Showing papers on "Lanosterol published in 2003"
TL;DR: These studies showed that erg11/erg11 mutants of a C. albicans strain harboring a defective erg11 allele can be obtained in vitro in the presence of amphotericin B, and could therefore be selected by similar mechanisms during antifungal therapy.
Abstract: The role of sterol mutations in the resistance of Candida albicans to antifungal agents has not been thoroughly investigated. Previous work reported that clinical C. albicans strains resistant to both azole antifungals and amphotericin B were defective in ERG3, a gene encoding sterol Δ5,6-desaturase. It is also believed that a deletion of the lanosterol 14α-demethylase gene, ERG11, is possible only under aerobic conditions when ERG3 is not functional. We tested these hypotheses by creating mutants by targeted deletion of the ERG3 and ERG11 genes and subjecting those mutants to antifungal susceptibility testing and sterol analysis. The homozygous erg3/erg3 mutant created, DSY1751, was resistant to azole derivatives, as expected. This mutant was, however, slightly more susceptible to amphotericin B than the parent wild type. It was possible to generate erg11/erg11 mutants in the DSY1751 background but also, surprisingly, in the background of a wild-type isolate with functional ERG3 alleles under aerobic conditions. This mutant (DSY1769) was obtained by exposure of an ERG11/erg11 heterozygous strain in a medium containing 10 μg of amphotericin B per ml. Amphotericin B-resistant strains were obtained only from ERG11/erg11 heterozygotes at a frequency of approximately 5 × 10−5 to 7 × 10−5, which was consistent with mitotic recombination between the first disrupted erg11 allele and the other remaining functional ERG11 allele. DSY1769 was also resistant to azole derivatives. The main sterol fraction in DSY1769 contained lanosterol and eburicol. These studies showed that erg11/erg11 mutants of a C. albicans strain harboring a defective erg11 allele can be obtained in vitro in the presence of amphotericin B. Amphotericin B-resistant strains could therefore be selected by similar mechanisms during antifungal therapy.
343 citations
TL;DR: The identification ofFXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.
Abstract: Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRβ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRβ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRα and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRβ. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.
171 citations
TL;DR: Analysis of the 14-α lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S in Cryptococcus neoformans.
Abstract: Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-α lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.
124 citations
TL;DR: The incorporation of the different sterols into the DPPC bilayer enhances the orientational order of TMA-DPH in the liquid-crystalline state of DPPC, whereas the orientation of the plant sterols is changed.
Abstract: Cholesterol, its precursor lanosterol, the plant sterols β-sitosterol and ergosterol, as well as three further sterols were added up to 50 mol % to vesicles of the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC). The aim of this study was to investigate the influence of the sterol side chain and ring structure on the acyl chain orientational order of the lipid bilayer by measuring the steady-state fluorescence anisotropy rss of the fluorophore 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and to establish the molecular basis underlying the changes in order parameter of the lipid bilayer system. Experiments were carried out in the temperature range from 30 to 60 °C, i.e., at temperatures below and above the gel to liquid-crystalline phase transition temperature Tm of DPPC bilayers. In general, the incorporation of the different sterols into the DPPC bilayer enhances the orientational order of TMA-DPH in the liquid-crystalline state of DPPC, whereas the orientationa...
123 citations
TL;DR: Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain.
Abstract: Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain. This strain had previously been deregulated by overexpressing a truncated HMG-CoA reductase (tHMG1) in the main bottleneck of the early ergosterol pathway. The overexpression of the gene ERG1 (squalene epoxidase) induced a significant decrease of the direct substrate squalene, a high increase of lanosterol, and a small increase of later sterols. The overexpression of the ERG11 gene encoding the sterol-14α-demethylase resulted in a decrease of lanosterol and an increase of downstream sterols. When these two genes were simultaneously overexpressed, later sterols from zymosterol to ergosterol accumulated and the content of squalene was decreased about three-fold, indicating that these steps had limited the transformation of squalene into sterols. The total sterol content in this strain was three-fold higher than in a wild-type strain.
114 citations
TL;DR: The studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s.
Abstract: The active site of lanosterol 14α-demethylase (CYP51) was investigated via MCSS functional group mapping and LUDI calculations. Several non-azole lead molecules were obtained by coupling structure-based de novo design with chemical synthesis and biological evaluation. All of the lead molecules exhibited a strong inhibitory effect on CYP51 of Candida albicans. They occupy the substrate-binding site and interfere with the binding of azole antifungal agents in a competitive manner. The mode of action of the lead molecules was validated by spectrophotomeric analysis and SAR studies. This is the first successful example reported for the inhibitor design of the cytochrome P450 superfamily using the de novo design strategy. Because the affinity of the lead molecules for CYP51 was mainly attributed to their nonbonding interaction with the apoprotein, the studies presented here afford the opportunity to develop novel antifungal agents that specifically interact with the residues in the active site and avoid the se...
