Showing papers on "Lanosterol published in 1998"

Beta-amyrin synthase--cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants.

Abstract: β-amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)-2,3-oxidosqualene. The enzyme, β-amyrin synthase, catalyzing the cyclization of oxidosqualene into β-amyrin, generates five rings and eight asymmetric centers in a single transformation. A homology-based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces oleanane saponins together with dammarane-type saponins. Two sets of degenerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCRs using these primers successfully amplified the core fragment which revealed the presence of two OSC clones PNX and PNY. Specific amplification of each clone by 3′-RACE and 5′-RACE was carried out to obtain the whole sequences. The two clones exhibited 60 % amino acid identity to each other. A full-length clone of PNY was ligated into the yeast expression vector pYES2 under the GAL1 promoter to give pOSCPNY . β-amyrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSCPNY contains an open reading frame of 2289 nucleotides which codes for 763 amino acids with a predicted molecular mass of 88 kDa. Sequence comparison with other OSCs showed a high level of similarity with lanosterol, cycloartenol and lupeol synthases. The other clone, pOSCPNX, was shown to be cycloartenol synthase by similar expression in yeast. The present studies have revealed that distinct OSC exists for triterpene formation in higher plants, and the high level of similarity with cycloartenol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis.

Elevated expression of lanosterol 14alpha-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats.

Abstract: Mammalian CYP51 encodes lanosterol 14alpha-demethylase (P45014DM) that is involved in the postsqualene part of cholesterol biosynthesis. This enzyme removes the 14alpha-methyl group from lanosterol and 24,25-dihydrolanosterol producing intermediates in cholesterol biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412. Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all tissues, with highest levels in the testis due to the presence of an additional shorter CYP51 transcript in this tissue. In situ hybridization shows the highest CYP51 mRNA levels in seminiferous tubules, with only background levels in Leydig cells. The rat testis-specific CYP51 mRNA arises from the use of an upstream polyadenylation site and is restricted to germ cells, being most abundant in elongating spermatids in stages VII-XIV, whereas somatic CYP51 transcripts are present in all cells. In contrast, the mRNA levels of squalene synthase are maximal in round spermatids, and no germ cell-specific transcript is observed. The rat male germ cell-specific CYP51 transcript is translated in vitro to two proteins of approximately 55 and 53.5 kDa. CYP51 activity is higher in protein extracts of testes and germ cells of sexually mature rats than in prepubertal animals, in which postmeiotic germ cells are not yet present. This shows increased capacity for the production of MAS sterols by male germ cells that have already completed meiosis, suggesting that they serve a role different from meiosis activation.

Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes

Abstract: The objective of this study was to investigate the mechanisms by which tamoxifen modifies cholesterol metabolism in cellular models of liver metabolism, HepG2 cells and rat hepatocytes. The effect of tamoxifen on cholesterol and triglyceride-palmitate synthesis was measured using isotopomer spectral analysis (ISA) and gas chromatography-mass spectrometry (GC-MS) and compared with the effects of progesterone, estradiol, the antiestrogen ICI 182,780, and an oxysterol, 25-hydroxycholesterol (25OHC). Cholesterol synthesis in cells incubated in the presence of either [1-(13)C]acetate, [U-13C]glucose, or [4,5-(13)C]mevalonate for 48 hours was reduced in the presence of 10 micromol/L tamoxifen and 12.4 micromol/L 25OHC in both HepG2 cells and rat hepatocytes. The ISA methodology allowed a clear distinction between effects on synthesis and effects on precursor enrichment, and indicated that these compounds did not affect enrichment of the precursors of squalene. Progesterone was effective in both cell types at 30 micromol/L and only in HepG2 cells at 10 micromol/L. Estradiol and ICI 182,780 at 10 micromol/L did not inhibit cholesterol synthesis. None of the compounds altered the synthesis of triglyceride-palmitate in either cell type. Treatment of cells with tamoxifen produced accumulation of three sterol precursors of cholesterol, zymosterol, desmosterol, and delta8 cholesterol. This pattern of precursors indicates inhibition of delta24,25 reduction in addition to the previously described inhibition of delta8 isomerase. We conclude that tamoxifen is an effective inhibitor of the conversion of lanosterol to cholesterol in cellular models at concentrations comparable to those present in the plasma of tamoxifen-treated individuals. Our findings indicate that this mechanism may contribute to the effect of tamoxifen in reducing plasma cholesterol in humans.

