pathways are well described in the literature; however, subcellular localization, activation, and transport of substrates and products are still somewhat unclear. Both α-oxidation and the first three cycles of β-oxidation are entirely peroxisomal processes (8Verhoeven N.M. Jacobs C. Prog. Lipid Res. 2001; 40: 453-466Crossref PubMed Scopus (76) Google Scholar, 11Van Veldhoven P.P. Casteels M. Mannaerts G.P. Baes M. Biochem. Soc. Trans. 2001; 29: 292-298Crossref PubMed Google Scholar, 12Mukherji M. Schofield C.J. Wierzbicki A.S. Jansen G.A. Wanders R.J.A. Lloyd M.D. Prog. Lipid Res. 2003; 42: 359-376Crossref PubMed Scopus (67) Google Scholar), with the possible exception of the last step of α-oxidation argued to take place either in the endoplasmic reticulum or peroxisomes (5Verhoeven N.M. Jacobs C. Carney G. Somers M.P. Wanders R.J.A. Rizzo W.B. FEBS Lett. 1998; 429: 225-228Crossref PubMed Scopus (57) Google Scholar, 13Jansen G.A. van den Brink D.M. Ofman R. Draghici O. Dacremont G. Wanders R.J.A. Biochem. Biophys. Res. Commun. 2001; 286: 674-679Crossref Scopus (39) Google Scholar). The importance of α-oxidation and β-oxidation is underscored by the diseases affecting either of the pathways, such as Refsum disease, AMACR deficiency, and multifunctional protein-2 deficiency (for review, see Refs. 8Verhoeven N.M. Jacobs C. Prog. Lipid Res. 2001; 40: 453-466Crossref PubMed Scopus (76) Google Scholar and 14Wanders R.J.A. Waterham H.R. Biochim. Biophys. Acta. 2006; 1763: 1707-1720Crossref PubMed Scopus (203) Google Scholar). Also, the importance of the breakdown of phytol to phytanic acid is demonstrated by the occurrence of a disease affecting the microsomal fatty aldehyde dehydrogenase catalyzing the oxidation of phytenal to phytenic acid, causing the Sjögren-Larsson syndrome (15Willemsen M.A.A.P. Ijlst L. Steijlen P.M. Rotteveel J.J. de Jong J.G.N. van Domburg P.H.M.F. Mayatepek E. Gabreëls F.J.M. Wanders R.J.A. Brain. 2001; 124: 1426-1437Crossref PubMed Scopus (114) Google Scholar). In this paper we describe the identification of a novel gene that encodes a peroxisomal acyl-CoA thioesterase specific for phytanoyl-CoA and pristanoyl-CoA that can hydrolyze these compounds to phytanic acid, pristanic acid, and coenzyme A. This phytanoyl-CoA/pristanoyl-CoA thioesterase (Acot6) gene is a member of a gene family of acyl-CoA thioesterases, localized in a condensed cluster on chromosome 12 D3 in mouse, which codes for acyl-CoA thioesterases with localizations in cytosol, mitochondria, and peroxisomes (16Hunt M.C. Nousiainen S.E.B. Huttunen M.K. Orii K.E. Svensson L.T. Alexson S.E.H. J. Biol. Chem. 1999; 274: 34317-34326Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Westin M.A.K. Alexson S.E.H. Hunt M.C. J. Biol. Chem. 2004; 279: 21841-21848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Westin M.A.K. Hunt M.C. Alexson S.E.H. J. Biol. Chem. 2005; 280: 38125-38132Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Localization of ACOT6 in Peroxisomes—The full-length open reading frame encoding ACOT6 was amplified by reverse transcription-PCR from mouse kidney using the following primers 5′-CAT ATG GCG GCG ACA CTG A-3′ and 5′-CAT ATG TTA CAG TTT GCT GTG-3′ (Cybergene AB, Huddinge, Sweden). The product was cloned into the pcDNA3.1/NT-GFP vector (Invitrogen), which expresses the protein as an N-terminal green fluorescent fusion protein, leaving the C-terminal SKL of ACOT6 accessible. The construct was transfected into human skin fibroblasts from a control subject and a Zellweger patient using the calcium phosphate method. Immunofluorescence microscopy was carried out as described previously (19Hunt M.C. Solaas K. Kase B.F. Alexson S.E.H. J. Biol. Chem. 2002; 277: 