Modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by azole antimycotics requires lanosterol demethylation, but not 24,25-epoxylanosterol formation

The lanosterol 14 alpha-methyl demethylase inhibitors miconazole and ketoconazole have been used to assess their effects upon cholesterol biosynthesis in cultured Chinese hamster ovary cells. In Chinese hamster ovary cells treated with either agent, an initial accumulation of lanosterol and dihydrolanosterol has been observed. At elevated concentrations, however, ketoconazole, but not miconazole, causes the preferential accumulation of 24,25-epoxylanosterol and squalene 2,3:22,23-dioxide. These metabolites accumulate at the expense of lanosterol, thereby demonstrating a second site of inhibition for ketoconazole in the sterol biosynthetic pathway. Both demethylase inhibitors produced a biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. The biphasic modulation is characterized by low levels of the drugs suppressing HMG-CoA reductase activity which is restored to either control or above control values at higher drug concentrations. This modulatory effect of the lanosterol demethylase inhibitors upon HMG-CoA reductase was not observed in the lanosterol 14 alpha-methyl demethylase-deficient mutant AR45. Suppression of HMG-CoA reductase activity is shown to be due to a decrease in the amount of enzyme protein consistent with a steroidal regulatory mechanism. Collectively, the results establish that lanosterol 14 alpha-methyl demethylation, but not 24,25-epoxylanosterol formation, is required to suppress HMG-CoA reductase in the manner described by lanosterol demethylase inhibitors.     

In situ accumulation of 3 beta-hydroxylanost-8-en-32-aldehyde in hepatocyte cultures. A putative regulator of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity

Biphasic modulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) has been demonstrated in primary hepatocyte cultures treated with the lanosterol 14 alpha-methyl demethylase inhibitor miconazole. At concentrations of the drug which lead to suppressed levels of reductase activity, the appearance of a polar, mevalonate-derived sterol is noted. Cochromatography of the identified sterol with 3 beta-hydroxylanost-8-en-32-aldehyde tentatively identified the metabolite as a lanosterol 14 alpha-methyl demethylation intermediate. Subsequent isolation and characterization of the metabolite by gas chromatography/mass spectroscopy confirmed this structural assignment. When the lanosterol 14 alpha-methyl demethylase-deficient mutant, AR45, was treated with authentic metabolite, a suppression of HMG-CoA reductase was observed. These results demonstrate that metabolism of the oxygenated biosynthetic intermediate is not required to suppress reductase activity. The results also strongly support the hypothesis that oxygenated 14 alpha-methyl demethylase intermediates are endogenously generated modulators of HMG-CoA reductase activity.     

Non-human primate platelets and arterial tissue cannot convert preformed [14C]lanosterol into[14C]cholesterol in vivo.

In contrast with the prevailing view, we report the inability of non-human primate platelets or arterial tissue to complete the biosynthesis of [14C]cholesterol from [14C]mevalonic acid in vitro or in vivo, or from performed [14C]lanosterol in vivo. The latter observation suggests that these tissues lack one or more components of the methyl sterol demethylase system.     

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.     

Effects of lysine deprivation and serum stimulation on the synthesis of non-histone chromosomal proteins by confluent chick fibroblasts

Cellular sterol content and sterol metabolism were studied in diploid human kidney cells in early passages in culture. Cholesterol, the main cellular sterol, was present at levels similar to those reported for other cultured mammalian kidneys. Cholesterol biosynthesis was characterized by a slow conversion of sterol precursors with accumulation of lanosterol and 27 carbon-atom sterols. In the absence of exogenous cholesterol, kidney cells grew slowly and intracellular cholesterol decreased; however, sterol formation from labelled acetate was stimulated. These results suggest that the cholesterol concentration in the culture medium influences the rate of sterol formation by the kidney cell. Furthermore, cholesterol appears to be essential to cultured human kidney and de novo synthesis by the cells in culture is not adequate to meet their requirements for growth.     

Low concentrations of the non-ionic detergent Nonidet P-40 interfere with sterol biogenesis and viability of the yeast Saccharomyces cerevisiae

Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1-1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol. Increased levels of sterol precursors (squalene, lanosterol) in hem1 cells grown in complex medium with cholesterol indicated general interference of NP-40 with sterol biosynthesis. NP-40 in the growth medium affected also cell viability estimated as the colony forming ability. More attention should be therefore paid to possible effects of mild detergents at low concentrations generally considered to be harmless, especially in cells with disturbed lipid biogenesis.     

Complete replacement of membrane cholesterol with 4,4',14-trimethyl sterols in a human T cell line defective in lanosterol demethylation.

