The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs.

The effect of low concentrations of a specifically designed sterol-24-transmethylase inhibitor, 25-aza-24, 25-dihydrozymosterol (10) on sterol production in Saccharomyces cerevisiae was examined. The synthesis of cholesta-5,7,22,24-tetraen-3beta-ol (4), its 7,22,24 analog (15) and the 7,24 analog (5) coupled with the availability of zymosterol (6) and cholesta-5,7,24-3beta-ol (3) derivatives facilitated a search for these sterols in cultures treated with this inhibitor. When S. cerevisiae was grown in the presence of 1.3 and 5 muM 10, it produced no ergosterol but accumulated zymosterol (6), cholesta-5,7,22,24-tetraen-3beta-ol (4) and related C27 sterols (3 and 5). These results indicate blockage of the side chain methylation that normally occurs during the biosynthesis of ergosterol in yeast by compound 10 is efficient. The cholesta-5,7,22,24-tetraen-3beta-ol is a close structural analog of provitamin D3 (7-dehydrocholesterol). The inhibited yeast thus provides a source of a potentially new provitamin D3 substitute.     

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.     

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.     

Resistance of yeasts to polyene antibiotics

The strains of Saccharomyces cerevisiae yeast with mutations in two genes NYS3 NYS4 were obtained by tetrad analysis. Sterol fraction of these mutants contains two sterols: ergosta-7-en-3 beta-ol (fungisterol) and ergosta-7,24-dien-3 beta-ol (episterol). The findings allowed to testify the sequence of the ergosterol biosynthesis reactions. Dehydrogenization of the sterol nucleus in C5(6) which is controlled by gene NYS3 occurs simultaneously with the introduction of double bond in C22(23) site of the side chain regulated by gene NYS4.     

A novel olefinic rearrangement. The enzymic conversion of cholesta-7,9-dien-3β-ol into cholesta-8,14-dien-3β-ol

1. [3alpha-(3)H]Cholesta-7,9-dien-3beta-ol is converted in high yield into cholesterol by a 10000g(av.) supernatant fraction of rat liver homogenate. 2. Incubation of cholesta-7,9-dien-3beta-ol with [4-(3)H]NADPH and rat liver microsomal fractions under anaerobic conditions resulted in (3)H being incorporated into the 14alpha-position of cholest-7-en-3beta-ol. 3. Under anaerobic conditions in the absence of NADPH cholesta-7,9-dien-3beta-ol was isomerized into cholesta-8,14-dien-3beta-ol by rat liver microsomal fractions.     

Ergosterol biosynthesis in yeast. Pathways in the late stages and their variation under various conditions.

[Methyl-14C]methionine was supplied to yeast cells under aerobic and anaerobic conditions for the investigation of the pathway for ergosterol biosynthesis after the methylation of the side-chain. Under aerobic conditions, the incorporation of radioactivity into ergosterol was high. With a limited oxygen supply, in contrast, the radioactivity was first accumulated in ergosta-7,24(28)-dien-3beta-ol and ergosta-8,24(28)-dien-3beta-ol, and then transferred to ergost-7-en-3beta-ol, ergost-8-en-3beta-ol and ergosta-7,22-dien-3beta-ol with time. Under strictly anaerobic conditions, a double bond was introduced neither to delta5 nor to delta22. The results of the tracer experiments suggested the operation of several pathways in the late stages of ergosterol biosynthesis. It was also suggested that the main pathways varied depending on the conditions such as oxygen supply and other factors. The above conclusion was supported by the results of the analyses of the sterol compositions of the cells grown under various conditions.     

A simple method for the isolation of zymosterol from a sterol mutant of Saccharomyces cerevisiae.

A simple method is described for the direct isolation of zymosterol (5 alpha-cholesta-8,24-dien-3 beta-ol) of high purity from a sterol mutant of Saccharomyces cerevisiae. This yeast strain, which is a double mutant of the ERG6 (sterol transmethylase) and ERG2 (C-8 sterol isomerase) genes, accumulates zymosterol as its major sterol component.     

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.     

Evaluation of the vitamin D activity of yeasts with altered sterol metabolism

The sterol content of Saccharomyces strains with altered ergosterol metabolism was studied by UV-spectrophotometry, thin-layer chromatography and chromatographic mass-spectroscopy. A technique for estimation of D-vitamin activity of the yeast strains is proposed. The irradiated biomass of the strains accumulated ergosta-5,7-dien-3 beta-ol and also cholesta-5,7,24-trien-3 beta-ol and cholesta-5,7,22,24-tetraen-3 beta-ol is characterized by high antirachitic activity.     