1128-1138Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Chemical Synthesis of Pristanoyl-CoA and Phytanoyl-CoA—Phytanoyl-CoA and pristanoyl-CoA were synthesized chemically from the respective free acids (Sigma-Aldrich) by first forming the anhydride and in the next step the CoA ester (20Chase J.F.A. Biochem. J. 1967; 104: 510-518Crossref PubMed Scopus (50) Google Scholar). Phytanoyl-CoA and pristanoyl-CoA were then purified by reversed phase high performance liquid chromatography using a C18 Ultrasphere ODS 5-μm (4.6 × 250 mm) column (Beckman Coulter, Inc., Fullerton, CA) with a mobile phase containing 50 mm potassium phosphate buffer, pH 5.4, and 38% isopropanol. After 10 min the mobile phase was changed to 58% isopropanol for 40 min. Purified products were then verified by mass spectrometry as described in Westin et al. (18Westin M.A.K. Hunt M.C. Alexson S.E.H. J. Biol. Chem. 2005; 280: 38125-38132Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Expression of Recombinant ACOT6 Protein—The full-length open reading frame for Acot6 was amplified by reverse transcription-PCR from mouse kidney using the One-Step RNA PCR kit (Takara Biomedicals, Shiga, Japan) using the primers 5′-GAA TTC ATG GCG GCG ACA CTG AGC G-3′ and 5′-GTC GAC TTA CAG TTT GCT GTG CCT G-3′ (Cybergene AB, Huddinge, Sweden). The addition of an EcoRI and a SalI site (indicated in bold) were used for cloning into the pMAL-C2x vector (New England Biolabs, Beverly, MA), which results in expression of the protein as a fusion protein with maltose-binding protein. The construct was transformed into BL21(DES3) pLysS (Novagen Inc., Madison, WI), and protein expression was induced by the addition of 0.3 mm isopropyl-β-d-galactopyranoside (Sigma-Aldrich Inc.) for 2 h at 37 °Cin LB-media supplemented with 2% glucose, 50 μg/ml ampicillin, and 34 μg/ml chloramphenicol. Bacteria were harvested by centrifugation at 5000 × g for 10 min, washed with 20 mm Tris-HCl, pH 7.4, and frozen overnight in 50 ml of column buffer (20 mm Tris, 200 mm NaCl, and 1mm EDTA, pH 7.4). Bacteria were lysed by sonication for 1 min at 5-s intervals and centrifuged at 16,000 × g for 30 min. Recombinant protein was purified by affinity chromatography using amylose resin (New England Biolabs), by elution with 10 mm maltose in column buffer. Purity and size of the eluted protein was checked by SDS-PAGE and Coomassie Brilliant Blue staining, and protein concentration was determined using the Bradford assay (21Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214321) Google Scholar). Measurement of Acyl-CoA Thioesterase Activity—Acyl-CoA thioesterase activity was measured spectrophotometrically at 232 nm in phosphate-buffered saline (PBS) pH 7.4, measuring the cleavage of the thioester bond as a decrease in the absorbance. Various acyl-CoAs were used as substrates, including acetyl-CoA, propionyl-CoA, acetoacetyl-CoA, butyryl-CoA, heptanoyl-CoA, decanoyl-CoA, lauroyl-CoA, myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, oleoyl-CoA, linoleoyl-CoA, linolenoyl-CoA, arachidoyl-CoA, arachidonoyl-CoA, behenoyl-CoA, branched-chain substrates including pristanoyl-CoA, phytanoyl-CoA, DMN-CoA, 2-methyloctadecanoyl-CoA, the bile acid intermediate trihydroxycholoyl-CoA, and the primary bile acid choloyl-CoA. The pristanoyl-CoA, phytanoyl-CoA, trihydroxycholoyl-CoA, and choloyl-CoA were synthesized in our laboratory, whereas 4,8-dimethylnonanoyl-CoA, behenoyl-CoA, and an aliquot of phytanoyl-CoA were a kind gift from Dr. Ronald Wanders. The other acyl-CoAs were obtained from Sigma-Aldrich. The effect of free CoASH on ACOT6 activity was tested at concentrations up to 500 μm. The kinetic parameters were calculated using Prism Enzyme Kinetics