UNIPROT:Q8NEX9 - FACTA Search

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.
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PMID:Subcellular localization of squalene synthase in human hepatoma cell line Hep G2. 131 47

Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation.
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PMID:Squalene synthase-deficient mutant of Chinese hamster ovary cells. 152 71

Certain enzymes of the mevalonate pathway have been investigated in persistent liver nodules induced in the rat by 2-acetylaminofluorene. In these nodules the dolichol level was increased 5-fold, the ubiquinone-9 content elevated 6-fold and the amount of cholesterol unchanged. Microsomal beta-hydroxy-beta-methylglutaryl-coenzyme A reductase activity was greatly increased compared to control liver tissue, which was also the case for the cytosolic farnesyl pyrophosphate synthase. A significant elevation of all-transgeranylgeranyl pyrophosphate synthase activity in the cytosol was also observed. The branch-point enzyme of microsomal dolichol synthesis, i.e. cis-prenyltransferase, was decreased in the nodules; whereas the activity of squalene synthase, the terminal regulating enzyme of cholesterol synthesis, remained unchanged. The dolichol species in nodular tissue were redistributed towards the longer chain length species. One factor regulating the chain length of the polyisoprene products formed in vitro was shown to be the ratio of the concentrations of isopentenyl pyrophosphate:farnesyl pyrophosphate employed. Other regulatory factors in the terminal steps of this biosynthetic pathway appear to determine the amounts and nature of the final isoprenoid compounds formed in vivo. In contrast to the microsomal trans-prenyltransferase activity, which was unchanged, the activity of nonaprenyl-4-hydroxybenzoate transferase, an enzyme participating in ubiquinone synthesis, was greatly elevated. The alterations observed in the activities of enzymes in the mevalonate pathway can at least partially explain the increased levels of dolichol and ubiquinone and the unchanged level of cholesterol found in liver nodules. It is reasonable to propose that this modified mevalonate metabolism will render nodular cells resistant to certain toxic factors and prone to cell proliferation.
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PMID:Enzymes of the mevalonate pathway in rat liver nodules induced by 2-acetylaminofluorene treatment. 769 19

Research on the ergosterol biosynthetic pathway in fungi has focused on the identification of the specific sterol structure required for normal membrane structure and function and for completion of the cell cycle. The pathway and its end product are also the targets for a number of antifungal drugs. Identification of essential steps in ergo-sterol biosynthesis could provide new targets for the development of novel therapeutic agents. Nine of the eleven genes in the portion of the pathway committed exclusively to ergosterol biosynthesis have been cloned, and their essentiality for aerobic growth has been determined. The first three genes, ERG9 (squalene synthase), ERG1 (squalene epoxidase), and ERG7 (lanosterol synthase), have been cloned and found to be essential for aerobic viability since their absence would result in the cell being unable to synthesize a sterol molecule. The remaining eight genes encode enzymes which metabolize the first sterol, lanosterol, to ultimately form ergosterol. The two earliest genes, ERG11 (lanosterol demethylase) and ERG24 (C-14 reductase), have been cloned and found to be essential for aerobic growth but are suppressed by mutations in the C-5 desaturase (ERG3) gene and fen1 and fen2 mutations, respectively. The remaining cloned genes, ERG6 (C-24 methylase), ERG2 (D8AE7 isomerase), ERG3 (C-5 desaturase), and ERG4 (C-24(28) reductase), have been found to be nonessential. The remaining genes not yet cloned are the C-4 demethylase and the C-22 desaturase (ERG5).
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PMID:Cloning of the late genes in the ergosterol biosynthetic pathway of Saccharomyces cerevisiae--a review. 779 29

The mechanisms by which sterols and nonsterols regulate hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene expression was investigated by measuring mRNA, protein, and enzyme activity in rats fed cholesterol or given drugs that deplete both endogenous sterols and nonsterols or that selectively deplete sterols. It was found that while dietary cholesterol had little effect on HMG-CoA reductase mRNA levels; immunoreactive protein was reduced to barely detectable levels, as was enzyme activity. Any possible effect on catalytic efficiency is thus ruled out. When rats were fed diets containing Lovastatin, a potent HMG-CoA reductase inhibitor which blocks synthesis of both nonsterols and sterols, similar 15- to 20-fold increases were observed for both HMG-CoA reductase mRNA and activity. However, HMG-CoA reductase immunoreactive protein was increased more than 200-fold. When endogenous sterols were selectively depleted by inhibiting squalene synthase with Zargozic acid A, the increase observed in reductase mRNA was similar to that seen in immunoreactive protein and enzyme activity. This is consistent with the concept that endogenous sterols exert their effects at the level of transcription while endogenous nonsterols act at the level of translation. The results suggest that in whole animals, sterols likely act at the level of mRNA while nonsterols exert their regulatory effects at the level of HMG-CoA reductase protein.
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PMID:Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression by sterols and nonsterols in rat liver. 810 70

The microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to rapid degradation when cells are incubated with sterols or mevalonic acid (MVA). It has been shown that this rapid degradation is dependent upon both a sterol and another MVA-derived metabolite (Nakanishi, M., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 258, 8929-8937). In the current study, inhibitors of the isoprene biosynthetic pathway were used to define further this mevalonic acid derivative involved in the accelerated degradation of HMG-CoA reductase. The accelerated degradation of HMG-CoA reductase in met-18b-2 cells, which is induced by the addition of MVA, was inhibited by the presence of the squalene synthase inhibitor, zaragozic acid/squalestatin, or the squalene epoxidase inhibitor, NB-598. Accelerated degradation of HMG-CoA reductase was observed when NB-598-treated cells were incubated with both MVA and sterols. In contrast, the addition of MVA and sterols to zaragozic acid/squalestatin-treated cells did not result in rapid enzyme degradation. This MVA- and sterol-dependent degradation of HMG-CoA reductase persisted in cells permeabilized with reduced streptolysin O. Finally, the selective degradation of HMG-CoA reductase was also observed in rat hepatic microsomes incubated in vitro in the absence of ATP and cytosol. We conclude that the MVA-derived component that is required for the accelerated degradation of HMG-CoA reductase is derived from farnesyl disphosphate and/or squalene in the isoprenoid biosynthetic pathway. We propose that this component has a permissive effect and does not, by itself, induce the degradation of HMG-CoA reductase. We also conclude that the degradation of HMG-CoA occurs in the endoplasmic reticulum, and, once the degradation of HMG-CoA reductase has been initiated by MVA and sterols, all necessary components for the continued degradation of HMG-CoA reductase reside in the endoplasmic reticulum.
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PMID:Mevalonic acid-dependent degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo and in vitro. 827 63

The possible difference between lovastatin (mevinolin, MK-803), simvastatin (MK-733) and pravastatin (CS-514), all chemically-related competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, were tested in the human hepatoma cell line Hep G2, which is often used as a model for the human hepatocyte. After an 18-hr incubation of the cells with the drugs, pravastatin (IC50 = 1900 nM) was less potent than simvastatin and lovastatin (IC50 = 34 and 24 nM, respectively) in inhibiting the sterol synthesis. As a consequence of this inhibition, the HMG-CoA reductase mRNA levels and squalene synthase activity, both negatively-regulated by sterols, were increased equally by simvastatin and lovastatin, whereas the induction by pravastatin was much less. In contrast, there were fewer differences between the compounds in inhibiting HMG-CoA reductase activity, when assayed directly in Hep G2 cell homogenates (IC50 values = 18, 61 and 95 nM for simvastatin, lovastatin and pravastatin, respectively). Moreover, in experiments with human hepatocytes in primary culture the IC50 values for inhibition of the cholesterol synthesis by simvastatin and pravastatin were of the same order of magnitude (23 and 105 nM, respectively). The results are therefore explained as follows: the three drugs act in the same way within the Hep G2 cell in terms of inhibiting HMG-CoA reductase and their subsequent effect on the feedback regulation of the cholesterol synthesis, i.e. increasing squalene synthase and HMG-CoA reductase mRNA. However, pravastatin seems to be less able to enter the cells compared with simvastatin and lovastatin, possibly because of the higher hydrophobicity of the latter compounds. The observation with human hepatocytes suggests that in Hep G2 cells a specific hepatic transporter is missing. On one hand the human hepatoma cell line Hep G2 has proved to be a good model for the study of the feedback regulation of enzymes of the cholesterol biosynthetic pathway such as HMG-CoA reductase and squalene synthase, but, on the other hand seems to be less suitable as a model for the study of specific uptake of drugs, e.g. the vastatins, in human hepatocytes.
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PMID:Pravastatin inhibited the cholesterol synthesis in human hepatoma cell line Hep G2 less than simvastatin and lovastatin, which is reflected in the upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and squalene synthase. 851 61

A recent report, in which cultured tumor cells were used, identified farnesol as the nonsterol mevalonate-derived metabolite required for the accelerated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (C. C. Correll, L. Ng, and P. A. Edwards, 1994, J. Biol. Chem. 269, 17390-17393). We examined this proposed linkage in animals by measuring hepatic farnesol levels and rates of HMG-CoA reductase degradation under conditions previously shown to alter the stability of the reductase. In normal rats, the hepatic farnesol level, quantified by high-pressure liquid chromatography, was 0.10 +/- 0.08 microgram/g and the half-life of HMG-CoA reductase was 2.5 h. Administration of mevalonolactone at 1 g/kg body wt to provide all nonsterol metabolites in addition to cholesterol increased farnesol levels 6-fold without significantly affecting the half-life of the reductase. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, raised hepatic farnesol levels 10-fold and decreased the half-life of HMG-CoA reductase to 0.25 h. However, feeding lovastatin to rats did not lower hepatic farnesol levels despite a marked stabilization of HMB-CoA reductase protein. Moreover, intubation of rats with 500 mg/kg body wt of farnesol failed to decrease the half-life of HMG-CoA reductase protein, alter the levels of enzyme activity, or change of the levels of immunoreactive protein despite an increase of 1000-fold in hepatic farnesol levels. These observations indicate that farnesol per se does not induce accelerated degradation of HMG-CoA reductase in rat liver.
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PMID:Farnesol is not the nonsterol regulator mediating degradation of HMG-CoA reductase in rat liver. 864 11

