Production of meiosis-activating sterols from metabolically engineered yeast

Meiosis-activating sterols (MAS), a class of potent regulators of reproductive processes, are difficult to obtain by chemical synthesis or isolation from natural sources. We demonstrate the development of metabolically engineered strains of Saccharomyces cerevisiae that accumulate MAS as the predominant sterol product. Homologous recombination was used to construct an erg24Delta erg25Delta hem1Delta mutant RXY4.3, which lacked sterol Delta14 reductase, C-4 oxidase, and delta-aminolevulinate synthase. The HEM1 deletion allowed sterol import and rendered RXY4.3 viable under aerobic conditions. This mutant accumulated 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), and a similar erg25Delta hem1Delta mutant produced 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (T-MAS). Based on consistent yields of approximately 5 mug of FF-MAS per mL of culture, fermentation of genetically modified yeast compares favorably with other approaches to produce MAS.     

Quantitation of lanosterol and its major metabolite FF-MAS in an inhibition assay of CYP51 by azoles with atmospheric pressure photoionization based LC-MS/MS

Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14alpha-demethylase (CYP51) is an enzyme inhibited by azoles. Only few data have been reported showing their inhibitory potency since an assay in an in vitro system is not available so far. In the present work an inhibition assay using human recombinant CYP51, coexpressed with human P450 oxido-reductase by the baculovirus/insect cell expression system, and LC-MS/MS as analytical method is described. Atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were used with a triple quadrupole mass spectrometer to compare quantitation of lanosterol (substrate) and 4,4-dimethyl-5alpha-cholesta-8,14,24-triene-3beta-ol (FF-MAS) (product of CYP51) with d(6)-2,2,3,4,4,6-cholesterol (d(6)-cholesterol) as internal standard. Optimization of analytical parameters resulted in a LC-APPI-MS/MS method with a LOQ of 10 pg on column for FF-MAS. The sensitivity of the method (LOD 0.5 ng/ml) makes it possible to analyze supernatants of inhibition experiments after precipitation of proteins by isopropanol without any sample enrichment. The coefficient of variation of the analytical method was <20% (n = 5) for FF-MAS, lanosterol and d(6)-cholesterol. The external calibration curve was linear from 1 to 10,000 ng/ml with R(2) >/= 0.999 and an accuracy of 94-115%. Compared with APCI, APPI provides a ten- to 500-fold increase in sensitivity for the analytes in this study. IC(50) values of epoxiconazole and miconazole-two widely used azole fungicides used in agriculture and in human medicine, respectively-were 1.95 microM and 0.057 microM.     

Carbon-13 NMR spectra of N-methylimidazole-substituted sterol esters

The ¹³C NMR spectra of two N-methylimidazole-substituted sterol esters are discussed. The calculated shifts are compared to those obtained experimentally. For one of the sterol esters the experimental and calculated data show good agreement, and the substituent increments can be evaluated. This does not apply in the case of the second sterol ester, where the steric and/or electronic effects of a further substituent—a hydroperoxy group in close proximity to the N-methylimidazole group—prohibits the additive calculation of substituent increments.     

Critical factors for detection of biphasic changes in membrane properties at specific sterol mole fractions for maximal superlattice formation

Here we use the excitation generalized polarization (GPex) of 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan) fluorescence in fluid cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine large unilamellar vesicles to explore the experimental conditions that would be required in order to detect a biphasic change in membrane properties at specific sterol mole fractions (Cr) (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol %) for maximal sterol superlattice formation. Laurdan's GPex changes with sterol content in an alternating manner, showing minima (termed as GPex dips) at approximately Cr. GPex dips are detectable if the vesicles are preincubated for a sufficient time period and protected from sterol oxidation. In most cases, vesicles with a higher lipid concentration take a longer time to show a GPex dip at Cr. The depth of the GPex dip increases with increasing incubation time and eventually reaches a plateau, at which the maximum area covered by superlattices is expected to be achieved. However, if the vesicles are not protected against sterol oxidation, the GPex dips are attenuated or obliterated. These effects can be attributed to the increased inter-bilayer lipid exchange and the increased vesicle-vesicle interactions present at high lipid (vesicle) concentrations as well as the decreased interactions between oxysterols and phospholipids. These possible explanations have been incorporated into a kinetic model that is able to calculate the effects of sterol oxidation and lipid concentration on the depth of the GPex dip. The depth of the GPex dip, the required incubation time for the dip formation, and the lipid concentration dependence of the GPex dip vary with Cr, suggesting different physical properties for different sterol superlattices. To detect a biphasic change in membrane properties at Cr, one should also use small sterol mole fraction increments over a wide range, keep all of the vesicles in the same sample set under the same thermal history, and consider lipid concentration, probe type, and Cr value. These results improve our mechanistic understanding of sterol superlattice formation and explain why some studies, especially those requiring high lipid concentrations, did not detect a biphasic change in membrane properties at Cr.     

