Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes.

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.

Low activity of mitochondrial HMG-CoA synthase in liver of starved piglets is due to low levels of protein despite high mRNA levels.

The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65-73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria.

3-Hydroxy-3-methylglutaryl coenzyme A synthase-1 of Blattella germanica has structural and functional features of an active retrogene.

Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase.

Regulation of the rat liver carnitine palmitoyltransferase I gene transcription by thyroid hormone.

L-CPT I isotype is the main locus of control for liver LCFA oxidation. T3 levels have been described as controlling L-CPT I gene expression, and in this paper we demonstrate that rat liver CPT I promoter responds to T3. Using deleted reporter constructs we located the thyroid hormone-responsive element between -2935 and -2918, consisting of a DR4. This response is mediated by the binding of the thyroid to this sequence as a monomer, homodimer, or heterodimer with RXR.

Contribution of steroidogenic factor 1 to the regulation of cholesterol synthesis.

Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family expressed in steroidogenic tissues, where it has an essential role in the regulation of the steroid hormone biosynthesis, adrenal and gonadal development and endocrine responses fundamental for reproduction. Here we show that SF-1 regulates the transcription of cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which is essential for the endogenous synthesis of cholesterol. We have identified an element located 365 bp upstream of the gene for cytosolic HMG-CoA synthase; SF-1 binds as a monomer to this element and confers SF-1 responsiveness to homologous and heterologous promoters. It has been shown that in tissues with a high demand for cholesterol to be used in steroid synthesis, there is a lack of correlation between the cholesterol levels and the activity of the limiting enzymes of the mevalonate pathway. In accord with those results, we observed that cholesterol synthesis from acetate and either cytosolic HMG-CoA mRNA expression or transcriptional activity were not changed in response to 25-hydroxycholesterol in the SF-1-expressing steroidogenic Leydig tumour MA-10 cells. Moreover, the overexpression of SF-1 in non-steroidogenic CV-1 cells renders them less sensitive to the regulatory effects of cholesterol. This observation led to the hypothesis that in steroidogenic tissues the expression of SF-1 permits high levels of endogenous synthesis of cholesterol irrespective of the intracellular levels of this metabolite.

Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element.

Low expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene during development correlates with an unusually low hepatic ketogenic capacity and lack of hyperketonaemia in piglets. Here we report the isolation and characterization of the 5' end of the pig mitochondrial HMG-CoA synthase gene. The 581 bp region proximal to the transcription start site permits transcription of a reporter gene, confirming the function of the promoter. The pig mitochondrial HMG-CoA synthase promoter is trans-activated by the peroxisomal proliferator-activated receptor (PPAR), and a functional response element for PPAR (PPRE) has been localized in the promoter region. Pig PPRE is constituted by an imperfect direct repeat (DR-1) and a downstream sequence, both of which are needed to confer PPAR-sensitivity to a thymidine kinase promoter and to form complexes with PPAR.retinoid X receptor heterodimers. A role of PPAR trans-activation in starvation-associated induction of gene expression is suggested.

Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor.

The expression of several genes involved in intra- and extracellular lipid metabolism, notably those involved in peroxisomal and mitochondrial beta-oxidation, is mediated by ligand-activated receptors, collectively referred to as peroxisome proliferator-activated receptors (PPARs). To gain more insight into the control of expression of carnitine palmitoyltransferase (CPT) genes, which are regulated by fatty acids, we have examined the transcriptional regulation of the human MCPT I gene. We have cloned by polymerase chain reaction the 5'-flanking region of this gene and demonstrated its transcriptional activity by transfection experiments with the CAT gene as a reporter. We have also shown that this is a target gene for the action of PPARs, and we have localized a PPAR responsive element upstream of the first exon. These results show that PPAR regulates the entry of fatty acids into the mitochondria, which is a crucial step in their metabolism, especially in tissues like heart, skeletal muscle and brown adipose tissue in which fatty acids are a major source of energy.

Catalytic properties of recombinant 3-hydroxy-3-methylglutaryl coenzyme A synthase-1 from Blattella germanica.

