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Molecular cloning and characterization of a mitochondrial peroxisome proliferator-induced acyl-CoA thioesterase from rat liver.

ABSTRACT: We have previously reported the purification and characterization of the peroxisome proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I) from rat liver mitochondria [L.T. Svensson, S.E. H. Alexson and J.K. Hiltunen (1995) J. Biol. Chem. 270, 12177-12183]. Here we describe the cloning of the corresponding cDNA. One full-length clone was isolated that contained an open reading frame of 1359 bp encoding a polypeptide with a calculated molecular mass of 49707 Da. The deduced amino acid sequence contains a putative mitochondrial leader peptide of 42 residues. Expression of the cDNA in Chinese hamster ovary cells, followed by immunofluorescence, immunoelectron microscopy and Western blot analyses, showed that the product was targeted to mitochondria and processed to a mature protein of 45 kDa, which is similar to the molecular mass of the protein isolated from rat liver mitochondria. The recombinant enzyme showed the same acyl-CoA chain-length specificity as the isolated rat liver enzyme. Sequence analysis showed no similarity to known esterases, but a high degree (approx. 40%) of identity with bile acid-CoA:amino acid N-acyltransferase cloned from human and rat liver. A putative active-site serine motif (Gly-Xaa-Ser-Xaa-Gly) of several carboxylesterases and lipases was identified. Western and Northern blot analyses showed that MTE-I is constitutively expressed in heart and is strongly induced in liver by feeding rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator, suggesting a role for the enzyme in lipid metabolism.

SUBMITTER: Svensson LT

PROVIDER: S-EPMC1219082 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

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Project description:Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from -758 bp to -198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBP?), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor ? (PPAR?), and PPAR? were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPAR? agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPAR? agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPAR? and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.

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Molecular cloning and characterization of a mitochondrial peroxisome proliferator-induced acyl-CoA thioesterase from rat liver.

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