Squalene synthetase. Stoichiometry and kinetics of presqualene pyrophosphate and squalene synthesis by yeast microsomes.
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Farnesol↗
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Plasma squalene: lipoprotein distribution and kinetic analysis.
Plasma squalene concentration is increased in hypertriglyceridemia. In 24 normotriglyceridemic and 12 hypertriglyceridemic subjects, whole plasma squalene correlated strongly with plasma triglyceride (r = 0.973, P < 0.001) in the latter. In normal postabsorptive plasma, squalene was found in each lipoprotein fraction, 50.8% in very low density lipoprotein, 25.6% in low density lipoprotein, and 23.6% in high density lipoprotein. When plasma triglyceride was increased by dietary intake in humans or by experimental diabetes in rats, plasma squalene increased correspondingly. Conversion of [(14)C]mevalonic acid into [(14)C]squalene and kinetic analysis of [(14)C]squalene die-away curves were studied in 17 subjects. Hypertriglyceridemia significantly increased the estimated metabolically active plasma squalene pool. This together with an increase in radioactivity of squalene (dpm/ml plasma) in hypertriglyceridemia suggested that squalene production was increased. Squalene specific activity curves in lipoprotein fractions from four chylomicronemic subjects demonstrated that each fraction had newly synthesized squalene and that total plasma squalene kinetics represent the composite of several individual die-away curves. We conclude that squalene in whole plasma and in lipoprotein fractions varies directly with triglyceride content. Hypertriglyceridemia expands the plasma pool of metabolically active squalene, and each lipoprotein fraction contains squalene that is metabolically active in cholesterol synthesis.
Adult↗
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↗
Measurement of cholesterol synthesis in man by isotope kinetics of squalene.
A method for measuring the rate of total daily cholesterol synthesis in man has been developed through isotope kinetic studies of squalene biosynthesis after intravenous administration of [14C]mevalonic acid. Plasma squalene becomes rapidly labeled, reaching maximal specific activity approximately 100 min after mevalonate administration and then decays exponentially to reach undetectable levels in 12 hr. The rate of daily squalene synthesis equals the percent dose of mevalonate converted to cholesterol divided by the area under the specific activity curve of squalene; the fraction of the dose of mevalonate converted to cholesterol is calculated by the simultaneous injection of [3H]- and [14C] cholesterol in plasma. The premise that squalene and cholesterol synthetic rates are equivalent was tested. In seven patients it was found that the mean daily cholesterol synthesis rates estimated simultaneously by sterol balance and by sqyalene kinetic methods agreed within 8%. In addition, fractional conversions of mevalonic acid to cholesterol were highly correlated with cholesterol synthesis rates. Maximum estimates of the pool sizes and half-lives of metabolically "active" squalene also were obtained. This measurement of daily cholesterol synthesis by squalene kinetics minimizes patient inconvenience, is suitable for outpatient studies, and yields results in 4 weeks or less. Because of the rapidity of the rate of squalene synthesis, the results obtained reflect cholesterol synthesis over a period of less than 10 hr and are therefore uniquely applicable to unsteady state situations.
Adult↗
Effect of a supernatant protein on microsomal squalene epoxidase and 2,3-oxidosqualene-lanosterol cyclase.
Squalene epoxidation catalyzed by rat liver microsomes requires oxygen NADPH, and the 105,000 x g supernatant (S105). The supernatant can be replaced by a partially purified S105 protein (SPF) and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). When washed microsomes are preincubated anaerobically with [14C]squalene and S105 without NADPH, followed by centrifugation and washing to remove the unbound squalene and S105, epoxidation in the presence of O2 and NADPH occurs subsequently at the same rate as in direct assays containing all required components from the start. Partially purified SPF (65-fold) shows the same effect. Washed microsomes preincubated anaerobically with squalene alone, or with bovine serum albumin instead of S105, also take up large amounts of squalene, but the squalene so incorporated is only poorly converted to epoxide. The epoxidation of endogenous squalene formed in liver homogenates from [14C]mevalonate is also stimulated by S105. The incorporation of squalene into microsomes is temperature dependent. 2,3-Oxidosqualene-lanosterol cyclase (cyclase) also requires S105 for optimal activity. It is suggested that the S105 protein acts internally within the microsomal membrane system facilitating the access of substrate to specific enzyme sites.
Animals↗
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↗
Effects of weight reduction on squalene, methyl sterols and cholesterol and on their synthesis in human adipose tissue.
