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At least 19 recordsLinked to original sources
PubMed1979

Squalene synthetase activity in human fibroblasts: regulation via the low density lipoprotein receptor.

Squalene synthetase (farnesyltransferase; farnesyl diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), the enzyme in the cholesterol biosynthetic pathway that converts farnesyl pyrophosphate into squalene, is subject to regulation in cultured human fibroblasts. When cholesterol-carrying low density lipoprotein (LDL) was removed from the serum of the culture medium, squalene synthetase activity increased 8-fold over 24 hr. When LDL was added back to the medium, squalene synthetase was slowly suppressed, 50% and 90% reduction occurring in 15 and 48 hr, respectively. Suppression of squalene synthetase required uptake of LDL via the LDL receptor; hence, it did not occur in mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack receptors. The addition of a mixture of 25-hydroxycholesterol and cholesterol suppressed squalene synthetase equally well in normal and mutant fibroblasts. Coupled with previous data, the current findings indicate that cholesterol derived from LDL regulates at least two enzymes in the cholesterol synthetic pathway in fibroblasts: (i) its primary action is to rapidly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase [mevalonate:NADP(+), oxidoreductase (CoA-acylating), EC 1.1.1.34], which reduces mevalonate production by 95% within 8 hr, and (ii) its secondary action is to slowly suppress squalene synthetase. The LDL-mediated suppression of squalene synthetase does not regulate de novo cholesterol synthesis; it occurs after 3-hydroxy-3-methylglutaryl coenzyme A reductase is already suppressed. Rather, we hypothesize that it may function to allow the pool size of farnesyl pyrophosphate to be maintained in the presence of LDL so that low levels of mevalonate can be shunted preferentially into nonsterol products, such as ubiquinone-10 and dolichol. This mechanism may explain the earlier observation that the synthesis of ubiquinone-10 in fibroblasts proceeds at a normal rate in the presence of LDL despite a 95% decrease in mevalonate production.

Cells, Cultured↗
PubMed1979

Squalene synthetase.

In the first part of the review the background to the discovery of the asymmetric synthesis of squalene from two molecules of farnesyl pyrophosphate and NADPH is described, then the stereochemistry of the overall reaction is summarized. The complexity of the biosynthesis of squalene by microsomal squalene synthetase demanded the existence of some intermediate(s) between farnesyl pyrophosphate and squalene. This demand was satisfied by the discovery of presqualene pyrophosphate, an optically active C30 substituted cyclopropylcarbinyl pyrophosphate, the absolute configuration of which at all three asymmetric centers of the cyclopropane ring was deduced to be R. Possible mechanisms for the biosynthesis of presqualene pyrophosphate and its reductive transformation into squalene are presented. In the second part of the review the nature of the enzyme is discussed. The question whether presqualene pyrophosphate is an obligate intermediate in the biosynthesis of squalene is examined, with the firm conclusion that it is. It is as yet uncertain whether the two half reactions of squalene synthesis, i.e. (i) 2 x farnesyl pyrophosphate leads to presqualene pyrophosphate; (ii) presqualene pyrophosphate + NADPH (NADH) leads to squalene, are catalyzed by one or two enzymes or by a large complex with two catalytic sites. Evidence is cited for the existence on the enzyme of two distinct binding sites with different affinities for the two farnesyl pyrophosphate molecules. The types of enzyme preparations available at present are described and types of experiments carried out with these are critically examined. The implications of the properties of a low molecular weight squalene synthetase solubilized with deoxycholate from microsomal membranes is discussed and a model for the enzyme in an organized membrane structure is presented.

Animals↗
PubMed1991

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase.

The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5 kb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51,600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1977

Ertosterol biosynthesis in Saccharomyces cerevisiae: mutants deficient in the early steps of the pathway.

Thermosensitive mutants, auxotrophic for ergosterol synthesis, have been isolated, analyzed genetically and their enzymatic deficiencies investigated. These mutants were classified into seven unlinked complementation groups. These groupes lack the following enzymatic activities: squalene epoxidase (erg 1), 2,3-oxidosqualene-lanosterol cyclase (erg 7), phosphomevalonic kinase (erg 8), mevalonic kinase (erg12) and squalene synthetase (erg 9, erg 10, erg 11).