RPR 101821 (trans-2-[4-(benzoxazol-2-yl)-phenylmethoxy] amino cyclohexane hydrochloride) is a potent cholesterol-lowering agent in rodents and marmoset. The compound inhibited rat liver microsomal squalene synthase (IC50 = 1 nM) and 7-dehydrocholesterol (7DHC) reductase (IC50 = 1 microM; Lewis et al. 1995). When RPR 101821 (10 mg/kg), the 7DHC reductase inhibitor BM 15.766 (4[2-[4-(4-chlorocinnamyl)piperazine-1-yl]ethyl] benzoic acid; 10 mg/kg) or the HMG-CoA reductase inhibitor lovastatin (30 mg/kg) was given orally to rats at -29 h, -21 h and -5 h, serum cholesterol was reduced by 56%, 46% or 15%, respectively. The reduction in cholesterol with RPR 101821 was associated with an accumulation of 7DHC in serum, suggesting an inhibition of 7DHC reductase. In the presence of BM 15.766, RPR 101821 reduced the serum accumulation of 7DHC in a dose-dependent manner, with complete inhibition at 30 mg/kg, p.o. In Balb-cJ mice, RPR 101821 and lovastatin (50 mg/kg, b.i.d., p.o., for 14 days) lowered serum cholesterol by 67% and 2%, respectively. In marmosets, RPR 101821 and lovastatin (both at a dose of 10 mg/kg, p.o., b.i.d., for 7 days) reduced cholesterol by 28% and 19%, respectively. In summary, RPR 101821 is an orally effective potent cholesterol-lowering agent in rodents and a small primate species. The suggested mechanism of hypocholesterolemic effect is the inhibition of squalene synthase and 7DHC reductase.
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PMID:RPR 101821, a new potent cholesterol-lowering agent: inhibition of squalene synthase and 7-dehydrocholesterol reductase. 871 65

Previously, we found that mevalonate-derived products together with an oxysterol regulated reductase synthesis at a posttranscriptional level. To determine which products were responsible for this regulation, either the squalene synthase inhibitor zaragozic acid A or the squalene cyclase inhibitor 4,4,10-beta-trimethyl-trans-decal-3beta-ol (TMD) was added to lovastatin-treated Syrian hamster cells in conjunction with mevalonate. Mevalonate alone decreased reductase synthesis 50% compared with lovastatin-treated cells. In contrast, when both zaragozic acid A and mevalonate were added to lovastatin-treated cells, there was no change in reductase synthesis. With either treatment, reductase mRNA levels did not change compared with lovastatin-treated cells. When both 25-hydroxycholesterol and mevalonate were added to lovastatin-treated cells, reductase synthesis and mRNA levels were decreased 95 and 50%, respectively. The 10-fold difference between changes in reductase synthesis and mRNA levels under these conditions reflects a specific effect of mevalonate-derived isoprenoids on reductase synthesis at the translational level. In contrast, coincubation of cells with mevalonate plus 25-hydroxycholesterol in the presence of zaragozic acid decreased reductase synthesis and mRNA levels 60 and 50%, respectively, compared with lovastatin-treated cells. Moreover, degradation of reductase was increased approximately 7-fold in cells treated with mevalonate alone but only 3-fold in cells treated with mevalonate and zaragozic acid A. These results indicate that isoprenoid products between mevalonate and squalene affect reductase at a posttranslational level by increasing degradation but do not regulate reductase synthesis at a posttranscriptional level. In contrast, when both TMD and mevalonate were added to lovastatin-treated cells, reductase synthesis was decreased approximately 50% with no corresponding decrease in reductase mRNA levels, similar to mevalonate only. Reductase degradation was increased approximately 7-fold under these conditions. Cellular incubation in TMD, mevalonate, and 25-hydroxycholesterol decreased reductase synthesis and mRNA levels 95 and 50%, respectively. From these results we concluded that mevalonate-derived nonsterols synthesized between squalene and lanosterol decrease reductase synthesis at a translational level-either alone or in combination with 25-hydroxycholesterol-and also increase reductase degradation.
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PMID:Inhibition of squalene synthase but not squalene cyclase prevents mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis at a posttranscriptional level. 901 20

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