Quantitation of cholesterol and its precursors from human adipose tissue biopsies revealed very high squalene and moderately high methyl sterol concentrations. The squalene and cholesterol values were correlated with each other. Weight reduction in obese subjects following a jejuno-ileal bypass resulted in a significant but transient increase in adipose tissue cholesterol. The squalene concentration was also increased postoperatively, the maximum being reached about 6 months later than that of cholesterol as if the mobilization of squalene from shrunken adipocytes had been slow. Weight reduction with a 2--14 day total fast significantly reduced the adipocyte size but had no consistent effect on adipose tissue squalene, methyl sterol and cholesterol concentrations or on their adipocyte contents. Incubation of adipose tissue with labelled acetate and mevalonate revealed that the bulk of the labels in non-saponifiable lipids stayed in the large intermediate pools of methyl sterols and squalene in particular, fairly little being found in the cholesterol fraction itself. The total fast inhibited the incorporation of both 14C-acetate and 3H-mevalonate to squalene, methyl sterols and cholesterol, suggesting that cholesterol synthesis was inhibited before and after the mevalonate step.
Adipose Tissue↗
Effects of prolactin, progesterone, and 17beta-hydroxy-5alpha-androstan-3-one on squalene production by the preputial gland of the immature female rat.
To examine further the previously demonstrated synergism between prolactin and progesterone on preputial glands of hypophysectomized, ovariectomized, immature rats, their effects on squalene production were determined and compared with the ability of 17beta-hydroxy-5alpha-androstan-3-one (DHT) and prolactin to increase the amount of squalene in the preputial glands. Glands from progesterone-treated rats incubated in vitro with [14C]mevalonic acid incorporated radioactivity into squalene (identified by chromatographic mobility) more rapidly than glands from controls or prolactin-treated rats. Using the same in vitro system, glands from prolactin-treated rats incorporated more [14C]acetate into squalene than those from progesterone-treated animals. In addition, results showed that prolactin and DHT increased nonradioactive squalene (identified by mass spectral analysis) content in the gland while progesterone had no effect. It is proposed that prolactin increases preputial gland squalene content by enhancing synthesis of mevalonic acid, while progesterone increases incorporation of mevalonic acid into squalene.
Animals↗
Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system.
Microsomal squalene epoxidase has previously been solubilized with Triton X-100 and resolved into fractions, FA and FB, by DEAE-cellulose chromatography (Ono T. and Bloch K (1975) J biol. Chem. 250, 1571-1579). It has now been found that FB is identical with NADPH-cytochrome c reductase (denoted FPT, EC 1.6.2.3). Although both NADPH and NADH served as electron donors, the former was preferred for squalene epoxidase activity in the reconstituted system of FA and FB. FB is characterized by its ability to reduce cytochrome c by NADPH. In place of FB, partially purified FPT was tested for its ability to support squalene epoxidation in the presence of FA. A stepwise purification of the deoxycholate-solubilized FPT yielded an increase in specific FPT activity with a parallel increase in squalene epoxidase activity. Bromelain-solubilized FPT was less effective. Rabbit antisera preparations to the purified FPT solubilized with trypsin were shown to inhibit concomitantly FPT activity and squalene epoxidase activity. These observations support the concept that squalene epoxidation is primarily mediated via a flavoprotein, NADPH-cytochrome c reductase, and a terminal oxidase, squalene epoxidase, which is distinct from cytochrome P-450.
Animals↗
Binding of squalene, lanosterol, desmosterol, and cholesterol to proteins in brain and liver 105,000 g supernatant fractions: evidence for specific binding sites.
The binding of squalene, lanosterol, desmosterol, and cholesterol to proteins in 105,000 g supernatant fraction (S105) from brain and liver of rats was investigated. The S105 fractions from both tissues contain specific binding sites for sterols, which are sensitive to trypsin. The dissociation constants for squalene and sterol protein complexes were in the range of 10(-6) M and were not appreciably different for proteins in brain and liver S105. Competition studies revealed that both brain and liver S105 contain one receptor protein which binds lanosterol and is specific for methyl sterols, and a second receptor which binds both desmosterol and cholesterol. Binding of 7-dehydrocholesterol reported by others must occur at a third independent site since this compound does not interfere with the binding of lanosterol, desmosterol, or cholesterol. Although binding of squalene to proteins in brain and liver S105 does occur, we were unable to show the specificity of squalene binding. The concentration of desmosterol and cholesterol binding sites, which ranged from 6 to 10 nmol/mg protein, was 3- to 5-fold higher than the concentration of squalene and lanosterol binding sites (1.6-2.3 nmol/mg protein). The brain S105 from suckling rats contained fewer binding sites for desmosterol and cholesterol than the brain S105 from weaned rats. However, the concentration of lanosterol binding sites in brain S105 did not show an age-dependent change. The receptor proteins in brain and liver appear to be identical.
