A second gene for peroxisomal HMG-CoA reductase? A genomic reassessment

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, the rate-limiting step of eukaryotic isoprenoid biosynthesis, and is the main target of cholesterol-lowering drugs. The classical form of the enzyme is a transmembrane-protein anchored to the endoplasmic reticulum. However, during the last years several lines of evidence pointed to the existence of a second isoform of HMGCR localized in peroxisomes, where mevalonate is converted further to farnesyl diphosphate. This finding is relevant for our understanding of the complex regulation and compartmentalization of the cholesterogenic pathway. Here we review experimental evidence suggesting that the peroxisomal activity might be due to a second HMGCR gene in mammals. We then present a comprehensive analysis of completely sequenced eukaryotic genomes, as well as the human and mouse genome drafts. Our results provide evidence for a large number of independent duplications of HMGCR in all eukaryotic kingdoms, but not for a second gene in mammals. We conclude that the peroxisomal HMGCR activity in mammals is due to alternative targeting of the ER enzyme to peroxisomes by an as yet uncharacterized mechanism.     

Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae

Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.     

The Inhibitory Effects of Bioactive Compounds of Tomato Juice Binding to Hepatic HMGCR: In Vivo Study and Molecular Modelling

The hypocholesterolemic effect of tomato juice has been investigated in an intervention study with rats, along with the possible inhibition effect of bioactive tomato compounds binding to the HMGCR enzyme. Two experimental groups (n = 8 Sprague-Dawley rats) were fed ad libitum for five weeks, with water or tomato juice provided to the control and intervention groups, respectively. Total, LDL and HDL cholesterol, and total triglycerides were analysed in plasma, and the lycopene content and the expression and activity of the enzyme HMGCR were determined in liver samples. A computational molecular modelling was carried out to determine the interactions between HMGCR and lycopene, chlorogenic acid and naringenin. Total, LDL and HDL cholesterol were significantly lower in the intervention group after the intake of tomato juice. In addition, a significant reduction in HMGCR activity was observed, although this was not accompanied by changes in gene expression. The molecular modelling showed that components of tomato can bind to the active site of the enzyme and compete with the ligand HMGCoA. Lycopene, from tomato juice, accumulates in the liver and can inhibit the activity of the rate-limiting enzyme of cholesterol biosynthesis, HMGCR.     

Sterol and Fatty Acid Metabolism in Fusarium oxysporum

The effects of temperatures ranging from 10°C to 35°C on sterol and fatty acid production and hydroxymethylglutaryl CoA reductase (EC 1.1,1.34, HMGCoA reductase) activity have been examined. Growth, based on dry weight, was maximal at 25°C to 30°C. Sterol production and the reductase activity were highest at 15°C after 28~32 hr incubation when the total fatty acids were minimal. Fatty acid unsaturation generally increased with decrease in temperature.     

Instructions for authors

The aim of the present study was to examine hypothesis that the enhanced cholesterologenesis, found in rats with experimental chronic renal failure (CRF) resulted from the increased gene expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – the rate limiting enzyme in the cholesterologenesis pathway, responsible for mevalonate synthesis. Wistar rats were used and experimental CRF was achieved by 5/6 nephrectomy model. We examined: (a) the changes in the rat liver microsomal HMG-CoA reductase activity, (b) the rat liver HMG-CoA reductase mRNA abundance in various times of day. Obtained data indicates that the increased activity of HMG-CoA reductase in the liver of rats with experimental CRF parallel enhanced mRNA level and suggests that enhanced cholesterol biosynthesis, observed in experimental CRF is at least in part due to the increased HMG-CoA reductase gene expression. The results also indicate that the physiological diurnal rhythm of HMG-CoA reductase activity is preserved in the course of experimental CRF.     

Mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase overaccumulation in three compactin-resistant cell lines

We have isolated three mammalian cell lines which are resistant to compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. The drug resistance in all three cell lines is due to an increase of HMG-CoA reductase activity. Two of the three cell lines overaccumulate HMG-CoA reductase messenger RNA when grown in the presence of compactin. DNA hybridization experiments indicate that both a baby hamster kidney-derived compactin-resistant cell line, C100, and a cell line derived from mouse 3T6 cells, 3T6-40, exhibit amplifications of the HMG-CoA reductase gene. A third compactin-resistant cell line derived from Chinese hamster ovary cells, ML100, does not exhibit an amplification of the HMG-CoA reductase gene, nor does it show an elevated level of HMG-CoA reductase mRNA, comparable to that seen in the other cell lines.     

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment

Aims:  Statins – inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs.
Methods and results:  We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins.
Conclusion:  The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes.
Significance and Impact of the Study:  These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.     

Subcutaneous administration of HMG-CoA reductase inhibitors in hyperlipidaemic and normal rats.