Epoxy Compounds↗
PubMed1992

Development of a radiometric spot-wash assay for squalene synthase.

The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1-14C]isopentenyl diphosphate as a precursor for squalene or [2-14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2-14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughout mode or in a qualitative, high-through-put mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.

Animals↗
PubMed1992

Subcellular localization of squalene synthase in human hepatoma cell line Hep G2.

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.

Androstenes↗
PubMed1977

Substrate selectivity of squalene synthetase.

Six 1-3H-labeled analogues of farnesyl pyrophosphate have been studied as potential substrates for yeast and rat liver squalene synthetases: 2-methylfarnesyl pyrophosphate (4), 3-demethylfarnesyl pyrophosphate (5), 7,11-dimethyl-3-ethyl-2,6,10-dodecatrienyl pyrophosphate (6), 6,7,10,11-tetrahydrofarnesyl pyrophosphate (7), 4-methylthiofarnesyl pyrophosphate (8), and 4-fluorofarnesyl pyrophosphate (9). Analogues 4 and 5 are enzymatically incorporated into 11-methylsqualene (10) and 10-demethylsqualene (11), respectively, even if no farnesyl pyrophosphate is added to the incubations. None of the other analogues gives nonpolar products with either the yeast or liver enzymes. No tritium is enzymatically released to the medium from any of the analogues, indicating that they are not accepted at the first (proton exchanging) site. The data rule out formation of dead-end presqualene pyrophosphate products with analogues as first, but not as second, substrates. Implications of these results for the enzyme active-site topology and mechanism are discussed.

Animals↗
PubMed1977

Inhibition of squalene synthetase by farnesyl pyrophosphate analogues.

The pyrophosphates of the following farnesol analogues have been synthesized: 2-methylfarnesol; 7,11-dimethyl-3-ethyl-2,6,10-dodecatrien-1-ol; 3-demethylfarnesol; 4-methylthiofarnesol; 7,11-dimethyl-3-iodo-2,6,10-dodecatrien-1-ol; 7,11-dimethyl02-iodo-2,6,10-dodecatrien-1-ol; 7,11-dimethyldodeca-6,10-dien-2-yn-1-ol; phytol; 3,7,11-trimethyl-2-dodecen-1-ol; 3,7,11-trimethyldodecan-1-ol; and geraniol. The double bonds in all the above compounds were in the E configuration, except phytol, which was a 7:3 mixture of 2E and 2Z isomers. Each of the pyrophosphates inhibits the incorporation of labeled farnesyl pyrophosphate into squalene by a yeast enzyme preparation. Free alcohols and monophosphates are inactive. The analogues, listed in order of decreasing inhibitory strength, are, by kinetic analysis, competitive or mixed inhibitors. Irreversible inhibition is not observed. The results suggest that binding to the enzyme is primarily mediated by the pyrophosphate moiety assisted by relatively nonspecific lipophilic interactions. Decreasing the chain length and saturating double bonds severely reduces binding, while substitution at the 2,3, and 4 positions, and lengthening of the chain, is well tolerated.

Diphosphates↗
PubMed1992

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. III. Biosynthesis.

The biosynthetic origin of the carbon and oxygen atoms of the novel fungal secondary metabolite 1 was studied. Incorporation studies with single and multiple labelled 13C precursors indicated that the major portion of the molecule was derived from two polyketide chains made up of acetate units. One of the chains had benzoic acid (which can be derived from phenylalanine) as a starter unit. The remaining carbons were derived from a four-carbon unit related to succinate and from methionine. Studies with [1-(13)C,18O2]acetate and 18O2 indicated that five of the oxygens, including both of the heterocyclic oxygens, were derived from atmospheric oxygen. The oxygens at the two ester carbonyls were derived from acetate.