Blattella germanica is the first organism in which two cytosolic HMG-CoA synthase genes have been described: HMGS-1 (Martínez-González et al., 1993b) and HMGS-2 (Buesa et al., 1994). The HMGS-1 gene showed special features, which led us to characterize the kinetic properties of the enzyme it encodes. Here we report the expression of recombinant HMGS-1, the protocol of enzyme purification, and the measurement of kinetic parameters. The K(m) for acetyl-CoA is 15.2 microM and the Ki for the other substrate, acetoacetyl-CoA, is 1.26 microM, both similar to that of yeast, ox, and chicken liver enzymes; the Vmax of HMGS-1 measured in this paper is 66 mU, which is the lowest Vmax of the HMG-CoA synthases reported to date.

Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet.

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2-3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling-weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.

Coordinated expression and activity of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase in the fat body of Blattella germanica (L.) during vitellogenesis.

Levels of mRNA for the two 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthases, (HMG-S1 and HMG-S2), and for HMG-CoA reductase (HMG-R) of Blattella germanica were analyzed in the fat body during the first gonadotrophic cycle. HMG-S2 and HMG-R showed the highest mRNA levels on day 0 and decreased thereafter, whereas HMG-S1, showed faint expression. Western blot using specific antibodies for HMG-S1 and HMG-S2 showed no detectable levels for HMG-S1 but a clear pattern for HMG-S2. Both results point to a very limited role for HMG-CoA synthase-1 in B. germanica fat body that the functional enzyme in this organ is HMG-CoA synthase-2. HMG-CoA reductase and synthase proteins shared a cyclic pattern (maximum levels at day 4 and minimum levels on days 0 and 8), which was coincident with the pattern of activity. The delay between gene transcription and protein synthesis suggests a finely regulated translation mechanism. Moreover, the pattern of mevalonate synthesis parallels that of vitellogenin production, suggesting a coordinate mechanism between the mevalonate pathway and the production of vitellogenin.

Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity.

Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthase show the presence of two aspartate rich highly conserved domains. The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transfer enzymes that utilize an allylic prenyl diphosphate as one substrate. In order to investigate the role of this second aspartate-rich domain in rat FPP synthase, we mutated the first or third aspartate to glutamate, expressed the wild-type and mutant enzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step. Approximately 12 mg of homogeneous protein was isolated from 120 mg of crude bacterial extract. The kinetic parameters of the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: Vmax = 0.84 mumol/min/mg; GPP Km = 1.0 microM; isopentenyl diphosphate (IPP) Km = 2.7 microM. Substitution of glutamate for the first aspartate (EDYLD) decreased the Vmax by over 90-fold. The Km for IPP increased, whereas the Km for GPP remained the same in this D243E mutant. Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in the D247E mutant. These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.

Characterization of the gene encoding the 10 kDa polypeptide of photosystem II from Arabidopsis thaliana.

We report the characterization of a 1388 bp genomic fragment from Arabidopsis thaliana that encompasses the entire transcription unit of the gene encoding the precursor of 10 kDa polypeptide of photosystem II, 495 bp of the 5' flanking region and 73 bp of the 3' boundary of the gene. The deduced protein shows 78% and 73% homology, respectively, with its homologues from potato and spinach. The transcription of the gene seems to be greatly enhanced by light and only transcribed in substantial amounts in leaves and stems. Analysis of the putative 5' regulatory region of the gene shows homology with several cis-elements involved in light regulation of the transcription.

Rat mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl-CoA synthases are encoded by two different genes.