Recent reports demonstrate a hypocholesterolaemic effect of daily subcutaneous injections of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in different rat models of hyperlipidaemia. However, this effect is not seen after oral administration of HMG-CoA reductase inhibitors in rats. We found that oral administration of the HMG-CoA reductase inhibitor Simvastatin also had no effect on plasma cholesterol in severely hyperlipidaemic Nagase analbuminaemic rats (NAR). Simvastatin (an apolar compound dissolved in propylene glycol) was infused continuously for 28 days into the subcutis of control Sprague-Dawley rats (SDR) and NAR using an implanted osmotic pump. All doses which were effective in reducing cholesterol in the NAR (reductions up to approximately 60%), reduced apolipoprotein AI but not apolipoprotein B and caused a severe inflammatory reaction in the dermis. Similar toxicity was observed in the SDR. Subcutaneous administration of the vehicle (propylene glycol) did not cause this reaction and did not affect plasma lipids. Administration of Lovastatin in osmotic pumps resulted in a similar inflammatory reaction. Incorporation of Simvastatin into liposomes did not diminish the toxic effect. On the other hand, infusion of Pravastatin (a polar HMG-CoA reductase inhibitor dissolved in isotonic saline) caused no changes in the dermis and had no effect on plasma lipids in NAR or SDR. Liver microsomes prepared from the Pravastatin-treated rats demonstrated a 3- to 4-fold increase in HMG-CoA reductase activity as compared to untreated rats, confirming uptake of the drug. We conclude that continuous subcutaneous administration of the HMG-CoA reductase inhibitors Simvastatin, Lovastatin and Pravastatin for 28 days may not reduce plasma cholesterol in rats by a mechanism which is related to inhibition of HMG-CoA reductase activity in the liver. The decrease of plasma cholesterol effected by subcutaneous infusion of Simvastatin or Lovastatin in NAR coincides with, and may be related to inflammatory changes caused by administering these compounds into the dermis.     

Differential regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, synthase, and low density lipoprotein receptor genes.

The ability of mitogenic stimulation of human T lymphocytes to alter the expression of genes involved in sterol metabolism was examined. Messenger RNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein (LDL) receptor were quantified in resting and mitogen-stimulated T lymphocytes by nuclease protection assay. Mitogenic stimulation increased HMG-CoA synthase mRNA levels by 5-fold and LDL receptor by 4-fold when cells were cultured in lipoprotein-depleted medium whereas HMG-CoA reductase gene expression was not significantly increased. When cultures were supplemented with concentrations of low density lipoprotein sufficient to saturate LDL receptors, expression of all three genes was inhibited in resting lymphocytes, as effectively as was noted with fibroblasts. Similarly, LDL down-regulated gene expression in mitogen-activated lymphocytes so that mitogenic stimulation did not increase either HMG-CoA reductase or synthase mRNA levels, although LDL receptor gene expression was enhanced. These results indicate that expression of three of the genes involved in sterol metabolism is differentially regulated by LDL and mitogenic stimulation. Moreover, the increase in rates of endogenous sterol synthesis and the activity of HMG-CoA reductase in mitogen-stimulated T lymphocytes cannot be accounted for by increases in HMG-CoA reductase mRNA levels.     

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta?(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH?6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.     

Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.     

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.     

Regulation of hepatic cholesterol biosynthesis by fatty acids: effect of feeding olive oil on cytoplasmic acetoacetyl-coenzyme A thiolase, beta-hydroxy-beta-methylglutaryl-CoA synthase, and acetoacetyl-coenzyme A ligase

We reported previously that, in the perfused rat liver, oleic acid increased the specific activity of cytosolic enzymes of cholesterol biosynthesis. In this study, we examined the effects of oral administration of olive oil on the activities of HMG-CoA synthase, AcAc-CoA thiolase, AcAc-CoA ligase and HMG-CoA reductase. Olive oil feeding increased the specific activity of hepatic HMG-CoA synthase by 50%, AcAc-CoA thiolase by 2-fold, and AcAc-CoA ligase by 3-fold. Olive oil had no effect on HMG-CoA reductase activity. These data suggest that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are regulated in parallel by a physiological substrate, fatty acid, independent of HMG-CoA reductase under these conditions.     

Thyroid-stimulating hormone decreases HMG-CoA reductase phosphorylation via AMP-activated protein kinase in the liver

Cholesterol homeostasis is strictly regulated through the modulation of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of cholesterol synthesis. Phosphorylation of HMGCR inactivates it and dephosphorylation activates it. AMP-activated protein kinase (AMPK) is the major kinase phosphorylating the enzyme. Our previous study found that thyroid-stimulating hormone (TSH) increased the hepatocytic HMGCR expression, but it was still unclear whether TSH affected hepatic HMGCR phosphorylation associated with AMPK. We used bovine TSH (bTSH) to treat the primary mouse hepatocytes and HepG2 cells with or without constitutively active (CA)-AMPK plasmid or protein kinase A inhibitor (H89), and set up the TSH receptor (Tshr)-KO mouse models. The p-HMGCR, p-AMPK, and related molecular expression were tested. The ratios of p-HMGCR/HMGCR and p-AMPK/AMPK decreased in the hepatocytes in a dose-dependent manner following bTSH stimulation. The changes above were inversed when the cells were treated with CA-AMPK plasmid or H89. In Tshr-KO mice, the ratios of liver p-HMGCR/HMGCR and p-AMPK/AMPK were increased relative to the littermate wild-type mice. Consistently, the phosphorylation of acetyl-CoA carboxylase, a downstream target molecule of AMPK, increased. All results suggested that TSH could regulate the phosphorylation of HMGCR via AMPK, which established a potential mechanism for hypercholesterolemia involved in a direct action of the TSH in the liver.