Animals↗
Evidence for carrier proteins in bile acid synthesis. The effect of squalene and sterol carrier protein and albumin on the activity of 12alpha-hydroxylase.
The possibility that carrier proteins are involved in bile acid synthesis was investigated using rat liver homogenates. The 105 000 X g supernatant fraction was found to contain heat stable proteins that bound the bile acid precursor, 7alpha-hydroxy-4-cholesten-3-one, and increased the amount of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one formed by the microsomal enzyme, 12alpha-hydroxylase. Subsequent studies were carried out to determine if squalene and sterol carrier protein or albumin, two lipid binding proteins present in the 105 00 X g supernatant fraction of rat liver homogenates, may be responsible for the effects seen with this fraction. Squalene and sterol carrier protein bound several water insoluble bile acid precursors, including 7alpha-hydroxy-4-cholesten-3-one, and increased the apparent activity of 12alpha-hydroxylase. Squalene and sterol carrier protein, however, did not bind either cholic acid or chenodeoxycholic acid. Rat serum albumin also bound 7alpha-hydroxy-4-cholesten-3-one and increased the apparent activity of 12alpha-hydroxylase. Kinetic analysis indicated that the apparent stimulation of 12alpha-hydroxylase by squalene and sterol carrier protein and albumin was due to increased solubilization of the substrate, 7alpha-hydroxy-4-cholesten-3-one. Thus, these studies indicate that bile acid precursor carrier proteins are present in the 105 000 Xg supernatant fraction of rat liver homogenates and suggest that squalene and sterol carrier protein or albumin may participate as carrier proteins in bile acid synthesis.
Animals↗
Squalene epoxidase and oxidosqualene lanosterol-cyclase activities in cholesterogenic and non-cholesterogenic tissues.
Squalene epoxidase and oxidosqualene lanosterol-cyclase activities have been studied in normal mammalian cholesterogenic and non-cholesterogenic tissues. This paper describes the kinetic conditions of measurement of these two enzymatic acitivities and their results. Oxidosqualene lanosterol-cyclase is a widespread enzyme, present in all cholesterogenic and non-cholesterogenic tissues. However, the level of squalene epoxidase is very low in non-cholesterogenic tissues. However, the level of squalene epoxidase is very low in non-cholesterogenic tissues. The effects of subcellular fractionizing and of the physico-chemical state of squalene incubated in vitro on squalene epoxidase activity are discussed.
Animals↗
Effects of a supernatant protein activator on microsomal squalene-2,3-oxide-lanosterol cyclase.
A soluble protein termed "supernatant protein factor" (SPF) that stimulates microsomal squalene epoxidase has been isolated in this laboratory (Ferguson, J.B., and Bloch, K. (1977) J. Biol. Chem. 252, 5381-5385). We now show that the purified protein also stimulates microsomal squalene-2,3-oxide leads to lanosterol cyclase but has no effect on the subsequent conversion of lanosterol to cholesterol. Phospholipid, specifically phosphatidylglycerol or phosphatidylethanolamine, is required for maximal stimulation of the cyclase by purified SPF. The response of microsomal squalene epoxide-lanosterol cyclase to SPF was abolished by pretreatment of the membranes with phospholipase A2 or by low concentrations of deoxycholate, indicating that an intact membrane system is required. Digestion of intact microsomes with trypsin had no effect on the SPF-stimulated cyclase activity. However, in the presence of 0.4% deoxycholate, trypsin completely inhibited microsomal squalene epoxide-lanosterol cyclase. We conclude that the cyclase is located on the luminal side of the microsomal membrane. SPF also significantly enhances the formation of lanosterol from squalene-2,3-oxide already bound to microsomes. This finding is constant with the proposal that SPF influences intramembrane events.
Animals↗
The novel transcriptional activator Bhr1 combining NTPase and Zn(II)2Cys6 DNA-binding domains controls (hemi-)cellulase response to mannose-rich substrates in the white-rot fungus Dichomitus squalens.