Thin-layer chromatography of radioactively labelled cholesterol and precursors from biological material

Summary The investigation methods of the action of xenobiotics on sterol biosynthesis from ¹⁴C-acetate in rat hepatocyte cultures can be developed, with regard to extraction using Extrelut and the separation of the sterol pattern by thin-layer chromatography, in such a way that they are suitable for wider application, e.g., screening. Good visualisation and recognition of changes in the sterol pattern are possible using autoradiography of the thin-layer chromatogram.

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Dünnschicht-Chromatographie von radioaktiv markiertem Cholesterin und Vorstufen aus biologischem Material. Eine einfache und empfindliche Methode zur Untersuchung einer Beeinflussung des Sterolbiosynthesewegs

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Dedicated to Dr. E. Roesch on occasion of his 65th birthday     

Mechanism-based enzyme inactivators of phytosterol biosynthesis

Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.     

Sterol Glycosyltransferases—The Enzymes That Modify Sterols

Sterols are important components of cell membranes, hormones, signalling molecules and defense-related biotic and abiotic chemicals. Sterol glycosyltransferases (SGTs) are enzymes involved in sterol modifications and play an important role in metabolic plasticity during adaptive responses. The enzymes are classified as a subset of family 1 glycosyltransferases due to the presence of a signature motif in their primary sequence. These enzymes follow a compulsory order sequential mechanism forming a ternary complex. The diverse applications of sterol glycosides, like cytotoxic and apoptotic activity, anticancer activity, medicinal values, anti-stress roles and anti-insect and antibacterial properties, draws attention towards their synthesis mechanisms. Many secondary metabolites are derived from sterol pathways, which are important in defense mechanisms against pathogens. SGTs in plants are involved in changed sensitivity to stress hormones and their agrochemical analogs and changed tolerance to biotic and abiotic stresses. SGTs that glycosylate steroidal hormones, such as brassinosteroids, function as growth and development regulators in plants. In terms of metabolic roles, it can be said that SGTs occupy important position in plant metabolism and may offer future tools for crop improvement.     

Variations in the condensing effect of cholesterol on saturated versus unsaturated phosphatidylcholines at low and high sterol concentration

In this work, we have investigated the condensing and ordering effect induced by cholesterol on phosphatidylcholines (PCs). To perform the studies systematically, for the experiments we have selected phospholipids differing only in the number of cis monounsaturated chains (1,2-distearoyl-sn-glycero-3-phosphocholine--DSPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine--SOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine--DOPC) or in the length (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine--POPC vs SOPC) of sn-1 acyl chain. Because the cholesterol concentration in mammalian membranes can be as high as 70 mol % of total lipids, the investigations were performed in a wide range of the sterol content. The results of the Langmuir monolayer experiments evidence that the relation between the structure of hydrophobic part of PC and the magnitude of the effects induced by cholesterol found at lower sterol content is different from that observed at higher sterol concentration. At a lower concentration of sterol (up to 30%), the condensing effect of cholesterol is stronger on saturated DSPC than on PCs containing monounsaturated chain(s), which is consistent with the conclusions drawn by other authors. However, at higher sterol content (≥50%), saturated DSPC is less susceptible to the influence of sterol than the investigated unsaturated PCs. To explain these irregularities, we have considered the strength of van der Waals interactions as well as the influence of sterol on the tilt of polar heads of PCs. It was also found that in the whole range of sterol concentration the ordering effect is stronger on saturated DSPC as compared to unsaturated phospholipids. However, at lower sterol content (up to 30%) the ordering effect induced on unsaturated PCs is rather weak, and the ordering does not change drastically in comparison with pure PCs film.     

