Induction of the three peroxisomal beta-oxidation enzymes is synergistically regulated by dexamethasone and fatty acids, and counteracted by insulin in Morris 7800C1 hepatoma cells in culture.
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This work describes the molecular mechanism of hormonal modulation of fatty-acid peroxisomal beta oxidation in liver. Morris 7800C1 hepatoma cells and isolated hepatocytes were cultured in the presence of myristic acid (1 mM) and tetradecylthioacetic acid, a 3-thia fatty acid (50 microM), separately or in combination with dexamethasone (0.25 microM) or insulin (0.4 microM). Myristic acid stimulated acyl-CoA oxidase and a synergistic action was observed with dexamethasone. Parallel changes were recognized in enzyme protein and mRNA levels as quantified from immunoblots and Northern analyses. Myristic acid and tetradecylthioacetic acid had similar effects on this enzyme, while insulin inhibited the basal activity and blocked all inductions by the fatty acids and dexamethasone. Parallel mRNA and immunoblot analyses of the subsequent enzymes in the peroxisomal beta-oxidation pathway, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/delta 3,delta 2-enoyl-CoA isomerase and 3-oxoacyl-CoA thiolase, showed an even stronger induction by tetradecylthioacetic acid and dexamethasone, while the counteraction by insulin was maintained in both 7800C1 hepatoma cells and hepatocytes. In hepatoma cells, the thiolase always showed the most pronounced induction (about 40-fold) after 14 days, with parallel changes in protein and mRNA levels. The results suggest that the changes in peroxisomal beta-oxidation enzymes in 7800C1 hepatoma cells are due to a major effect on steady-state mRNA levels giving rise to corresponding alterations in enzyme protein. These results may be explained by regulation at the level of transcription of corresponding genes, but mRNA stability changes and/or translational effects may also be of importance. (+info)
Further analysis of mutant thiolase protein in fibroblasts from a Japanese boy with 3-ketothiolase deficiency.
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We examined the mutant protein of mitochondrial acetoacetyl-CoA thiolase (mutant T2) in fibroblasts from a Japanese boy with 3-ketothiolase deficiency. The molecular size of the mutant T2 protein, determined by pulse labeling and SDS/PAGE, was intermediate between the mature subunit and the precursor of T2. To characterize the mutant T2 protein, pulse-labeling and rhodamine 6G inhibition of mitochondrial transport in fibroblasts, cell-free translation experiments, and family studies by thiolase assay, immunoblotting, and pulse-labeling were carried out. The mutant T2 was detectable as early as a 10-min pulse. The probable precursor of the mutant T2 was not detectable in either the rhodamine 6G inhibition or cell-free translation experiments. In the parents, the K+ ion dependency of acetoacetyl-CoA thiolase activity was low and the T2 bands in immunoblots were faint. It would thus appear that the parents are heterozygotes of this disease. In pulse-labeling, only a band for the mutant T2 was detected in the patient and a single band for the normal mature subunit of T2 in the father; both bands were detected in the mother. These findings suggested that the mutant T2 in the patient was inherited from the mother, and that the expression of another mutant allele of the father may be either abolished or scanty. (+info)
Peroxisomal acetoacetyl-CoA thiolase of an n-alkane-utilizing yeast, Candida tropicalis.
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Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambda EMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41,898 Da for thiolase IA and 41,930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto. (+info)
Physiological roles of acetoacetyl-CoA thiolase in n-alkane-utilizable yeast, Candida tropicalis: possible contribution to alkane degradation and sterol biosynthesis.
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The presence of two types of thiolases, acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase, was demonstrated in peroxisomes of n-alkane-grown Candida tropicalis [Kurihara, T., Ueda, M., & Tanaka, A. (1989) J. Biochem. 106, 474-478], while acetoacetyl-CoA thiolase was also shown to be present in cytosol. The activity of the enzyme in cytosol was constant irrespective of culture conditions, while the peroxisomal enzyme was inducibly synthesized in the alkane-grown yeast cells. These results indicate that peroxisomal acetoacetyl-CoA thiolase participates in alkane degradation, while the cytosolic enzyme is associated with other fundamental metabolic processes, probably sterol biosynthesis, because this enzyme can catalyze the first step of the sterol biosynthesis. 3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase, a key regulatory enzyme of sterol biosynthesis, was found to be localized exclusively in microsomes of the alkane-grown yeast cells. These results suggest that yeast peroxisomes do not contribute to sterol biosynthesis, unlike the case of mammalian cells. (+info)
Regulation of sterol carrier protein gene expression by the forkhead transcription factor FOXO3a.