program using an extinction coefficient of E232 = 4.25 mm–1 cm–1 to calculate the specific activities. Subcellular Fractionation and Isolation of Peroxisomes—For subcellular fractionation experiments, adult male mice on a pure C57Bl6 background (The Jackson Laboratory, Bar Harbor, ME) were used. Mice were fed either a normal chow diet or a diet containing 0.5% (w/w) clofibrate (ICI Pharmaceuticals, Macclesfield, Cheshire, UK) for 1 week, and all mice were fasted overnight before sacrifice. After sacrifice by CO2 asphyxiation and cervical dislocation, liver and kidneys were excised and used directly for subcellular fractionation. Tissues were weighed and placed in ice-cold buffer containing 0.25 m sucrose, 1 mm EDTA, 15 mm MES, pH 6.5, minced finely, homogenized, and diluted to a 10% homogenate. Homogenates were centrifuged for 10 min at 500 × gav, and pellets were rehomogenized and re-centrifuged. Post-nuclear supernatants were combined and centrifuged at 4000 × gav for 10 min to sediment mitochondria, which were saved for subsequent measurements. The supernatants were centrifuged at 30 000 × gav for 20 min to obtain the light mitochondrial fraction containing the bulk of peroxisomes. The pellets were washed once by centrifugation and finally dissolved in ice-cold 0.25 m sucrose and 15 mm Hepes, pH 7.4. These fractions were then layered on top of linear 20–45% Optiprep (Sigma-Aldrich) gradients, resting on a 50% Optiprep cushion, and centrifuged for 90 min at 40,000 × gav in a fixed angle rotor, and 1-ml fractions were collected from the bottom of the gradient. The fractions were analyzed for the peroxisomal marker catalase as described in Baudhuin et al. (22Baudhuin P. Beaufay H. Rahman-Li Y. Sellinger O.Z. Wattiaux R. Jacques P. De Duve C. Biochem. J. 1964; 92: 179-184Crossref PubMed Scopus (484) Google Scholar). Supernatants obtained after preparation of the light-mitochondrial fractions were centrifuged at 40,000 × gav for 2 h, supernatants were saved as cytosolic fractions, and pellets were dissolved in 0.25 m sucrose and 15 mm Hepes, pH 7.4 and saved as microsomal fractions for subsequent measurements. Immunoprecipitation of ACOT6 in Purified Peroxisomes— ACOT6 anti-sera were produced in rabbits immunized with a peptide with the sequence CQKYLNGEKPARH, which corresponds to amino acids 406–417 of the ACOT6 protein and an N-terminal Cys for coupling of the peptide to keyhole limpet hemocyanin. The antibody was affinity-purified by chromatography as described previously (17Westin M.A.K. Alexson S.E.H. Hunt M.C. J. Biol. Chem. 2004; 279: 21841-21848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Pre-immune serum or affinity-purified ACOT6 antibody in various concentrations (in PBS, 0.1% Triton) was added to protein A-Sepharose beads and incubated at 4 °C overnight. The protein A-Sepharose suspension was centrifuged at 10 000 × gav in a microcentrifuge for 10 min, the supernatants were removed, and 100 μg of peroxisomal protein in PBS, 0.1% Triton was added and incubated for 2 h at 4 °C in an orbital shaker. The protein A-Sepharose suspension was spun down, and the supernatants were used for acyl-CoA thioesterase activity measurements as described above. Animals and Treatments for Investigation of Tissue Expression and Regulation of Expression—Adult male wild type (+/+) mice or peroxisome proliferator-activated receptor α (PPARα)-null (–/–) mice on a pure Sv/129 genetic background (23Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1494) Google Scholar) (kindly provided by Dr. Frank Gonzalez and Dr. Jeffrey Peters) were used for RNA isolation and for preparation of protein homogenates. Mice were fed either a standard chow diet or a diet containing 0.1% Wy-14,643 (Calbiochem-Novabiochem) for 1 week before sacrifice. Mice were sacrificed by CO2 asphyxiation, and cervical dislocation and tissues were excised, frozen in liquid nitrogen, and stored at –70 °C. Isolation of Total RNA and Real-time PCR (Q-PCR)—Total RNA from various mouse tissues was isolated using TRIzol Reagent (Invitrogen) and was DNase-treated using RQ1 RNase-free DNase (Promega Corp., Madison, WI). The quality of the RNA was checked on a 1% agarose-formaldehyde gel. Tissue expression was investigated in liver, kidney, heart, lung, spleen, brain, proximal (first 10 cm of small intestine) and distal intestine (last 10 cm of small intestine), brown adipose tissue and white adipose tissue using total RNA pooled from three individual animals. For regulation by Wy-14,643 treatment in liver, three individual animals in each group were used. Synthesis of cDNA was performed with 1 μg of total RNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). For Acot6, a specific amplicon was designed using the Primer Express software spanning the exon 2/exon 3 boundary using the primers 5′-ACG CCA TCC TCA GGT GAA AG-3′ and 5′-TCA GGA AAG CAG CCA TCG A-3′ and a probe with a 5′-6-carboxyfluorescein and 3′-dabcyl with the sequence 5′-TGT CTC CAA AGG TGC TGA TCT GTG CC-3′. Q-PCR was performed in single-plex and in triplicate with 18 S as an endogenous control as described previously (18Westin M.A.K. Hunt M.C. Alexson S.E.H. J. Biol. Chem. 2005; 280: 38125-38132Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). For tissue expression of Phyh, Amacr, and Acox3, SYBR Green (Applied Biosystems) was used as well as for Acot6 in the comparative tissue expression experiment. Specific primers for Phyh were designed over the exon 4/exon 5 boundary, for Amacr over the exon2/exon3 boundary, and for Acox3 over the exon 5/exon 6 boundary using the primers shown in Table 1. As an endogenous control, a specific amplicon of mouse hypoxanthine guanine phosphoribosyltransferase (Hprt1) spanning the exon6/exon7 boundary was used, with primers also shown in Table 1. Q-PCR was run as described in Westin et al. (18Westin M.A.K. Hunt M.C. Alexson S.E.H. J. Biol. Chem. 2005; 280: 38125-38132Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) with the change of master mix to SYBR Green Power master mix (Applied Biosystems) and with the addition of a dissociation step to check the specificity of the products. The PCR products were checked by agarose gel electrophoresis. The efficacy of all primer pairs was checked by running Q-PCR on dilutions of the template cDNA, verifying that tissue expression was analyzed in the linear range of the PCR. The average threshold (Ct) values per triplicate were used to calculate the relative amounts of mRNA using the 2–ΔΔCt method according to Applied Biosystems guidelines.TABLE 1Sequences of SYBR Green primers for mouse phytanoyl-CoA hydroxylase (Phyh), α-methylacyl-CoA racemase (Amacr), acyl-CoA oxidase 3 (Acox3), acyl-CoA thioesterase 6 (Acot6), and hypoxanthine guanine phosphoribosyltransferase (Hprt1) for Q-PCRPhyhForward 5′-ACT GCC TTC TCC CCG AGA TT-3′Reverse 5′-TGG GTC CAG TGA AAC ACT CCA-3′AmacrForward 5′-CTA TTT GGC TTT ATC AGG CGT TC-3′Reverse 5′-TTC TCA CCG CTT CTG CCA AT-3′Acox3Forward 5′-TTG AGA AGA TCT ATA GCC TGG AGA TTT-3′Reverse 5′-AGT TCG GTG AGA GCA AAACAG C-3′Acot6Forward 5′-GGT GAA AAG GAC CTC TCG AAG TG-3′Reverse 5′-ATA GTC AAG GGC ATA TCC AAC AAC A-3′Hprt1Forward 5′-GGT GAA AAG GAC CTC TCG AAG TG-3′Reverse 5′-ATA GTC AAG GGC ATA TCC AAC AAC A-3′ Open table in a new tab Western Blot Analysis—Liver homogenates were prepared from control and Wy-treated male mouse, and 50 μg of protein was used for SDS-PAGE and Western blotting as described