Stereochemical Aspects of Acid-Catalyzed Cyclopropane Ring-Opening Reactions. A Stereospecific Pathway to Crinosterol and Brassicasterol

It is shown that the acid-catalyzed ring-opening of the two diastereoisomeric 23:24-methylenecholesterols 3 and 5 on treatment with gaseous HCl in acetic acid leads stereospecifically to the naturally occurring crinosterol ( 4 ) and brassicasterol ( 6 ), respectively (Scheme 1). This isomerization can be viewed as a biomimetic model of an in vivo methylation process of the type already known in plant sterol metabolism (cf. cycloeucalenol → obtusifoliol, 1 → 2 ). The synthetic application of this method provides a convenient labelling of sterol side chains for tracer experiments. The mechanistic features of the reaction with respect to its particular stereospecificity are discussed.     

Phase behavior of palmitic acid/cholesterol/cholesterol sulfate mixtures and properties of the derived liposomes

The phase behavior of mixtures formed by palmitic acid (PA), cholesterol (Chol), and sodium cholesteryl sulfate (Schol) has been characterized by differential scanning calorimetry and infrared and 2H NMR spectroscopy. It is reported that it is possible to form, with PA/sterol mixtures, fluid lamellar phases where the sterol content is very high (a sterol mole fraction of 0.7). As a consequence of the rigidifying ability of the sterols, the PA acyl chains are very ordered. The stability of these self-assembled bilayers is found to be pH-dependent. This property can be controlled by the Chol/Schol molar ratio, and it is proposed that this parameter modulates the balance between the intermolecular interactions between the constituting species. A phase-composition diagram summarizing the behavior of these mixtures as a function of pH, at room temperature, is presented. It is also shown that it is possible to produce large unilamellar vesicles (LUVs) from these mixtures, using standard extrusion techniques. The resulting LUVs display a very limited passive release of the entrapped material. In addition, these LUVs constitute a versatile vector for pH-triggered release.     

A ROLE FOR STEROLS IN THE PORPHYRIN MEDIATED PHOTOSENSITIZATION OF YEAST

The yeast Saccharomyces cerevisiae was used as a model system to determine the role of sterols in the porphyrin mediated photosensitization of yeast. A sterol auxotroph, RD5-R, was grown on sterols with different levels of unsaturation and assayed for photosensitivity in the presence of either protoporphyrin IX or hematoporphyrin (both at 100 micrograms/ml). Cells grown on the completely saturated sterol (stanol), cholestanol, were substantially more resistant to the photosensizing effects of the porphyrin. We hypothesize that this resistance arises from the inability of the porphyrin to mediate the oxidation of the membrane sterol. Our results indicate that photodegradation of the native yeast sterol, ergosterol, can account for substantial losses of cell viability.     

Determination of sterol and triterpene alcohol acetates in natural products by reversed-phase liquid chromatography and gas chromatography-mass spectrometry

A partial separation of nine sterol acetates and seven triterpene alcohol acetates by reversed-phase liquid chromatography is described. Good results are obtained using acetonitrile-water (90:10, v/v) as mobile phase with an UV detector at 205 nm. The variation in sterol sensitivity shows that this technique is not suitable for quantitative analyses. A combination of this technique for the fractionation of the natural sterol mixture, gas-liquid chromatography for quantitation and gas chromatography-mass spectrometry for identification is necessary for the determination of sterol compounds contained in natural products. An example of the separation, identification and quantitation of sterol acetates from sunflower seed oil is given.     