Sterol synthesis: studies of the metabolism of 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol

[3 alpha-3H]14 alpha-Methyl-5 alpha-cholest-7-en-3 beta-ol has been prepared by chemical synthesis. The metabolism of this compound has been studied in the 10,000 g supernatant fraction of liver homogenates of female rats. Efficient conversion to cholesterol was observed. Other labeled compounds recovered after incubation of [3 alpha-3H]14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol with the enzyme preparations include the unreacted substrate, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, cholesta-5,7-dien-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, and 5 alpha-cholest-7-en-3 beta-ol. In addition, significant amounts of incubated radioactivity were recovered in steryl esters. The steroidal components of these esters were found to contain labeled 14 alpha-methyl-5 alpha-cholest-7-en-3 beta-ol, 5 alpha-cholesta-8,14-dien-3 beta-ol, 5 alpha-cholesta-7,14-dien-3 beta-ol, 5 alpha-cholest-8-en-3 beta-ol, 5 alpha-cholest-7-en-3 beta-ol, and cholesterol.     

Novel nuclear methylation of sterols by the nematode Caenorhabditis elegans

Caenorhabditis elegans possesses a unique sterol methylation pathway not reported to occur in any other organism and also removes the C-24 ethyl group of sitosterol (a plant sterol). This nematode produced substantial quantities of 4 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol and smaller amounts of lophenol from dietary cholesterol, desmosterol or sitosterol. When C. elegans was propagated in media containing sitosterol plus 25-azacoprostane hydrochloride (25-aza-5 beta-cholestane hydrochloride), an inhibitor of delta 24-sterol reductase in insects, its 4 alpha-methylsterol fraction largely consisted of equal amounts of 4 alpha-methyl-5 alpha-cholesta-7,24-dien-3 beta-ol and 4 alpha-methyl-5 alpha-cholesta-8(14),24-dien-3 beta-ol. Thus 25-azacoprostane hydrochloride inhibited both a delta 24-sterol reductase and a delta 7-sterol isomerase in C. elegans.     

Detection of the ergosterol and episterol isomers lichesterol and fecosterol in nystatin-resistant mutants of Neurospora crassa

Wild-type Neurospora crassa is completely inhibited by 5 ppm nystatin. Ultraviolet-induced mutants have been isolated that grow in the presence of 60 ppm of the antibiotic. Gas-liquid chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses showed the wild-type sterols to be ergosterol (ergosta-5,7,22-trien-3β-ol) and episterol (ergosta-7,24(28)-dien-3β-ol) in a 3:1 ratio. The mutants contained lichesterol (ergosta-5,8,22-trien-3β-ol) and fecosterol (ergosta-8,24(28)-dien-3β-ol) in a 2:1 ratio, differing from the wild type only in the position of the B-ring unsaturation. A deficiency of an ergosta-8,24 (28)-dien-3β-ol:ergosta-7,24(28)-dien-3β-ol isomerase is indicated.     

Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells.

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.     

Mode of formation of cholesta-5,7-dien-3β-ol from cholest-5-en-3β-ol by larvae of Calliphora erythrocephala

1. The conversion of cholest-5-en-3beta-ol (cholesterol) into cholesta-5,7-dien-3beta-ol by axenic Calliphora erythrocephala larvae was demonstrated. 2. The transformation is probably direct (Delta(5)-->Delta(5,7)) and does not involve a Delta(0) intermediate (Delta(5)-->Delta(0)-->Delta(7)--> Delta(5,7)). 3. Delta(7)-bond formation involves the stereospecific elimination of the 7beta hydrogen atom. 4. The relative amounts of free and esterified sterols were determined in larvae grown on cholesterol as sole sterol source and on 5alpha-cholestan-3beta-ol supplemented with minimal amounts of cholesterol. 5. The significance of the results is assessed in relation to the probable role of cholesta-5,7-dien-3beta-ol as an intermediate in the biosynthesis of ecdysones.     

Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

Phospholipid studies of marine organisms: 26. Interactions of some marine sterols with 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) in model membranes.