100 citations
TL;DR: Increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR- and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis.
Abstract: Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux. In this study, we tested the novel hypothesis that stimulation of endogenous oxysterol synthesis would block foam cell formation induced by atherogenic lipoproteins. Macrophage synthesis of 24(S),25-epoxycholesterol, a potent LXR ligand, increased 60-fold by partial inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), a microsomal enzyme in both the cholesterol biosynthetic pathway and the alternative oxysterol synthetic pathway. When macrophages were challenged with human hypertriglyceridemic VLDL (HTG-VLDL), cellular cholesteryl ester accumulation increased 12-fold. This was reduced dramatically, by 65%, after preincubation with an OSC inhibitor (OSCi). The HTG-VLDL-induced accumulation of macrophage TG (70-fold) was unaffected by the OSCi or exogenous 24(S),25-epoxycholesterol, an effect associated with suppression of SREBP-1 processing. By contrast, TO901317, a synthetic LXR agonist, increased cellular TG significantly and markedly increased SREBP-1 processing. OSC inhibition decreased HTG-VLDL uptake through downregulation of LDL-receptor expression, despite substantial inhibition of cholesterol synthesis. Furthermore, OSC inhibition significantly upregulated ABCA1 and ABCG1 expression, which led to enhanced macrophage cholesterol efflux, an effect mediated through LXR activation. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR- and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis.
98 citations
TL;DR: While sterol regulatory element binding protein (SREBP)-dependent transcriptional regulation of CYP51 contributes to synthesis of cholesterol, the germ-cell-specific cAMP/CREMtau-dependent upregulation might contribute to increased production of MAS.
95 citations
TL;DR: In this article, the authors investigated if a causal relation exists between serum concentrations of precursors and metabolites of cholesterol and cognitive performance in a healthy aging population and found that the levels of lathosterol and lanosterol at baseline correlated with performance on the Stroop test and Word Learning tests over the 6-year follow-up period.
84 citations
TL;DR: The first mammalian cholesterol biosynthetic enzyme unequivocally localized at the surface of intracellular lipid storage droplets is reported, adding to the growing notion that the lipid droplet is an organelle endowed with more complex roles in various biological phenomena.
81 citations
TL;DR: The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation.
TL;DR: Evidence is presented that Erg27p interacts with Erg7p, facilitating the association of Erg6p with lipid particles (LPs) and preventing digestion of ErG7p both in the endoplasmic reticulum (ER) and LPs, and it is demonstrated that ErG27p is required for oxidosqualene cyclase (Erg 7p) activity in LPs.
TL;DR: Ab initio calculations were performed on a cyclohexane derivative to elucidate the mechanism of the formation of the five-membered C ring in the biosynthesis of lanosterol from squalene and indicated that the conformer that should give rise to the cyclized C ring is not a minimum on the potential surface.
TL;DR: Given the ability of alpha-tocopherol to down-regulate the expression of two scavenger lipoprotein receptors, SR-A and CD36, these observations raise some interesting questions regarding the role of SPF/TAP and vitamin E in cholesterol metabolism.
Abstract: Supernatant protein factor (SPF) is a recently cloned member of a family of cytosolic lipid-binding proteins that includes Sec14p, α-tocopherol transfer protein, and cellular retinal-binding protein. SPF stimulates the conversion of squalene to lanosterol in the downstream pathway for cholesterol biosynthesis, and overexpression of cloned SPF in hepatoma cells increases cholesterol synthesis. The mechanism of this stimulation has yet to be defined, but SPF appears to facilitate the transfer of squalene into and between intracellular membranes. The recent identification of SPF as α-tocopherol-associated protein (TAP) has called into question its long-standing association with cholesterol biosynthesis. TAP binds α-tocopherol, but not other isomers of tocopherol, with high affinity; in the presence of α-tocopherol TAP translocates to the nucleus and activates reporter gene transcription. Given the ability of α-tocopherol to down-regulate the expression of two scavenger lipoprotein receptors, SR-A and CD36, these observations raise some interesting questions regarding the role of SPF/TAP and vitamin E in cholesterol metabolism.
TL;DR: It is reported that sterol intermediates in cholesterol synthesis, beginning with the initial post-cyclization sterol, lanosterol, continuing with zy mosterol, and ending with desmosterol are also substrates for CYP27A1, and the retention times and major mass fragments of these novel metabolites are characterized.
TL;DR: A new lanostane triterpenoid with a cyano-enone functionality in ring A was synthesized in two steps from the corresponding [2,3-d]isoxazole, which is interesting from the perspective of biological activity because lanosterol is the biogenetic precursor of steroids.