Solid-phase extraction-thin-layer chromatography-gas chromatography method for the detection of hazelnut oil in olive oils by determination of esterified sterols

The sterol composition of extra virgin olive oil is very characteristic and, thus, has become a helpful tool to detect adulterations with other vegetable oils. Special attention has been addressed to the separate determination of the free and esterified sterol fractions, since both have different compositions and can thus provide more precise information about the actual origin of the olive oil. In the case of admixtures with small amounts of hazelnut oil, this approach can be extremely useful, because the similarity between the fatty acid compositions of both oils hampers the detection of the fraud. A hyphenated chromatographic method was developed for a sensitive and precise determination of esterified sterols in olive oils. The oil was subjected to silica solid-phase extraction (SPE) fractionation, cold saponification of the collected fraction and purification on silica TLC. The sterol band was then injected into an SPB-5 (30 m x 0.25 mm I.D., 0.25 microM film thickness) and the ratio [% campesterol x (% 7-stigmastenol)2]/(% 7-avenasterol) was calculated. The method was tested on extra virgin olive oil; good sterol recoveries and repeatability were obtained. The results were compared with another method. which has a different sample preparation sequence (silica column chromatography, hot saponification and silica TLC). Similar results were achieved with both methods; however, the SPE-cold saponification-TLC-capillary GC was faster, required less solvent and prevented sterol decomposition. The SPE-method was applied to an admixture with 10% of hazelnut oil and to a screening of 11 oils (husk oil, virgin and refined olive oils) from different Mediterranean countries.     

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.     

Sterol composition of a cultured marine dinoflagellate,Heterocapsa circularisquama

The composition of sterols was determined in a cultured marine dinoflagellate Heterocapsa circularisquama. Ten kinds of the sterol in H. circularisquama were identified by capillary gas chromatography-mass spectrometry. The major sterol was a 4-methyl sterol, 4alpha,23,24-trimethyl-5alpha-cholest-22E-en-3beta-ol (dinosterol) which is the common sterol in many dinoflagellates.     

Selective sterol-phospholipid associations in fluid bilayers

The nearest-neighbor preferences of three exchangeable lipid monomers (two phospholipids that differ in length, A and B, and a derivative of cholesterol, C) have been quantified in the fluid bilayer state by use of the nearest-neighbor recognition method (Davidson, S. K. M.; Regen, S. L. Chem. Rev. 1997, 97, 1269). Thus, an analysis of the equilibrium dimer distributions has shown that (i) the sterol favors both phospholipids as nearest neighbors relative to other sterol molecules, (ii) that this recognition is selective (i.e., the sterol favors the longer phospholipid as a nearest neighbor over the shorter one, especially when the sterol concentrations in the bilayer is high (e.g., 40 mol %), and (iii) the phospholipids, themselves, are unable to recognize each other. Taken together, these findings indicate that the probable mechanism by which cholesterol induces homoassociation of A and B in analogous bilayers is one in which the sterol "pulls" two or more of the longer phospholipid monomers (B) out of a "sea" of randomly mixed A and B. These findings also lend support for the notion of cholesterol-phospholipid complexation in fluid bilayers. The biological implications of these findings are briefly discussed.     

Synthèse de l'hydroxy-3β-méthano-22,23-nor-24-cholestène-5 pour comparaison avec le cystostérol

Synthesis of 22,23-methano-24-nor-cholest-5-en-3β-ol and comparison with cystosterol 22,23-Methano-24-nor-cholest-5-en-3β-ol, a novel cyclopropane-containing sterol was synthesized from stigmasterol and found to be different from the isomeric, naturally occurring marine sterol cystosterol.     

Detection of unusual lipid mixing in cholesterol-rich phospholipid bilayers: the long and the short of it

Nearest-neighbor recognition studies have revealed that favored sterol-phospholipid associations can be reversed in a fluid bilayer that contains relatively long (high melting) and short (low melting) phospholipids, when the sterol content is sufficiently high; that is, like-lipids now become favored nearest-neighbors. A possible origin of this effect is briefly discussed.     

Peridinosterol - a new Δ17-unsaturated sterol from two cultured marine algae

X-ray diffraction analysis of peridinosterol p-bromobenzoate has shown the parent sterol to be E-4α,23R,24R-trimethylcholest-17(20)-en-3β-ol - a new member of the rare Δ¹⁷-unsaturated sterol class. Its possible biosynthetic origin is discussed.     

Tilt: major factor in sterols' ordering capability in membranes

Using extensive atomistic simulations, we show that there is a single experimentally accessible parameter--the sterol tilt--that can be used to determine a sterol's capability to induce order, and thus to promote, e.g., formation of lipid rafts. The observations also facilitate designing new effective sterols.