A3.01 is a hypoxanthine/aminopterin/thymidine-sensitive, human immunodeficiency virus-susceptible, human T cell line derived by Folks et al. (Folks, T., Benn, S., Rabson, A., Theodore, T., Hoggan, M. D., Martin, M., Lightfoote, M., and Sell, K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4539-4543) following exposure of CEM cells to 8-azaguanine. In the present study, it is shown that A3.01 also contains a heretofore unrecognized mutation in cholesterol biosynthesis. A3.01 cells grown in the presence of 10% fetal bovine serum (FBS) contain primarily cholesterol in their membranes, but based on [14C]acetate labeling, synthesize only lanosterol and 24,25-dihydrolanosterol. Reduction in the amount of FBS provided resulted in decreased cellular levels of cholesterol with corresponding increases in the two 4,4',14-trimethyl sterols. In A3.01 cells cultured in 1% FBS medium, lanosterol and 24,25-dihydrolanosterol accounted for 7 and 45%, respectively, of total cellular sterols. Following dilution of the 1% FBS-grown cells into serum-free media, the level of membrane cholesterol gradually declined, such that after three passages it became virtually undetectable, whereas the proportions of lanosterol and 24,25-dihydrolanosterol rose to 25 and 75%, respectively. Even after eight passages in the serum-free media, A3.01 cells displayed a complete absence of cholesterol with no obvious effect on cell growth. Membranes isolated from A3.01 cells grown in the presence or absence of 10 micrograms/ml of cholesterol displayed similar phospholipid:sterol ratios, but membranes from the unsupplemented cells contained only approximately 5% as much cholesterol as the supplemented cell membranes. Finally, A3.01 cells grown in the absence of cholesterol were extremely resistant to the cytotoxic effects of amphotericin B, whereas cells cultured in the combined presence of 1% FCS and 10 micrograms/ml of cholesterol were sensitive to the drug. Collectively, these results demonstrate that 4,4',14-trimethyl sterols can effectively replace cholesterol in a human T cell lineage, indicating that not all mammalian cells have a requirement for cholesterol, per se. The A3.01 T cell lineage should prove useful in defining the role of cholesterol in membrane fusion and human immunodeficiency virus-mediated syncitia formation and cytopathic effects.     

The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from [2-14C]mevalonic acid in vitro

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-(14)C]mevalonic acid under gas phases consisting of either N(2)+O(2) (90:10) or CO+O(2) (90:10). CO inhibits cholesterol biosynthesis from [2-(14)C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N(2)+O(2) as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.-mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [(14)C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [(14)C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Delta(24)-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.     

Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3-oxidosqualene.

Mutants of Saccharomyces cerevisiae were isolated which were blocked in heme biosynthesis and required heme for growth on a nonfermentable carbon source. They were rho+, and grew fermentatively on ergosterol or cholesterol and Tween 80, as a source of oleic acid. Cells grown on ergosterol and Tween 80 lacked cytochromes and catalase which were restored by growth on heme. The mutants comprised five nonoverlapping complementation groups. Tetrad analysis showed that the pleiotropic properties of each of the mutants resulted from a single mutation in one of five unlinked loci (hem1 to hem5) affecting heme biosynthesis. Biochemical studies confirmed that each mutation resulted in loss of a single enzyme activity. hem1 mutants grew on delta-aminolevulinate and lacked delta-aminolevulinate synthase activity, hem2 mutants lacked delta-aminolevulinate dehydratase, and hem3 mutants uroporphyrin I synthase. Mutants in hem1, hem2, and hem3 had an additional requirement for methionine on synthetic medium supplemented with either heme or ergosterol and Tween 80, owing to a lack of sulfite reductase which contains siroheme, a modified uroporphyrin III. Since hem4 and hem5 mutants have sulfite reductase activity under all growth conditions, they are blocked after uroporphyrin III. Cell extracts of a hem4 mutant incubated with delta-aminolevulinate accumulated coproporphyrin III suggesting a block in coproporphyrinogenase, the enzyme which converts coproporphyrinogen III to protoporphyrinogen. Cells and extracts of a hem5 mutant accumulated protoporphyrin IX. Since it was the only mutant that grew on heme but not on protoporphyrin IX, a block in ferrochelatase was suggested for this strain. Mutant strains grown on heme had the sterol composition of wild type cells, whereas without heme only squalene, small amounts of lanosterol, and added sterol was observed. A heme product therefore participates in the transformation of lanosterol to ergosterol. A hem3 mutant was isolated which was also blocked between 2,3-oxidosqualene and lanosterol (erg12). When grown on lanosterol or ergosterol (with Tween 80) it accumulated a compound which was identified as 2,3-oxidosqualene by comparison with the synthetic compound in thin layer and gas-liquid chromatography, and by proton magnetic resonance and mass spectroscopy. Supplementation with heme did not remove the requirement for sterol, but it enabled the mutant to convert lanosterol to ergosterol.     

Speculations on the evolution of sterol structure and function.