previously (24Hunt M.C. Lindquist P.J.G. Peters J.M. Gonzalez F.J. Diczfalusy U. Alexson S.E.H. J. Lipid Res. 2000; 41: 814-823Abstract Full Text Full Text PDF PubMed Google Scholar). ACOT6 antibody was produced and purified as described above, and the antibody reactivity was checked against recombinant ACOT6 protein. Identification of ACOT6 as a Novel Peroxisomal Acyl-CoA Thioesterase—We have previously identified a cluster of genes on mouse chromosome 12 D3, which codes for acyl-CoA thioesterases. To date we have characterized 5 of these genes, named Acot1-5. Acot3, Acot1, and Acot2 encode long-chain acyl-CoA thioesterases localized in peroxisomes, cytosol, and mitochondria, respectively (17Westin M.A.K. Alexson S.E.H. Hunt M.C. J. Biol. Chem. 2004; 279: 21841-21848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 25Lindquist P.J.G. Svensson L.T. Alexson S.E.H. Eur. J. Biochem. 1998; 251: 631-640Crossref PubMed Scopus (60) Google Scholar, 26Svensson L.T. Engberg S.T. Aoyama T. Usuda N. Alexson S.E.H. Hashimoto T. Biochem. J. 1998; 329: 601-608Crossref PubMed Scopus (65) Google Scholar). Acot5 and Acot4 encode a medium-chain acyl-CoA thioesterase and a succinyl-CoA thioesterase, respectively (17Westin M.A.K. Alexson S.E.H. Hunt M.C. J. Biol. Chem. 2004; 279: 21841-21848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 18Westin M.A.K. Hunt M.C. Alexson S.E.H. J. Biol. Chem. 2005; 280: 38125-38132Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). This cluster contains a further gene, named Acot6, which shows an identical gene organization to the other genes in this cluster. Based on the genomic sequence, primers were designed to amplify the open reading frame of Acot6 by PCR, and the product was completely sequenced and deposited under GenBankTM accession number AY999300. The resulting protein contains 419 amino acids, with a calculated molecular mass of 46.7 kDa and shows ∼70% sequence identity to the other ACOTs in this gene family. Notably the C-terminal ends serine, lysine, leucine (SKL) strongly suggesting a peroxisomal localization. To examine whether this SKL results in targeting of the protein to peroxisomes, we expressed the protein as an N-terminal fusion protein with green fluorescent protein (GFP), leaving the C-terminal SKL accessible. The ACOT6-GFP plasmid was transfected into human skin fibroblasts and showed a punctate expression indicative of a peroxisomal localization (Fig. 2A). A peroxisomal localization was then confirmed by transfection of the same plasmid into fibroblasts from a Zellweger patient. These fibroblasts lack functional peroxisomes since they have a generalized import defect, and in these cells ACOT6-GFP expression showed a diffuse cytosolic localization (Fig. 2B), confirming that the punctate expression visible in the control fibroblasts is indeed peroxisomal. Identification of ACOT6 as a Peroxisomal Phytanoyl-CoA/Pristanoyl-CoA Thioesterase—Expression of ACOT6 as a fusion protein with a histidine tag or as a fusion protein with thioredoxin failed to produce soluble protein. However, expression of ACOT6 as a fusion protein with maltose-binding protein resulted in soluble protein. After purification by affinity chromatography using an amylose resin, acyl-CoA thioesterase activity was measured on a variety of acyl-CoAs (see “Materials and Methods”), which showed ACOT6 to be active only on pristanoyl-CoA (2,6,10,14-tetramethylpentadecanoyl-CoA) with a Vmax of 3.20 μmol/min/mg and a Km of 24 μm (Fig. 3). The enzyme was not active on any other methyl-branched substrates, i.e. 2-methyloctadecanoyl-CoA and DMN-CoA, although at this stage phytanoyl-CoA was not available, demonstrating that ACOT6 is highly specific for pristanoyl-CoA. The 2-methyl group in pristanic acid is present in both the 2R and 2S configuration, and it has been shown that the peroxisomal pristanoyl-CoA oxidase (ACOX3) is only active on the 2S stereoisomer (8Verhoeven N.M. Jacobs C. Prog. Lipid Res. 2001; 40: 453-466Crossref PubMed Scopus (76) Google Scholar). Therefore, the AMACR is required as an auxiliary enzyme for complete oxidation of pristanic acid (6Schmitz W. Fingerhut R. Conzelmann E. Eur. J. Biochem. 