Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae

Abstract

Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S.cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation.

Keywords: Saccharomyces cerevisiae metabolism; diselenide; methylseleninic acid; methylselenol; protein aggregation; redox equilibrium; thiol/disulfide exchange; toxicity.

Figure 1

UV absorption spectra of various Se-containing species and effects of reducing agents. (A) Reduction of methylseleninic acid (MSeA) by glutathione (GSH). UV absorption spectra in 100 mM potassium phosphate, pH 7.4, of MSeA (250 µM) mixed with increasing amounts of GSH as indicated in the figure. (B) UV absorption spectra of various Se-containing species. The sample cuvette contained 100 µM MSeA in the absence () or presence () of 500 µM TCEP, 100 µM MeSeSG (), or 100 µM DMDSe () in 100 mM potassium phosphate, pH 7.4; spectra in the red box are extended in the inset. (C) Reaction of MeSeSG with methylselenol (MeSeH). MeSeH was produced by mixing 25 µM DMDSe with 25 µM TCEP in 100 mM potassium phosphate, pH 7.4. UV absorption spectra were recorded before () and after the addition of 50 µM MeSeSG () to this sample.

Figure 2

Reduction of DMDSe by GSH. 100 µM DMDSe was mixed with 1 mM GSH (containing 15 µM GSSG) in the absence (black trace) or presence (blue trace) of 50 µM added GSSG in 100 mM potassium phosphate, pH 7.4. The absorbance at 252 nm was recorded as a function of time starting 7 to 8 s after mixing. The absorbance of a solution of 100 µM DMDSe alone, measured with a control sample, was 0.016. The experimental data were fitted to the kinetic model described in Supplementary Materials (red lines).

Figure 3

Reduction of MeSeSG by GSH. 100 µM MeSeSG was mixed with 10 mM GSH (containing 150 µM GSSG) in 100 mM potassium phosphate, pH 7.4, and UV absorption spectra were recorded after 20 s (blue line); 60 s (orange); 100 s (grey); 140 s (yellow); 180 s (light blue); 220 s (green); 260 s (dark blue); 300 s (brown). The spectrum of a solution of 100 µM MeSeSG alone is displayed in black. Inset: The spectrum of MeSeSG (black solid line) was subtracted from that of the products of the reaction after 5 min (dotted line) to obtain the difference spectrum (red line).

Figure 4

Redox titration of DMDSe with DTT. 50 µM DMDSe was mixed with increasing concentrations of DTT (0 to 2400 µM) in 100 mM potassium phosphate, pH 7.4 and UV absorption spectra were recorded. Inset: The absorbance at 252 nm was plotted against DTT concentration and fitted to the theoretical equation.

Figure 5

Equilibrium distribution of MeSeH, MeSeSG, and DMDSe as a function of the GSH/GSSG ratio for a GSH concentration of 10 mM and a total Se concentration of 5 µM ([total Se] = [MeSeH] + [MeSeSG] + 2 [DMDSe]). The curves were obtained using values of 1350 for 1/K1 and 1000 for K2.

Figure 6

Reduction of MeSeSG or DMDSe by S. cerevisiae glutathione reductase. (A) NADPH-dependent reduction of MeSeSG (☐) or GSSG (). The reaction mixtures contained 100 µM NADPH, and 50 µM of the substrate under study in 100 mM potassium phosphate, pH 7.4. The reactions were started by adding 1.5 nM GR to the GSSG mixture or 73 nM GR to the MeSeSG mixture. (B) Aerobic () and anaerobic (, +) consumption of NADPH in the presence of GR and 100 µM DMDSe (+) or 100 µM MeSeSG (, ). The reaction mixtures contained 200 µM NADPH, 100 nM GR in 100 mM potassium phosphate, pH 7.4, at 22 °C. Anaerobic reactions were performed in a glove box under nitrogen atmosphere. (C) Stoichiometry of NADPH consumption as a function of MeSeSG concentration in anaerobic conditions. NADPH consumption was followed in anaerobic conditions with 0 to 200 µM MeSeSG added to the reaction mixture as in B). The amount of NADPH consumed after 125 min was calculated from the ∆A₃₄₀ after subtraction of the value in the absence of substrate.

Figure 7

Reduction of MeSeH precursors by the TRX/TRR system. The reaction mixtures contained 100 µM NADPH, 2 µM TRX and 0.5 µM TRR from S. cerevisiae in 100 mM potassium phosphate, pH 7.4, 22 °C. The reactions were started by adding buffer (), or 50 µM of DMDSe (), MeSeSG(☐), or MSeA(∆).

Figure 8

Cell growth inhibition by MeSeH precursors. BY4742 and mutant strains were grown overnight at 30 °C in SD medium or SD medium supplemented as indicated. Exponentially growing cells were diluted to 0.04–0.08 OD₆₀₀ in the same medium and grown for 20–24 h at 30 °C in various concentrations of MeSeH precursors. Growth was monitored by change in OD₆₀₀ and plotted as % of the growth in the absence of toxic. (A) BY4742 cells grown in SD medium in the presence of increasing concentrations of MSeA. (B) BY4742 cells grown in SD () or SD supplemented with 100 µM methionine () in the presence of increasing concentrations of a mixture containing MSeA with a three-fold excess of GSH. (C) BY4742 cells grown in SD () or SD supplemented with 100 µM methionine () in the presence of increasing concentrations of DMDSe. (D) BY4742 () and ∆met17 (), and ∆cys3 (■) isogenic mutants cells grown in SD medium supplemented with 100 µM cysteine in the presence of increasing concentrations of DMDSe.

Figure 9

DMDSe promotes protein aggregation in a MET17-dependent manner. (A) Induction of Hsp104-GFP by SeMet or DMDSe. Exponentially growing BY4742-Hsp104-GFP (dark bars) or BY4741-Hsp104-GFP (light bars) cells were incubated in SC + 100 µM methionine for 2 h at 30 °C in the absence or presence of the indicated concentration of SeMet or DMDSe. The fluorescence in whole cell extracts was recorded at 508 nm and normalized to the optical density of the extracts at 280 nm. The fluorescence intensity in the absence of toxic (control) was set as 1. (B) Localization of Hsp104-GFP in cells exposed to SeMet or DMDSe. Hsp104-GFP localization was monitored by fluorescence in living cells after 1 h of exposure to the indicated concentrations of SeMet or DMDSe. BY4742 and BY4742-∆cys3 cells were grown in SD supplemented with 100 µM cysteine, BY4741 cells were grown in SD supplemented with 100 µM methionine. Representative images obtained after maximum intensity z-projection, bar equals 10 µm. The fraction of cells containing at least one Hsp104-GFP focus was determined by manual inspection of 100–200 cells in each condition.