The thermotropic behavior of multilamellar vesicles (MLV) composed of different mole fractions of various marine sterols and 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) was examined by differential scanning calorimetry (DSC), and was compared to pure SOPC as well as their mixtures with cholesterol. The marine sterols investigated were capable of interacting with the phospholipid bilayers. Upon addition of marine sterols, the apparent transition temperature (Tm) of SOPC decreased significantly. Desmosterol (cholesta-5,24-dien-3 beta-ol) had the least interaction with SOPC, as reflected by the larger delta H values of its mixtures with the phospholipid. Fucosterol (24-ethylcholesta-5,24(28)-dien-3 beta-ol) showed a non-linear trend as the mole percent of the sterol increased. Mixtures of sutinasterol (24R-24-ethyl-26,26-dimethylcholesta-7,25(27)-dien-3 beta-ol) with SOPC had similar enthalpy values to cholesterol. The shape of the SOPC/marine sterol endotherm and their delta H values were not identical when liposomes prepared by dialysis were compared to MLV.     

Zymosterol is located in the plasma membrane of cultured human fibroblasts

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.     

Defective sterol C5-6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals

Two azole resistant isolates of Saccharomyces cerevisiae carried mutations allelic to erg 3 and were blocked to differing degrees at the C5-6 desaturation step of ergosterol biosynthesis. When treated with the sterol 14 alpha-demethylation inhibitor fluconazole the wild-type sensitive strain accumulated lanosterol and 14 alpha-methyl-erogosta-8,24(28)-dien-3 beta, 6 alpha-diol (14-methyl-3,6 diol). The stringent desaturase mutant, A2, accumulated 14 alpha-methyl-8,24(28)-dien-3 beta-ol (14-methyl fecosterol) and lanosterol as the major sterol components when treated with fluconazole. Resistant isolate A3 accumulated 14-methyl-3,6-diol, 14-methyl fecosterol, and lanosterol and was only partially blocked at sterol C5-6 desaturation. We conclude that functional sterol C5-6 desaturase is required for the synthesis of 14-methyl-3,6-diol under conditions of azole inhibition. We present a new hypothesis for the mode of action of azole antifungals based on the inability of 14-methyl-3,6-diol to support growth, and suggest that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation.     

Zymogen secretion and phospholipid metabolism in the pancreas. Phospholipids of the zymogen granule

1. The metabolism of [4-(14)C]pregnenolone to androst-16-enes has been studied in short-term incubations of boar testis tissue. With fresh tissue androsta-5,16-dien-3beta-ol (8%) and 5alpha-androst-16-en-3beta-ol (2%) were formed. Tissue that had been stored at -20 degrees C was still capable of metabolizing pregnenolone to androsta-5,16-dien-3beta-ol. 2. NADPH was essential for the formation of androsta-5,16-dien-3beta-ol from pregnenolone; NADH had less activity and ATP was not necessary for the reaction. 3. [4-(14)C]Androsta-5,16-dien-3beta-ol, prepared biosynthetically from [4-(14)C]pregnenolone, was shown to be converted by boar testis preparations into androsta-4,16-dien-3-one (31%) if NAD(+) was present or into 5alpha-androst-16-en-3beta-ol (4%) if NADPH was present. 4. 17alpha-Hydroxyandrost-4-en-3-one and 3beta,17alpha-dihydroxypregn-5-en-20-one were considered as possible precursors for androst-16-ene formation, but both were shown to be ineffective. 5. No radioactivity was incorporated into androst-5-en-3beta-ol used to trap any corresponding (14)C-labelled compound formed from [4-(14)C]pregnenolone.     

1,25-Dihydroxyvitamin D3 stimulated increase of 7,8-didehydrocholesterol levels in rat skin

A convenient, accurate assay was developed for determining skin cholesta-5,7-dien-3 beta-ol (7,8-didehydrocholesterol) concentrations. Ultraviolet spectrophotometry provided quantitation of the sterol from rat skins following saponification and chromatography on Lipidex and high-performance liquid chromatography. Correction for recoveries was accomplished by using 7,8-didehydro[3 alpha-3H]cholesterol as an internal standard. Chronic dosing of vitamin D-deficient rats with 1,25-dihydroxyvitamin D3 caused a 4-fold increase in skin 7-dehydrocholesterol content. This rise was not the result of changes in food consumption, body weight, or plasma calcium. Cholesterol concentrations were not significantly elevated although some of the other nonsaponifiable lipid components found in the high-performance liquid chromatogram appeared to be increased by the treatment. These results suggest that the vitamin D hormone 1,25-(OH)2D3 may exert a positive feedback regulation on the production of vitamin D3 in skin.