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PubMed2003

A functional cytochrome P450 lanosterol 14 alpha-demethylase CYP51 enzyme in the acrosome: transport through the Golgi and synthesis of meiosis-activating sterols.

Mammalian lanosterol 14 alpha-demethylase (CYP51) is a microsomal cytochrome P450 that demethylates lanosterol to FF-MAS, an oocyte meiosis-activating sterol and late intermediate of cholesterol biosynthesis. Herein we report CYP51 unequivocally localized to acrosomal membranes of male germ cells in mouse, bull, and ram, in which it synthesizes FF-MAS in the presence of the acrosomal form of nicotinamide adenine dinucleotide phosphate reduced-P450 reductase. In the mouse, CYP51 (53 kDa) resides in endoplasmic reticulum (ER) and Golgi during all phases of acrosome development, indicating an intracellular transport from ERs through the Golgi to the acrosome. CYP51 (50 kDa) also resides on acrosomal membranes of bull- and ram-ejaculated sperm. In mouse liver, a 53-kDa CYP51 is no longer detected in trans Golgi, suggesting retrieval back to the ER and no further transport to other organelles. Glycosylated high-molecular-mass CYP51-immunoreactive proteins in acrosomal membranes of bull and ram and Golgi-enriched fractions of mouse liver indicate that mammalian CYP51s are subjected to posttranslational modifications in the Golgi. In conclusion, CYP51 is the first cytochrome P450 enzyme to be detected on acrosomal membranes. It exhibits a unique, cell-type-specific intracellular transport that is in agreement with its cell-type-specific physiological role: production of cholesterol in the liver and sterols with signaling properties in sperm. Demethylation of lanosterol to FF-MAS by the acrosomal lanosterol 14 alpha-demethylase enzyme complex demonstrates for the first time the ability of ejaculate sperm to synthesize meiosis-activating sterols.

Acrosome↗
PubMed1992

Active site topology of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase (CYP51) and its G310D mutant (cytochrome P-450SG1).

Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.

Binding Sites↗
PubMed1988

A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity.

A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B resistance after treatment with the mutagen ethyl methanesulfonate. The mutant is a cholesterol auxotroph with a deficiency in cholesterol biosynthesis. Whole cell experiments demonstrate that the mutant accumulates the C30 sterols, lanosterol and dihydrolanosterol, under culture conditions which promote active sterol biosynthesis. Metabolic studies show that the C29 sterol demethylation product of lanosterol, but not lanosterol itself, is actively converted to end product cholesterol by whole cells as well as by microsomal preparations derived from the mutant. Detectable amounts of several cytochromes can be observed spectrally in the AR45 demonstrating that it is not a general heme-deficient mutant. Collectively, these results characterize the AR45 mutant cells as being lanosterol 14 alpha-methyl demethylase-deficient. The cell line should prove useful in studying regulation of the demethylase enzyme and the putative endogenous regulatory oxysterol. It should also be a useful tool in the molecular cloning and elucidation of genetic properties of the demethylase.

Amphotericin B↗
PubMed1981

Detection and quantification of lanosterol and dihydrolanosterol esters by measurements of metastable decompositions. Application to their direct analysis in wool wax.

The fatty acyl moiety in lanosterol and dihydrolanosterol esters was determined by measurement of the metastable transition [M--15]+ leads to [M--15-fatty acid]+ which occurs in the first field free region of a double focusing mass spectrometer. The analysis was made by accelerating voltage scanning. Quantification of the esters was made by single reaction monitoring, using calibration curves measured in the presence of the corresponding matrix. The detection limit for a selected lanosterol ester was below 300 pg. using this technique, each individual lanosterol and dihydrolanosterol ester was detected and quantified in a sample of wool wax. Interference from dihydroagnosterol esters is very likely, but interference from sterol esters which do not have a 4-methyl group is unlikely since the corresponding metastable peak is of very low intensity.

Animals↗
PubMed1995

Purification and characterization of a cytochrome P450 isozyme catalyzing lanosterol 14 alpha-demethylation (P45014DM) in hamster liver.