1994; 38: 2991-2999Google Scholar). Because the commercial pristanic acid was chemically synthesized, it is presumed to contain a racemic mixture of 2R and 2S stereoisomers. We, therefore, tested if ACOT6 was active on both stereoisomers by incubation of ACOT6 with the presumed racemic mixture of synthesized pristanoyl-CoA for 30 min, and results showed that ACOT6 hydrolyzed almost 100% of the added substrate, suggesting that ACOT6 has no stereo-specificity for the 2R and 2S isomers (data not shown). Increasing concentrations of free CoASH up to 500 μm showed that ACOT6 activity is not regulated by free CoASH (data not shown), similar to the other members of this gene family. Even using the maltose-binding protein expression system it was very difficult to obtain active ACOT6 protein, probably due to improper folding. To verify that ACOT6 is indeed a peroxisomal pristanoyl-CoA thioesterase, we also performed complementary experiments utilizing purified peroxisomes from mouse liver and kidney from control and clofibrate-treated animals. Peroxisomes were isolated using standard procedures with the final step being gradient centrifugation in Optiprep. When performing the biochemical characterization of recombinant ACOT6 (described in Fig. 3), we did not have access to phytanoyl-CoA, which is not commercially available. Based on the structural similarity to pristanoyl-CoA, we therefore subsequently synthesized phytanoyl-CoA and tested the activity in the purified organelle fractions. Indeed, the activity in purified peroxisomes is very similar with phytanoyl-CoA and pristanoyl-CoA, with Km values of 32 and 35 μm and Vmax values of 176 and 215 nmol/min/mg, respectively, suggesting that ACOT6 could be similarly active on both these substrates (Fig. 4, A and B). To establish whether the peroxisomal phytanoyl-CoA and pristanoyl-CoA thioesterase activities in peroxisomes are catalyzed by ACOT6, we immunoprecipitated the peroxisomal fraction with an ACOT6 peptide antibody using preimmune serum as a control. The anti-ACOT6 IgG immunoprecipitated almost all of the peroxisomal phytanoyl-CoA and pristanoyl-CoA thioesterase activity, demonstrating that ACOT6 is the major phytanoyl-CoA/pristanoyl-CoA thioesterase in peroxisomes (Fig. 4C). As expected, no lauroyl-CoA thioesterase activity was immunoprecipitated with the ACOT6 antibody. Taken together, these data strongly suggest that ACOT6 is a novel peroxisomal thioesterase with a function in regulation of phytanic acid and pristanic acid metabolism. Phytanoyl-CoA and Pristanoyl-CoA Thioesterase Activity Is Highest in Peroxisomes—The important role of peroxisomes in the metabolism of phytanic acid and pristanic acid has been well established (for review, see Ref. 27van den Brink D.M. Wanders R.J.A. Cell. Mol. Life Sci. 2006; 63: 1752-1765Crossref PubMed Scopus (103) Google Scholar). If the physiological function of ACOT6 is to regulate phytanic acid and prista" @default.
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• W1991882913 title "Peroxisomes Contain a Specific Phytanoyl-CoA/Pristanoyl-CoA Thioesterase Acting as a Novel Auxiliary Enzyme in α- and β-Oxidation of Methyl-branched Fatty Acids in Mouse" @default.
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