Bridged Bicyclo Compounds↗
PubMed1992

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity.

During the screening of fungi for inhibitors of squalene synthase, Phoma sp. C2932 was found to produce three structurally related novel inhibitors. These compounds, designated the squalestatins, exhibited potent activity against both mammalian (rat liver) and fungal (Candida albicans) squalene synthase. Furthermore, they also had broad spectrum in vitro antifungal activity.

Animals↗
PubMed1992

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. II. Structure elucidation.

Three novel fungal metabolites 1-3 isolated from cultures of a Phoma sp. C2932, are potent and selective inhibitors of squalene synthase. Their structures have been determined by a combination of spectroscopic, X-ray crystallographic and chemical methods; these natural products incorporate the highly functionalised bicyclic core, [1S-(1 alpha, 3 alpha, 4 beta, 5 alpha, 6 alpha, 7 beta]-4,6,7-trihydroxy- 2,8-dioxabicyclo-[3.2.1]octane-3,4,5-tricarboxylic acid.

Antifungal Agents↗
PubMed1992

Molecular cloning, expression, and characterization of the cDNA for the rat hepatic squalene synthase.

Amino acid sequence information was obtained for the NH2 terminus, and for endogenous peptides generated by trypsin digestion, of a purified, truncated form of rat hepatic squalene synthase (RSS, EC 2.5.1.21) (Shechter, I., Klinger, E., Rucker, M. L., Engstrom, R. G., Spirito, J. A., Islam, M. A., Boettcher, B. R., and Weinstein, D. B. (1992) J. Biol. Chem. 267, 8628-8635). Degenerate primers, based on the amino acid sequences, were synthesized and used for the amplification and sequencing of a 1708-base pair (bp) cDNA for RSS from the rat hepatoma cell line H35. An open reading frame of 1248 bp encoding 416 amino acids (M(r) = 48,103) was detected for RSS. We have constructed a pRSS1327 expression vector by molecular cloning of a 1327-bp cDNA, which includes sequences of the entire coding region for RSS, into pBluescript. Expression in Escherichia coli of a functional, full-length RSS was confirmed by immunoblot analysis and enzymatic activity. We present and evaluate a model for the secondary structure of RSS and its possible membrane orientation. The model predicts a 315-residue domain at the center of the protein that contains the catalytic site and is released in a soluble form by partial proteolysis. The 33-residue NH2-terminal and 98-residue COOH-terminal sections are not involved in catalysis. Sequence analysis of the catalytic domain of RSS indicate three regions with high homology to sequences in a number of functionally distinct proteins that utilize polyprenyl diphosphate substrates.

Amino Acid Sequence↗
PubMed1992

Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis.

Some bisphosphonates used for the treatment of bone disorders are also potent inhibitors of squalene synthase, a critical enzyme for sterol biosynthesis. Among seven drugs tested, YM 175 (cycloheptylaminomethylene-1,1-bisphosphonic acid) was the most potent inhibitor of rat liver microsomal squalene synthase (Ki = 57 nM) and sterol biosynthesis from [14C]mevalonate in rat liver homogenate (IC50 = 17 nM). EB 1053 (3-(1-pyrolidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) and PHPBP (3-(1-piperidino)-1-hydroxypropylidene-1,1-bisphosphonic acid) were less potent inhibitors in both these assays. Pamidronate and alendronate were poor inhibitors of squalene synthase (IC50 > 10 microM) but were potent inhibitors of sterol biosynthesis from mevalonate (IC50 = 420 and 168 nM, respectively), suggesting that the latter two agents may have inhibited other enzymes involved in the synthesis of farnesyl pyrophosphate from mevalonate. Etidronate and clodronate were inactive in both these assays. YM 175 also inhibited sterol biosynthesis in mouse macrophage J774 cells (IC50 = 64 microM) and in rats, when administered acutely, it inhibited cholesterol biosynthesis in the liver (ED50 = 30 mg/kg, s.c.). Structural modifications on YM 175 to enhance cell permeability may result in a new class of cholesterol-lowering agents.

Animals↗