To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450 lanosterol 14 alpha-demethylase (P45014DM), which catalyzes the initial step in the biosynthesis of cholesterol from lanosterol, was purified and its mode of induction by microsomal enzyme inducers was characterized. P450450DM was purified from hamster livers by chromatography using aminooctyl-Sepharose CL-6B columns, to a specific content of 12.8 nmol/mg-protein. The purified protein displayed a single band on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 52,000. The absorption spectra of the oxidized form of the purified protein showed a Soret peak at 417 nm in a low-spin state and a Soret peak of reduced CO-binding complex at 448 nm. In a reconstituted system, the purified protein catalyzed 14 alpha-demethylation of 24,25-dihydrolanosterol (1.58 nmol/min/nmol-P450), although it did not show any activities toward testosterone and 7-ethoxyresorufin, marker substrates of other P450 families. Immunoblot analysis using an antibody against porcine P45014DM, which inhibited the activity of lanosterol 14-alpha-demethylation in the hamster liver microsomes, demonstrated that the level of this isozyme protein was markedly decreased in dexamethasone-treated hamster livers. This was accompanied by a decrease in the enzyme activity. In contrast, the levels and the activity in the phenobarbital- and 3-methylcholanthrene-treated hamsters were almost equal to that in the untreated animals.

Animals↗
PubMed1977

Evidence for the early reduction of the 24,25 double bond in the conversion of lanosterol to cholesterol in cerebrotendinous xanthomatosis.

The metabolism of lanosterol and 24,25-dihydrolanosterol (DL) was examined in a patient with cerebrotendinous xanthomatosis after intravenous pulse labeling with a mixture of DL-2-14C and 3S,4S,3R,4R-(4-3H)mevalonate. Sterols were isolated from the feces and purified by silver nitrate thin-layer chromatography, and their identities were confirmed by gas-liquid chromatography and mass spectrometry. Their specific activities were then determined and plotted as a function of time. These isotope ratio measurements and specific activity decay curves were consistent with 24,25-dihydrolanosterol and delta7-cholestenol being intermediates in the synthesis of cholesterol from mevalonate and lanosterol, and they suggested that reduction of the lanosterol side chain may occur as an early step in the synthesis of cholesterol. These results are in contrast to the results reported after the administration of triparanol, a delta24-reductase inhibitor.

Brain Diseases, Metabolic↗
PubMed2005

Insig-mediated degradation of HMG CoA reductase stimulated by lanosterol, an intermediate in the synthesis of cholesterol.

Feedback control of cholesterol synthesis is mediated in part by sterol-induced binding of HMG CoA reductase to Insig proteins in the endoplasmic reticulum (ER). Binding leads to ubiquitination and proteasomal degradation of reductase, a rate-controlling enzyme in cholesterol synthesis. Using in vitro and in vivo assays, we show that lanosterol, the first sterol intermediate in cholesterol synthesis, potently stimulates ubiquitination of reductase, whereas cholesterol has no effect at 10-fold higher concentrations. Lanosterol is not effective in mediating the other action of Insigs, namely to promote ER retention of SCAP-SREBP complexes, a reaction that is mediated directly by cholesterol. A pair of methyl groups located in the C4 position of lanosterol confers this differential response. These data indicate that buildup of cholesterol synthesis intermediates represses the pathway selectively at reductase and reveal a previously unappreciated link between feedback inhibition of reductase and carbon flow through the cholesterol synthetic pathway.

Cell Line, Transformed↗
PubMed2004

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.

Azoles↗
PubMed2004

Efficient routes to epimerically-pure side-chain derivatives of lanosterol.

A technically simple route is described to individual epimers of side-chain derivatives of lanosterol (3beta-hydroxy-5alpha-lanosta-8,24-diene). Epimerically pure 24,25-epoxy-, 24,25-dihydroxy- and 24-bromo-25-hydroxy-lanosterol have been prepared in good yield from commercial (50-60%) lanosterol. Hypophosphorous acid was used as a catalyst for the cohalogenation of the Delta24(25) bond and also for the efficient conversion of 24,25-epoxy- and 24-bromo-25-hydroxylanosterol to epimerically pure 24(R) or 24(S)-24,25-dihydroxylanosterols.

Acetylation↗
PubMed1986

Evidence for the contribution of a sterol 14-reductase to the 14 alpha-demethylation of lanosterol by yeast.