Lanosterol 14alpha-demethylase (CYP51) and spermatogenesis.

Abstract: CYP51 is the only gene of the cytochrome P450 (P450, or CYP) superfamily that is expressed in prokaryotes and eukaryotes. In animals, the gene product, P45014DM, catalyzes the lanosterol 14α-demethylase reaction, an essential step in cholesterol biosynthesis. P45014DM serves a housekeeping role, and it was surprising to find the highest level of CYP51 expression in the testes. This is a result of very high-level CYP51 expression in postmeiotic, haploid spermatids and results in elevated P45014DM activity in these cells. It is proposed that the elevated level of 14α-demethylase activity leads to production of signaling sterols by haploid germ cells, although the function of such sterols in males is unknown.

Cholesterol biosynthesis from lanosterol: differential inhibition of sterol delta 8-isomerase and other lanosterol-converting enzymes by tamoxifen.

Abstract: The fact that administration of tamoxifen (Tam) to humans and laboratory animals (e.g., rats and monkeys) results in both a drastic reduction in cholesterol and a marked accumulation of certain sterol intermediates in their serum led us to undertake more direct biochemical studies on the mechanism of Tam's inhibitory action on the cholesterogenic enzymes. Of the five rat hepatic lanosterol-converting enzymes examined, the enzyme most sensitive to inhibition by Tam was sterol delta 8-isomerase (delta 8-SI) (a 208-fold inhibition relative to lanosterol 14 alpha-methyl demethylase), followed by sterol delta 24-reductase (13-fold) and sterol delta 14-reductase (5.2-fold). The inhibition patterns of all four affected enzymes were found to be noncompetitive, despite widely different inhibition constants (Ki) of 0.21 to 23.5 microM. The inhibitory activity of Tam on delta 8-SI was not affected by detergent-mediated solubilization of the microsomes. In Chinese hamster ovary cells, inhibition of delta 8-SI activity (IC50 = 0.15 microM) was paralleled by a decreased rate of [14C]-mevalonate incorporation into cholesterol (IC50 = 0.70 microM). Our results should provide more insight into an underlying mechanism of Tam's cardioprotective role by interfering the operation of the pathway of cholesterol biosynthesis from lanosterol in mammals.

Efflux and plasma transport of biosynthetic sterols

Abstract: The pathway of cholesterol biosynthesis has many steps, producing several intermediates. Some of the intermediates, such as lanosterol, zymosterol and desmosterol, are delivered to the plasma membrane, and are available for efflux and entry into the pathway of reverse sterol transport. Our studies with CHO and other extrahepatic cells suggest that these intermediates constitute the majority of biosynthetic sterol released to HDL; however, we see very little release from liver-derived cell lines, which make mainly cholesterol. Interestingly, we find that one of the intermediates, desmosterol, is released from cells three times more efficiently than cholesterol to extracellular sterol acceptors. This release may be an important mechanism in extrahepatic cells to prevent the pathology that is associated with the build-up of intermediates in cells and in tissues. In this chapter, we review what is known about biosynthetic sterols and their transport within cells, and summarize our data on their release to extracellular acceptors.

Synthesis and Enzymatic Cyclization of (3S)11-Fluoro-2,3-oxidosqualene.

Abstract: A convergent asymmetric synthesis provided (3 S )11-fluoro-2,3-oxidosqualene (11-FOS, 14 ), which was cyclized by bacterial squalene:hopane cyclase to a bridged ether. 11-FOS was neither a substrate nor an inhibitor for vertebrate oxidosqualene:lanosterol cyclase.