phytosterols and mevalonolactone

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (

Topics: Arabidopsis; Carbon Isotopes; Chromatography, Gas; Dolichols; Erythritol; Isotope Labeling; Metabolic Networks and Pathways; Mevalonic Acid; Models, Theoretical; Osmotic Pressure; Phytosterols; Sorbitol; Spectrometry, Mass, Electrospray Ionization; Sugar Phosphates; Terpenes; Xylulose

Genes encoding sterol ester-forming enzymes were recently identified in the Arabidopsis (Arabidopsis thaliana) genome. One belongs to a family of six members presenting homologies with the mammalian Lecithin Cholesterol Acyltransferases. The other one belongs to the superfamily of Membrane-Bound O-Acyltransferases. The physiological functions of these genes, Phospholipid Sterol Acyltransferase1 (PSAT1) and Acyl-CoA Sterol Acyltransferase1 (ASAT1), respectively, were investigated using Arabidopsis mutants. Sterol ester content decreased in leaves of all mutants and was strongly reduced in seeds from plants carrying a PSAT1-deficient mutation. The amount of sterol esters in flowers was very close to that of the wild type for all lines studied. This indicated further functional redundancy of sterol acylation in Arabidopsis. We performed feeding experiments in which we supplied sterol precursors to psat1-1, psat1-2, and asat1-1 mutants. This triggered the accumulation of sterol esters (stored in cytosolic lipid droplets) in the wild type and the asat1-1 lines but not in the psat1-1 and psat1-2 lines, indicating a major contribution of the PSAT1 in maintaining free sterol homeostasis in plant cell membranes. A clear biological effect associated with the lack of sterol ester formation in the psat1-1 and psat1-2 mutants was an early leaf senescence phenotype. Double mutants lacking PSAT1 and ASAT1 had identical phenotypes to psat1 mutants. The results presented here suggest that PSAT1 plays a role in lipid catabolism as part of the intracellular processes at play in the maintenance of leaf viability during developmental aging.

Topics: Arabidopsis; Arabidopsis Proteins; Flowers; Homeostasis; Mevalonic Acid; Mutagenesis, Insertional; Mutation; Phytosterols; Plant Leaves; Seeds; Squalene

Epidemiological studies suggest that dietary phytosterols may offer protection form some types of cancer including breast cancer. In an attempt to investigate the mechanism by which phytosterols offer this protection, we investigated the effect of the two most common dietary phytosterols, beta-sitosterol and campesterol, on the mevalonate and MAP Kinase (MAPK) pathways in MDA-MB-231 cells. These pathways play a role in cell growth and apoptosis. MDA-MB-231 cell line was used in this study since it is a hormone-insensitive tumor cell line which represents the majority of advanced breast cancer cases. Cells grown in the presence of 16 microM beta-sitosterol or campesterol for 3 days exhibited a 70% and 6% reduction in cell growth, respectively, while cholesterol treatment had no effect on growth as compared to the control. Studies investigating the effect of sterol supplementation on the relative and total sterol composition of cells, showed that cells supplemented with cholesterol contained 23% more cholesterol than the control. Cells supplemented with campesterol had almost one-half the cholesterol of controls but accumulated campesterol to account for 40% of the total sterols. In the case of cells supplemented with beta-sitosterol, cells had only 25% of their sterols as cholesterol and the rest was in the form of beta-sitosterol. All sterols tested equally inhibited de novo cholesterol synthesis using 14C-acetate as substrate. beta-Sitosterol supplemented cells had reduced cholesterol synthesis when using 3H-mevalonolactone as substrate, which suggests that the inhibition in this pathway is downstream of mevalonate where processes such as isoprenylation of proteins may take place. Mevalonate supplementation to cells treated with beta-sitosterol did not completely correct the observed growth inhibition by beta-sitosterol. There was no effect of sterols on the concentrations of both low (21-26 kDa) or high (44-74 kDa) molecular weight isoprenylated proteins in these cells. On the other hand, both the quantity and activity of MAPK was elevated in the cells supplemented with beta-sitosterol. These data suggest that the down regulation of cholesterol synthesis from mevalonate and stimulation of the MAPK pathway may play roles in the inhibition of MDA-MB-231 cell growth by beta-sitosterol.

Topics: Breast Neoplasms; Cell Division; Cholesterol; Humans; Mevalonic Acid; Mitogen-Activated Protein Kinases; Phytosterols; Protein Prenylation; Sitosterols; Tumor Cells, Cultured