We report the isolation and characterization of a 1994-base-pair cDNA that encompasses the entire transcription unit of the mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5.) gene from rat. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 508 residues and 56,918-Da molecular mass. Identify of the cDNA clone isolated as mitochondrial HMG-CoA synthase was confirmed by the following criteria: (i) Amino acid residues are 65% homologous with hamster cytosolic HMG-CoA synthase. (ii) A 19-amino acid sequence probably corresponding to the catalytic site is highly homologous (90%) to that reported for chicken liver mitochondrial HMG-CoA synthase. (iii) The expression product of the cDNA in Escherichia coli has HMG-CoA synthase activity. (iv) The protein includes a sequence of 37 amino acid residues at the N terminus that is not present in the cytosolic enzyme. The predominantly basic, hydrophobic, and hydroxylated nature of the residues of this sequence suggests that it is a leader peptide to target HMG-CoA synthase inside mitochondria. These data plus the hybridization pattern in genomic Southern blot analysis, the different transcript size (2.0 kilobases versus 3.4 kilobases for the cytosolic enzyme), and the different expression pattern shown in RNA blot experiments suggest the presence of two HMG-CoA synthase genes, one for the cytosolic and another for the mitochondrial enzyme.

Identification of a cholesterol-regulated 180-kDa microsomal protein in rat hepatocytes.

The immunoprecipitation by antibodies to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of extracts of [35S]methionine-pulse-labelled isolated hepatocytes, followed by electrophoresis and fluorography, showed the presence not only of 97-kDa HMG-CoA reductase, but also of another protein of 180 kDa. Boiling the immunoprecipitates both in the presence and in the absence of 2-mercaptoethanol, followed by SDS/polyacrylamide gel electrophoresis both in the presence and in the absence of 8 M urea, was not found to change the ratio of 180-kDa/97-kDa proteins. These facts suggest that the 180-kDa protein is not an aggregated form of HMG-CoA reductase. A different batch of antibodies obtained from a newly purified HMG-CoA reductase fully titrated the reductase activity, but did not immunoprecipitate the 180-kDa protein, showing that there is no cross-reactivity between these proteins. The 180-kDa polypeptide is a glycoprotein of N-linked high-mannose oligosaccharide chains, which is not processed on the Golgi system. The apparent molecular mass of the carbohydrate is 16 kDa. The incubation of rat hepatocytes with sterols produces, on the one hand, a decrease in the rate of synthesis, and on the other hand, an acceleration in the turnover rate of the 180-kDa protein. In addition, mevalonate is known to decrease its rate of synthesis. The carbohydrate-free 164-kDa protein was found to degrade only a tenth as fast as the glycoprotein and, furthermore, the degradation was no longer accelerated by sterols. These results support the notion that the 180-kDa protein is not a modified form of 97-kDa reductase, but probably a different protein related to cholesterol metabolism, and also that the N-linked, high-mannose chains, which are bound to the glycoprotein, are required for rapid and controlled degradation of the protein.

Phosphorylation of HMG-CoA reductase induced by mevalonate accelerates its rate of degradation in isolated rat hepatocytes.

Incubation of rat hepatocytes with 10 mM mevalonate produces a decrease in HMG-CoA reductase activity and in the rate of synthesis of both monomeric and dimeric HMG-CoA reductase, and an increase in the rate of degradation of the monomeric form without significant change in that of the dimeric form. Since mevalonate promotes a short-term phosphorylation of the monomeric form without affecting the dimeric form, it is suggested that the mechanism of degradation of reductase is controlled by its phosphorylation state.

Rat liver HMG CoA reductase, a glycoprotein of the endoplasmic reticulum, is in equilibrium between monomeric and dimeric forms.

Incubation of rat hepatocytes with [35S]methionine in pulse and pulse-chase experiments followed by immunoprecipitation of the HMG CoA reductase and SDS-PAGE results in two labelled polypeptides of 104 and 180 Kdaltons. These two polypeptides have half lives of 80 and 46 minutes respectively. When hepatocytes are incubated with mevalonolactone, and a pulse of [35S]methionine is given, the rate of synthesis of both the 180 and 104 Kd peptides is strongly diminished. After treatment of the [35S] labelled immunoprecipitates with endoglycosidase H, the 180 Kd reductase splits into two labelled peptides of 110 and 97 Kd. We suggest that in addition to the 104 Kd reductase, the endoplasmic reticulum contains the dimer of two reductases linked by a carbohydrate chain. The equilibrium monomer-dimer probably regulates the rate of degradation of reductase.