Antioxidant Properties of Ergosterol and Its Role in Yeast Resistance to Oxidation

Abstract

Although the functions and structural roles of sterols have been the subject of numerous studies, the reasons for the diversity of sterols in the different eukaryotic kingdoms remain unclear. It is thought that the specificity of sterols is linked to unidentified supplementary functions that could enable organisms to be better adapted to their environment. Ergosterol is accumulated by late branching fungi that encounter oxidative perturbations in their interfacial habitats. Here, we investigated the antioxidant properties of ergosterol using in vivo, in vitro, and in silico approaches. The results showed that ergosterol is involved in yeast resistance to tert-butyl hydroperoxide and protects lipids against oxidation in liposomes. A computational study based on quantum chemistry revealed that this protection could be related to its antioxidant properties operating through an electron transfer followed by a proton transfer mechanism. This study demonstrates the antioxidant role of ergosterol and proposes knowledge elements to explain the specific accumulation of this sterol in late branching fungi. Ergosterol, as a natural antioxidant molecule, could also play a role in the incompletely understood beneficial effects of some mushrooms on health.

Keywords: antioxidant; lipids; oxidation; plasma membrane; sterol; yeast.

Figure A1

Schematic representation of the ergosterol biosynthesis pathway from acetyl CoA. Plants, animals, and fungi have a common early sterol synthetic pathway up to squalene epoxide. After this compound, cycloartenol is produced in plants, whereas squalene epoxide gives lanosterol in animals and fungi. The end-products of the sterol pathways are sitosterol, cholesterol, and ergosterol for plants, animals, and fungi, respectively. The final enzymes involved in the ergosterol biosynthesis (ergXp) are described.

Figure A2

Tolerance of S. cerevisiae to various concentrations of t-BOOH in the growth medium. WT BY4742 () and erg6Δ () strains of S. cerevisiae are grown for 48 h at 25 °C in a yeast extract–peptone–dextrose (YPD) medium supplemented with various concentrations of t-BOOH (0, 0.1, 0.5, or 1 mM). Cells were spotted at different ten-fold dilutions (from 10⁻¹ to 10⁻⁴) from the initial suspension at OD_{600 nm} = 0.5. Data are presented as mean values ± standard deviation of three independent experiments.

Figure 1

Lipid composition of the different S. cerevisiae strains. BY4742 WT (), RH448 WT (), erg6Δ (), and erg2Δerg6Δ () strains of S. cerevisiae. (a) Analysis of the sterol composition and the relative abundance. (b) Analysis of the fatty acid composition and the relative abundance. Vertical bars represent standard deviation.

Figure 2

Resistance of S. cerevisiae to treatment with t-BOOH. After growth in the early stationary phase, BY4742 WT (), RH448 WT (), erg6Δ (), and erg2Δerg6Δ () strains of S. cerevisiae were washed, adjusted to a concentration corresponding to DO_{600 nm} = 0.5, and placed in PBS containing t-BOOH (4 or 6 mM) for 1 or 2 h. (a) After treatment, the cells were washed and spotted at different ten-fold dilutions (from 10⁻¹ to 10⁻⁴) to assess the cell viability. (b) Plasma membrane integrity was assessed by PI staining of cells and flow cytometry analysis. Data are presented as mean values ± standard deviation of three independent experiments. ANOVA was performed on R v3.6.1 software and if it was significant (p < 0.01), Tukey’s HSD (Honest Significant Difference) test was performed to observe significant differences among conditions. Letters a, b, c, d, and e represent significantly different groups.

Figure 3

Effect of the sterol content of the liposome properties. Liposomes were made from yeast lipid extracts without sterol (), with a zymosterol/phospholipid molar ratio of 1/3 (), with a cholesta-5,7,24-trienol/phospholipid molar ratio of 1/3 (), or with an ergosterol/phospholipid molar ratio of 1/3 (). Control experiments were performed with lipososmes with a tocopherol/phospholipid molar ratio of 1/3 (). (a) Phospholipid oxidation induced by cumene hydroperoxide and haemin was assessed by the oxidation kinetics of BP-C11 in liposomes with different sterol compositions. Oxidation was expressed as the intensity ratio (I_{520 nm})_oxidized/(I_{590 nm})_non-oxidized. Oxidation was started at t = 0 s. (b) Fluidity of liposomes was assessed by steady-state DPH fluorescence anisotropy measurement as a function of temperature in the range 4–48 °C. The results are presented with error bars corresponding to the standard deviation calculated from three repeated experiments.

Figure 4

Assessment of the anti-oxidant activity of sterols using the stable free radical diphenylpicrylhydrazyl (DPPH). (a) Kinetics of the decrease of the absorbance at 515 nm for different zymosterol/DPPH^● ratios (R). Error bars represent the 95% confidence interval. (b) Kinetics of the decrease of the absorbance at 515 nm for different ergosterol/DPPH^● ratios (R). Each of the kinetics were modelled by polynomial law (30 points per kinetic). With the Pearson’s table (critical values of correlation coefficient), using a value of level of significance for a two-tailed test of 0.001, and with 30 points (freedom degree = 28), the critical r value is 0.570. The values of regression coefficients reflect a suitable fit.

Figure 5

Computational study of the antioxidant properties of ergosterol, zymosterol, and cholesta-5,7,24-trienol. (a) B3LYP/6-311G(2d,p)-calculated properties for ergosterol, zymosterol, and cholesta-5,7,24-trienol species. (b) Representation of spin density of the radical cations of ergosterol, zymosterol, and cholesta-5,7,24-trienol. (c) Electrostatic potential maps of sterols. Blue indicates more positive regions; red indicates more negative regions. (d) Relative Gibbs free energies (at 298 K) in kJ/mol of the main intermediates for the reaction of DPPH radicals with ergosterol, zymosterol, and cholesta-5,7,24-trienol species. The activation barrier for the electron transfer was estimated using the Marcus theory.