Lanosterol was converted to a 14-demethylated metabolite, 4,4-dimethylzymosterol by Saccharomyces cerevisiae microsomes. This metabolism was mediated by a cytochrome P-450 (P-450/14DM). However, a reconstituted system consisting of P-450/14DM and its reductase converted lanosterol to the 14-desaturated derivative of 4,4-dimethylzymosterol, 4,4-dimethyl-5 alpha-cholesta-8, 14,24-trien-3 beta-ol (trienol). When AY-9944 was added to the reaction system with the microsomes, the trienol was formed with corresponding decrease in 4,4-dimethylzymosterol. These observations indicate that the 14 alpha-demethylation of lanosterol by yeast microsomes occurs sequentially via the trienol. Reduction of the trienol to 4,4-dimethylzymosterol is mediated by an AY-9944-sensitive reductase.

Cyclohexanes↗
PubMed2001

Molecular dynamics simulation of the structure of dimyristoylphosphatidylcholine bilayers with cholesterol, ergosterol, and lanosterol.

Five molecular dynamics computer simulations were performed on different phospholipid:sterol membrane systems in order to study the influence of sterol structure on membrane properties. Three of these simulated bilayer systems were composed of a 1:8 sterol:phospholipid ratio, each of which employed one of the sterol molecules: cholesterol, ergosterol, and lanosterol. The two other simulations were of a bilayer with a 1:1 sterol:phospholipid ratio. These simulations employed cholesterol and lanosterol, respectively, as their sterol components. The observed differences in simulations with cholesterol and lanosterol may have their implication on the form of the phospholipid/sterol phase diagram.

Carbon↗
PubMed2002

From lanosterol to cholesterol: structural evolution and differential effects on lipid bilayers.

Cholesterol is an important molecular component of the plasma membranes of mammalian cells. Its precursor in the sterol biosynthetic pathway, lanosterol, has been argued by Konrad Bloch (Bloch, K. 1965. Science. 150:19-28; 1983. CRC Crit. Rev. Biochem. 14:47-92; 1994. Blonds in Venetian Paintings, the Nine-Banded Armadillo, and Other Essays in Biochemistry. Yale University Press, New Haven, CT.) to also be a precursor in the molecular evolution of cholesterol. We present a comparative study of the effects of cholesterol and lanosterol on molecular conformational order and phase equilibria of lipid-bilayer membranes. By using deuterium NMR spectroscopy on multilamellar lipid-sterol systems in combination with Monte Carlo simulations of microscopic models of lipid-sterol interactions, we demonstrate that the evolution in the molecular chemistry from lanosterol to cholesterol is manifested in the model lipid-sterol membranes by an increase in the ability of the sterols to promote and stabilize a particular membrane phase, the liquid-ordered phase, and to induce collective order in the acyl-chain conformations of lipid molecules. We also discuss the biological relevance of our results, in particular in the context of membrane domains and rafts.

Calorimetry, Differential Scanning↗
PubMed1991

Effect of cholesterol and lanosterol on the structure and dynamics of the cell membrane of Mycoplasma capricolum. Deuterium nuclear magnetic resonance study.