The regulatory landscape responsible for lignocellulose degradation in white-rot basidiomycete fungi remains largely unexplored. In this study, we characterize a novel transcriptional activator, Bhr1, in the white-rot fungus Dichomitus squalens. Bhr1 exhibits an unusual domain architecture that combines a septin-like P-loop NTPase fold with Zn(II)2Cys6 DNA-binding domains and plays a critical role in activating (hemi-)cellulase enzyme production when D. squalens is exposed to mannose-rich substrates. Using CRISPR/Cas9-mediated gene editing, we generated a bhr1 disruption mutant that displayed distinct phenotypes and enzyme activity profiles on mannose and guar gum compared to the wild type. RNA sequencing data indicate that Bhr1 induces specific (hemi-)cellulase-encoding genes without altering the expression of genes encoding sugar transporters or sugar metabolic enzymes. Phylogenetic analyses show that Bhr1 is basidiomycete specific and largely restricted to saprotrophic and plant-associated Agaricomycetes fungi. Based on the domain architecture of Bhr1 and the effects of its disruption in D. squalens, our findings reveal a lineage-specific regulatory innovation in basidiomycetes that is distinct from those described in ascomycetes. Elucidating the function and evolutionary conservation of Bhr1 advances our understanding of lignocellulose degradation at the molecular level in basidiomycete fungi and may inform studies of their ecological adaptation and the development of biotechnological applications.IMPORTANCEUnderstanding the transcriptional regulatory mechanisms in white-rot fungi, such as Dichomitus squalens, is crucial for advancing our knowledge of lignocellulose degradation. This study identifies D. squalens Bhr1 as a key regulator of (hemi-)cellulase production on mannose-rich substrates and further distinguishes basidiomycete transcription factors involved in plant biomass degradation from their ascomycete counterparts. Our findings highlight the significance of lineage-specific regulators in facilitating adaptive enzyme production for efficient biomass utilization, which is critical to carbon cycling in terrestrial ecosystems. This work establishes a foundation for exploring novel regulatory strategies among wood-degrading fungi, potentially enabling targeted strain engineering in biotechnological applications.
Mannose↗
Effect of alimentation on human serum squalene levels.
Five subjects consumed a meal containing ca. 1.5 mg squalene. Postprandial serum samples showed that in three of the subjects squalene and lipid levels reached maxima at 3 hr. Squalene levels in the low density lipoproteins of these subjects also increased. The results indicate that alimentation is a major factor in the variation in serum squalene levels.
Diet↗
Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-i-ethylhexyl phthalate.
Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the art. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-14C and mevalonate-5-3H into squalene, C30 sterols, and C27 sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of 3H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% control values in 11 days, whereas 14C-acetate conversion was reduced to ca. 35% of control values during the same period. 3H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as 14C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from 14C-acetate and 3H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were similar in control and DEHP-fed rats.
Acetates↗
Oxidation of linear terpenes and squalene variants by Arthrobacter sp.
Cells of Arthrobacter sp. that had been isolated from soil were used to study oxidation of some linear terpenes and squalene variants. The cells oxidized geraniol, nerol, and farnesol to the corresponding aldehydes, with partial conversion of the geometrical isomerism of the alpha,beta-double bond. The squalene variant, squalene-2,3-oxide, was cleaved to 9,10-epoxygeranylacetone and geranylacetone. Squalene-2,3-22,23-dioxide was cleaved to 9,10-epoxygeranylacetone. These products were optically active, and their stereochemistry and optical purity were determined.
Arthrobacter↗
Cholesterol metabolism in diabetes: the effect of insulin on the kinetics of plasma squalene.
The turnover of isotopically labeled squalene formed in plasma from [14C]mevalonate has been used to measure cholesterol synthesis in diabetics over a 7-h period. Five patients were studied while in poor diabetic control (mean daytime glycemia, 349 mg/dl) and at a later date once improved control was established by multiple daily insulin injections (mean glycemia, 175 mg/dl). This degree of diabetic control resulted in an increase in the fractional conversion of [14C]mevalonic acid to [14C]squalene from 55.2 +/- 1% to 67.5 +/- 4% (P less than 0.025). These data together with the area under the squalene specific activity curve yeild an estimated rate of cholesterol synthesis based on the likely assumption that mevalonate pool size did not decrease. Insulinization increased this calculated mean rate of cholesterol synthesis from 961 +/- 151 to 1206 +/- 223 mg/day (P less than 0.025). The use of squalene kinetics to evaluate changes in cholesterol synthesis deserves further study, particularly in metabolically unstable states such as diabetes in which conventional methods for measuring cholesterol synthesis are difficult to apply and to interpret.
Adult↗