The essential oxygen requirement for sterol biosynthesis dates this molecule as a relative latecomer in cellular evolution. Structural details of the cholesterol molecule and related sterols can be rationalized in terms of optimal hydrophobic interactions between the planar sterol ring system and phospholipid acyl chains in the membrane bilayer. The prediction that the cholesterol precursor lanosterol (4,4',14 trimethyl cholastadienol) is incompetent for membrane function is verified by in vivo experiments with eucaryotic sterol auxotrophs and microviscosity measurements of sterol-containing artificial membranes. For procaryotic cells the sterol specificity is very much broader. Methylococcus capsulatus produces 4,4-dimethyl- and 4-monomethyl sterols, but not sterols of the cholesterol type. Similarly lanosterol and its partially demethylated derivatives satisfy the sterol requirement of Mycoplasma capricolum. A more primitive but unspecified role of cyclized squalene derivatives is therefore postulated for procaryotic membranes. The finding that cholesterylmethyl ether satisfies the sterol requirement of certain microbial systems is at variance with current views on the role played by the sterol hydroxyl group in membrane organization and function.     

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.     

Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.     

Inhibition of cholesterol biosynthesis in ovine ovarian follicles in vitro by human chorionic gonadotrophin

Cholesterol biosynthesis from DL-[2-14C]mevalonic acid ([14C]MVA) was demonstrated in ovine ovarian follicles and isolated thecal tissues and granulosal cells incubated in vitro. Thecal tissues more readily synthesized cholesterol than did granulosal cells when incubated separately, but in the intact follicle the newly synthesized cholesterol distributed evenly between the two tissue layers, indicating that the theca could act as a supplementary source of cholesterol for the granulosal cells. Human chorionic gonadotrophin (hCG) added to the incubation medium was found to inhibit cholesterol biosynthesis from [14C]MVA by intact follicles and isolated thecal tissues, but not granulosal cells. This hCG-induced inhibition was evident in whole follicles incubated for 12--48 h, but not at 3--6 h, and was demonstrated in thecal tissues incubated for 3 h. In all cases where inhibition of cholesterol biosynthesis was observed, 14C label accumulated in a product characterized by thin layer and vapour phase chromatography as lanosterol, implying that the hCG block lies between lanosterol and cholesterol. Treatment of follicles with hCG also reduced the amount of 14C label incorporated into the cholesteryl ester fraction. These changes were accompanied by a corresponding reduction in the tissue content of cholesteryl ester, but there were no changes in the specific activities to indicate that newly synthesized cholesteryl ester was used selectively as a substrate for progestin biosynthesis.     

Effectors of rapid homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoA reductase

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.     

Isolation of NPC1-deficient Chinese hamster ovary cell mutants by gene trap mutagenesis

Chinese hamster ovary cell mutants defective in the NPC1 gene (NPC1-trap) were generated by retrovirus-mediated gene trap mutagenesis from a parental cell line JP17 expressing an ecotropic retrovirus receptor. Insertion of the gene trap vector in the NPC1 gene and the absence of the gene product were verified by 5'RACE and immunological analyses, respectively. NPC1-trap cells showed intracellular accumulation of low-density lipoprotein (LDL)-derived cholesterol and had an increased level of unesterified cellular cholesterol. Cholesterol biosynthesis through the mevalonate pathway was upregulated in the mutant cells as assessed by [(14)C]acetate incorporation into cellular sterols. When JP17 cells were depleted of lipoproteins and then loaded with LDL, cell surface LDL receptors were promptly downregulated and the mature form of the sterol regulatory element-binding protein-1 disappeared from the nucleus. These responses to LDL were obviously retarded in NPC1-trap cells, suggesting an impaired response of the cholesterol-regulatory system to LDL. NPC1-trap cells will be a useful tool to study the regulation of cellular cholesterol homeostasis and the pathogenesis of Niemann-Pick disease type C.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

Effect of estradiol and antiestrogens on cholesterol biosynthesis in hormone-dependent and -independent breast cancer cell lines

The effects of estradiol and/or antiestrogens on cholesterol biosynthesis were studied in two breast cancer cell lines. Cholesterogenic activity was evaluated after labeling cells with sodium [14C]acetate for increasing periods of time (up to 24 h) and measuring the incorporation of the radioactivity into nonsaponifiable lipids and into cholesterol, after separation from other labeled metabolites. We compared the effects of estradiol on cholesterogenesis with the well-known effects of this hormone on cell proliferation: estradiol stimulated both cholesterol synthesis and cell growth in MCF-7 cells, but stimulated neither in BT20 cells. The stimulation affected both the 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase step and the post-HMGCoA steps. Only the key enzyme step appeared to be mediated by the estrogen receptor. The hydroxytamoxifen and LY 117018 antiestrogens strongly inhibited cellular cholesterol production in both cell lines. Under the same conditions, cell growth is affected in MCF-7 cells, but not in BT20 (as shown by groups from other laboratories). This demonstrates that de novo synthesis of cholesterol is not essential for cell growth when cells are cultured in the presence of whole serum. The inhibition of cholesterol synthesis by antiestrogens mainly affected the lanosterol demethylation step and the C-27 sterol to cholesterol conversion. This inhibiting effect of antiestrogens was not mediated by the estrogen receptor.