Deuterium nuclear magnetic resonance (NMR) techniques were employed to study the effect of sterols on the composition and dynamics of the membrane lipids of Mycoplasma capricolum, a natural fatty acid auxotroph that requires sterols for growth. The membrane lipids of cells grown in modified Edwards medium supplemented with cholesterol, oleic acid (OA), and palmitic acid (PA) were composed primarily of phosphatidylglycerol (PG) (60%) and cardiolipin (CL) (35%). The incorporation of cholesterol and the cellular OA/PA ratio increased nonlinearly with increases in exogenous cholesterol level, whereas the levels of phospholipid increased only slightly. At the growth temperature, 37 degrees C, the residual deuterium quadrupole splittings were found to be 43-46 kHz for cells grown with (7,7,8,8-2H4) PA and 1.25 micrograms/ml (30 mol%) to 10 micrograms/ml (50 mol%) cholesterol, respectively, similar to that found in the cholesterol/lecithin binary dispersions of similar cholesterol contents. Deuterium T2e of these samples were found to be 170 +/- 10 microseconds and were independent of cellular cholesterol content. In comparison, T2e of the corresponding lipid extracts were longer (320-420 microseconds) and dependent on cholesterol content. Thus, lipid-protein interactions in the cell membrane is the dominant mechanism responsible for the reduced T2e. At lower temperatures, spectra indicative of the coexistence of gel and liquid-crystalline states were observed for cells having low cholesterol levels. For both cell membrane and membrane lipid extract containing 50 mol% cholesterol, T2e was found to be constant at the temperature range from 15 to 40 degrees C. On the other hand, T2e of cell membrane containing 30 mol% cholesterol decreased linearly at 3.2 microseconds/degrees C. T2e of the corresponding lipid extract showed much stronger temperature variation. Cells containing 39 mol% lanosterol were found to have a quadrupole splitting of 39 kHz, broader than that of the cholesterol-free lecithin dispersion (less than 30 kHz) but less than that of cell membrane containing 30 mol% cholesterol (43 kHz). T2e of the lanosterol sample was found to be 130 +/- 10 microseconds which decreased linearly at a slope similar to that observed for the low cholesterol sample. Therefore, although lanosterol appeared to be capable of modulating cell membrane physical properties it is less effective than cholesterol. When growth rates were correlated with NMR parameters, we found that the membranes of faster growing cells were also more ordered. In contrast, the T2e of the cells of M. capricolum seemed to be maintained at a relatively constant value around 170 microseconds.

Biophysical Phenomena↗
PubMed1998

Induction of apoptosis in p53-null HL-60 cells by inhibition of lanosterol 14-alpha demethylase.

To determine the role of cholesterol deprivation in cell proliferation and, eventually, in apoptosis, HL-60 promyelocytic cells were incubated in a cholesterol-depleted medium in the presence of SKF 104976, a specific inhibitor of lanosterol 14-alpha demethylase. As expected, SKF 104976 efficiently blocked the [14C]-acetate incorporation into cholesterol, whereas it induced the accumulation of both lanosterol and, especially, dihydrolanosterol. As a consequence, cell proliferation was greatly depressed at 24 h of treatment with the drug, and clear signs of apoptosis--annexin V binding, condensed and fragmented nuclei and DNA ladder--were observed thereafter. Provided that the HL-60 cell line does not express p53, it may be concluded that apoptosis induced by cholesterol deprivation is not dependent on this tumor suppressor protein. Supplementing the incubation medium with LDL-cholesterol or pure free cholesterol, fully prevented cell growth inhibition and apoptosis induction, whereas mevalonate was ineffective. These results indicate that cholesterol plays a specific role in cell proliferation, a function that is not shared by its precursors lanosterol and dihydrolanosterol.

Apoptosis↗
PubMed1982

Further characterization of a Chinese hamster ovary cell mutant defective in lanosterol demethylation.

Sensitive in vitro lanosterol 14 alpha- and 4 alpha-methylsterol oxidase assays, particularly suitable for cell extracts of tissue culture cells, were developed and validated. Using these assays, we showed that the biochemical lesion of mutant 215, a cholesterol-requiring Chinese hamster ovary cell auxotroph isolated and partially characterized previously [Chang, T. Y., Telakowski, C., Vanden Heuvel, W., Alberts, A. W., & Vagelos, P. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 832-836], was localized at the 4 alpha-methylsterol oxidase enzyme system. The defect in 4 alpha-methylsterol oxidase activity in mutant 215 cells could be demonstrated by using either 4,4-dimethylcholestanol or 4 alpha-methylcholestanol as the substrate, suggesting that the enzyme systems responsible for 4 alpha-methyl- and 4,4-dimethylsterols may share a common component. However, demethylation of the C-14 alpha methyl group was found to occur at identical rates in wild-type and mutant 215, suggesting that C-14 alpha demethylation and C-4 alpha demethylation may occur by separate enzyme systems. A [3H]dihydrolanosterol incorporation experiment in intact cells of wild-type and mutant 215 supported these conclusions. Despite these results, a [14C]acetate pulse experiment indicated that [14C]lanosterol, instead of its 14C-labeled 14-demethylated sterol derivative(s), accumulated in intact cells of mutant 215. Possible implications of these findings for the mechanisms of lanosterol demethylation reactions are discussed.