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The SCP gene encodes two proteins, sterol carrier protein X (SCPx) and SCP2, that are independently regulated by separate promoters. SCPx has been shown to be the thiolase involved in the breakdown of branched-chain fatty acids and in the biosynthesis of bile acids. The in vivo function of SCP2 however remains to be established. The transcriptional regulation of SCPx and SCP2 is unclear, but their promoter regions contain several putative regulatory domains. We show here that both SCPx and SCP2 are upregulated by the daf-16-like Forkhead transcription factor FOXO3a (also known as FKHRL1) on the level of promoter activity. It was recently described that Forkheads regulate protection against (oxidative) stress in both Caenorhabditis elegans and mammalian cells. We looked into a role for SCP2 in the cellular defense against oxidative damage and found that a fluorescent fatty acid analog bound to SCP2 is protected against H2O2/Cu2+-induced oxidative damage. We propose a model for the way in which SCP2 could protect fatty acids from peroxidation. (+info)
Telomere attachment, meiotic chromosome condensation, pairing, and bouquet stage duration are modified in spermatocytes lacking axial elements.
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During the extended prophase to the meiosis I division, chromosomes assemble axial elements (AE) along replicated sister chromatids whose ends attach to the inner nuclear membrane (NM) via a specialized conical thickening. Here, we show at the EM level that in Sycp3(-/-) spermatocyte chromosomes lack the AE and the conical end thickening, but still they attach their telomeres to the inner NM with an electron-dense plate that contains T(2)AG(3) repeats. Immunofluorescence detected telomere proteins, SCP2, and the meiosis-specific cohesin STAG3 at the Sycp3(-/-) telomere. Bouquet stage spermatocytes were approximately threefold enriched, and the number of telomere but not centromere signals was reduced to the haploid in advanced Sycp3(-/-) spermatocytes, which indicates a special mode of homolog pairing at the mammalian telomere. Fluorescence in situ hybridization with mouse chromosome 8- and 12-specific subsatellite probes uncovered reduced levels of regional homolog pairing, whereas painting of chromosomes 13 revealed partial or complete juxtapositioning of homologs; however, condensation of Sycp3(-/-) bivalents was defective. Electron microscopic analysis of AE-deficient spermatocytes revealed that transverse filaments formed short structures reminiscent of the synaptonemal complex central region, which likely mediate stable homolog pairing. It appears that the AE is required for chromosome condensation, rapid exit from the bouquet stage, and fine-tuning of homolog pairing. (+info)
Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I.
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SCP3 is a meiosis-specific structural protein appearing at axial elements and lateral elements of the synaptonemal complex. We have analysed the behaviour of SCP3 and the cohesin subunit Rad21 in mouse spermatocytes by means of a squashing technique. Our results demonstrate that both proteins colocalize and are partially released from chromosome arms during late prophase I stages, although they persist at the interchromatid domain of metaphase I bivalents. Thus, Rad21 cannot be considered a 'mitotic'-specific variant, but coexists with Rec8. During late prophase I SCP3 and Rad21 accumulate at centromeres, and together with the chromosomal passenger proteins INCENP and aurora-B kinase, show a complex 'double cornet'-like distribution at the inner domain of metaphase I centromeres beneath the associated sister kinetochores. We have observed that Rad21 and SCP3 are displaced from centromeres during telophase I when sister kinetochores separate, and are not present at metaphase II centromeres. Thus, we hypothesise that Rad21, and the superimposed SCP3 and SCP2, are involved in the monopolar attachment of sister kinetochores during meiosis I, and are not responsible for the maintenance of sister-chromatid centromere cohesion during meiosis II as previously suggested. (+info)
ACAT1 deficiency disrupts cholesterol efflux and alters cellular morphology in macrophages.
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OBJECTIVE: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) converts intracellular free cholesterol (FC) into cholesteryl esters (CE) for storage in lipid droplets. Recent studies in our laboratory have shown that the deletion of the macrophage ACAT1 gene results in apoptosis and increased atherosclerotic lesion area in the aortas of hyperlipidemic mice. The objective of the current study was to elucidate the mechanism of the increased atherosclerosis. METHODS AND RESULTS: CE storage and FC efflux were studied in ACAT1(-/-) peritoneal macrophages that were treated with acetylated low-density lipoprotein (acLDL). Our results show that efflux of cellular cholesterol was reduced by 25% in ACAT1-deficient cells compared with wild-type controls. This decrease occurred despite the upregulated expression of ABCA1, an important mediator of cholesterol efflux. In contrast, ACAT1 deficiency increased efflux of the cholesterol derived from acLDL by 32%. ACAT1-deficient macrophages also showed a 26% increase in the accumulation of FC derived from acLDL, which was associated with a 75% increase in the number of intracellular vesicles. CONCLUSIONS: Together, these data show that macrophage ACAT1 influences the efflux of both cellular and lipoprotein-derived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1(-/-) macrophages. (+info)