Abstract: It was previously reported that methyl oleanonate (5) and lanost-8-en-3-one (10) give predominantly [3,2-c]isoxazoles. On the contrary, we have confirmed that both compounds 5 and 10 do not give [3,2-c]isoxazoles but rather afford regioselectively [2,3-d]isoxazoles in good yields. Consequently, a new lanostane triterpenoid with a cyano-enone functionality in ring A was synthesized in two steps from the corresponding [2,3-d]isoxazole, which is interesting from the perspective of biological activity because lanosterol is the biogenetic precursor of steroids.
TL;DR: RT-PCR results show that three SREBP2 immunoreactive proteins (72, 63 and 55kDa), that are not present in mouse liver nuclei, reside in testis nuclei of prepubertal and adult mice and seem to be insensitive to the level of cholesterol.
TL;DR: Pneumocystis carinii causes severe pneumonia in immunocompromised patients and the susceptibility of ERG11 deletion strains of Saccharomyces cerevisiae expressing PCERG11, PCERG 11-SDM, and wild-type SCERG11 to three azole antifungals is tested.
Abstract: Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised patients. PC is intrinsically resistant to treatment with azole antifungal medications. The enzyme lanosterol 14α-demethylase (Erg11) is the target for azole antifungals. We cloned PCERG11 and compared its sequence to Erg11 proteins present in azole-resistant organisms, and performed chromosomal and Northern blot analysis for PCERG11. Of 13 potential sites which could confer resistance to azoles, two were identical to azole-resistant Candida. By site-directed mutagenesis we changed these two sites in PCERG11 to those present in azole-sensitive Candida to generate PCERG11-SDM (E113D, T125K). We tested the susceptibility of ERG11 deletion strains of Saccharomyces cerevisiae (SC) expressing PCERG11, PCERG11-SDM, and wild-type SCERG11 to three azole antifungals: fluconazole, itraconazole, and voriconazole. PCERG11 required a 2.2-fold higher dose of voriconazole and 3.5-fold higher dose of fluconazole than SCERG11 for a 50% reduction in gro...
TL;DR: The T. cruzi lanosterol 14α-demethylase (Tc14DM) gene was cloned by degenerate PCR and found to be expressed in both insect and mammalian life-cycle stages of the parasite.
TL;DR: In this article, the authors performed density functional calculations on a model system of squalene oxide to study the mechanism of the formation of ring A in the biosynthesis of lanosterol.
Abstract: Density functional calculations were performed on a model system of squalene oxide to study the mechanism of the formation of ring A in the biosynthesis of lanosterol from squalene. When (2 Z )-6,7-epoxy-3,7-dimethyloct-2-ene was protonated, it was calculated to undergo a very facile ring opening of the oxirane in concert with the formation of the six-membered ring of the 4-(hydroxymethyl)-1,2,3,3-tetramethy1cyclohexyl cation. A study of the reaction pathway (IRC) indicates a very early transition structure in which the carbon- carbon double bond participates anchimerically in the ring-opening of the protonated oxirane. It is suggested that the primary role of the enzyme in this first step of the biosynthesis of lanosterol is protonation of the oxirane ring along with holding the substrate in the proper conformation for the concerted ring-closure to occur. The similarity between this mechanism and that recently proposed for concerted C-ring expansion and D-ring formation in the biosynthesis of lanosterol is discussed.
Patent•
22 Jan 2003
TL;DR: In this article, a method for the production of zymosterol, the biosynthetic intermediate or subsequent products thereof by cultivation of organisms, in particular yeasts, which have an increased lanosterol C14-demethylase activity and an increased HMG-CoA reductase activity, was described.
Abstract: The invention relates to a method for the production of zymosterol the biosynthetic intermediate or subsequent products thereof by cultivation of organisms, in particular yeasts, which have an increased lanosterol C14-demethylase activity and an increased HMG-CoA reductase activity, the nucleic acid constructs necessary for the production of the genetically-modified organisms and the genetically modified organisms, in particular the yeasts themselves.
Journal Article•
Patent•
07 Mar 2003
TL;DR: In this article, a novel nuclear receptor called "npFXRB" or also npFXR-β a homologue of the NPFXRα, a prototypical type 2 nuclear receptor is described.
Abstract: The present invention relates to a novel nuclear receptor called 'npFXRB' or also npFXR-β a homologue of the npFXR-α, a prototypical type 2 nuclear receptor. The invention also relates to the isolated nucleic acid sequence of npFXRB and the isolated protein thereof. The invention further relates to processes for isolating and/or producing the nucleic acid or the protein as well as methods of use of the receptor npFXRB.