Animals↗
PubMed1977

Yeast mutants blocked in removing the methyl group of lanosterol at C-14. Separation of sterols by high-pressure liquid chromatography.

Sterols of a nystatin resistant mutant of the wild type parent of Saccharomyces cerevisiae were separated by a newly developed procedure involving high-pressure liquid chromatography and were identified. The mutant contained larger amounts of squalene and lanosterol (I) than the wild type, as well as 4,14-dimethylcholesta-8,24-dien-3beta-ol (II), 4,14-dimethylergosta-8,24(28)-dien-3beta-ol (III), and 14-methylergosta-8,24(28)-dien-3beta-ol (IV), which were not hitherto found in yeast. These results indicated a block in removal of the methyl group at C-14 of lanosterol. An ergosterol requiring derivative of the mutant which carried in addition a mutation in heme biosynthesis had the same sterols as the parent, but at one-third the concentration. The low level of sterols may be due to a requirement for a heme or cytochrome in oxygenation reactions between lanosterol and ergosterol.

Chromatography, High Pressure Liquid↗
PubMed2002

Anisotropic motion and molecular dynamics of cholesterol, lanosterol, and ergosterol in lecithin bilayers studied by quasi-elastic neutron scattering.

Quasi-elastic neutron scattering (QENS) was employed to study the molecular dynamics of three structurally related sterols, namely, cholesterol, lanosterol, and ergosterol. Oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) were investigated at 40 mol % sterol content and at three temperatures (20, 36, and 50 degrees C) for two energy resolutions. Data analysis was concentrated on a direct comparison of the out-of-plane and the in-plane high-frequency motions of the three sterols in terms of their rates and amplitudes. The (spatially restricted) diffusive motion of the three sterols in the two directions was characterized by diffusion constants in the range of (5-30) x 10(-12) x m(2) x s(-1), with a significantly faster rate of diffusion along the membrane normal, resulting in a diffusional anisotropy, D(a). At low temperature (20 degrees C), cholesterol showed the highest value (D(a) = 4.5), while lanosterol gave the lowest one (D(a) = 2.0). At high temperature (50 degrees C), ergosterol diffusion had the highest diffusion anisotropy (D(a) = 2.0) compared to lanosterol (D(a) = 1.8) and cholesterol (D(a) = 1.6). Most interestingly, cholesterol showed at all three temperatures an amplitude of its out-of-plane-motion of 1.0-1.1 nm, more than a factor of 3 higher than measured for the other two sterols. This finding suggests that the short alkyl chain of the cholesterol molecule may cross at high frequency the bilayer midplane, while the other two sterols remain confined within the geometrical limits of each monolayer leaflet. The results provide an example of how slight structural alterations of sterols can affect their molecular dynamics in bilayers, which in turn may be relevant to the membrane micromechanical properties.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

A relationship between the activities of hepatic lanosterol 14 alpha-demethylase and 3-hydroxy-3-methylglutaryl-CoA reductase.

At 1-2 h after intragastric administration of ketoconazole, a cytochrome P-450 inhibitor, to rats, there was a 50-60% decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Inhibition reached a maximum at 6-12 h after the drug was given, but after 24 h enzyme activity was stimulated by 60%. The rates of synthesis of hepatic non-saponifiable lipids in vivo showed a similar time-dependent pattern of change. During the first few hours after drug administration, the hepatic cytochrome P-450-dependent metabolism of lanosterol was suppressed in vivo. However, 24 h after treatment, this activity was stimulated, an effect which was also observed by pre-treatment of the rats with the drug for several days. Suppression of hepatic HMG-CoA reductase and lanosterol 14 alpha-demethylase activities was accompanied by a relative increase in the accumulation of labelled polar sterols in the liver in vivo. In the intestine, ketoconazole also resulted in a rapid decline in the rate of synthesis of non-saponifiable lipids and an inhibition of lanosterol 14 alpha-demethylation in vivo. However, in contrast with the liver, there was no stimulation of non-saponifiable lipid synthesis after 24 h.

Animals↗