Lanosterol

About: Lanosterol is a research topic. Over the lifetime, 1239 publications have been published within this topic receiving 36737 citations. The topic is also known as: (3β)-lanosta-8,24-dien-3-ol & (3β,20R)-lanosta-8,24-dien-3-ol.

Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors

Abstract: Cytochrome P450 14α-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14α-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-Å crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences—a bent I helix and an open conformation of BC loop—that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species

Abstract: Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC50) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.

Candida albicans Mutations in the Ergosterol Biosynthetic Pathway and Resistance to Several Antifungal Agents

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.

Lanosterol reverses protein aggregation in cataracts

Abstract: The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people1, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase

Abstract: In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.