Tomonori Takami is employed by the commercial company JCL Bioassay Corporation. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: YF TK TT. Performed the experiments: TT YF XZ WJ YM. Analyzed the data: TT YF XZ WJ YM TK. Contributed reagents/materials/analysis tools: YF YM TK. Wrote the paper: YF TT TK.
Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In
The isoprenoid pathway is essential for all organisms. Regulation of the isoprenoid pathway has been extensively studied in mammals for many years, because this pathway produces such critical end-products as steroid hormones, cholesterol and bile acids
Statins are selective inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which inhibit the biosynthesis of cholesterol and thereby reduce serum cholesterol levels in humans. In addition to the inhibition of cholesterol synthesis, statins have been shown to possess anti-inflammatory and immune-modulatory pleiotropic effects, even in patients with normal cholesterol levels
In unicellular eukaryotes such as
Here, we quantified the final product (ergosterol) and the pathway intermediates (squalene, FPP, GGPP, and lanosterol) in various isoprenoid pathway mutants, treated with statins or antifungals using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results showed that compounds such as pravastatin, allylamine terbinafine, and miconazole inhibit Hmg1, squalene epoxidase (Erg1), and lanosterol demethylase (Erg11), respectively, and the inhibition was associated with significant changes in the levels of the pathway products and intermediates. Notably, the ergosterol level showed substantial changes but the changes were smaller in magnitude when compared with FPP and GGPP in response to these drugs.
Selected reaction monitoring (SRM) chromatograms of squalene, lanosterol, ergosterol, pyrene (used as an internal standard), FPP, and GGPP in the standard solution are shown in
The SRM chromatograms of ergosterol and pyrene in the blank sample, the zero sample, and the lower limit of quantification (LLOQ) sample are shown in
The
The quantification results of squalene and ergosterol in wild-type cells and Δ
(A) Levels of squalene and ergosterol in wild-type cells and Δ
Enzymes predicted to be involved in ergosterol biosynthesis are shown in parentheses with key intermediates in gray boxes. Inhibitors are shown by red letters. The
The
Next, we measured levels of squalene and ergosterol in
Wild-type and
We also measured levels of FPP and GGPP in
(A) Levels of FPP and GGPP in wild-type cells and
We first examined the effects of terbinafine and miconazole in wild-type cells. As expected, terbinafine significantly decreased lanosterol level and increased squalene level in wild-type cells (
Wild-type cells were grown to saturation at 27°C in liquid YPD medium. Cells were further incubated at 27°C for 10 h in the absence or presence of terbinafine (0.1, 0.3 µg/ml) or miconazole (0.5, 2.0 µg/ml) as indicated, and then ergosterol, lanosterol, and squalene of the strains were extracted and determined. Error bars represent standard deviations (n = 3 experiments).
Then, we examined the effects of these drugs on the levels of ergosterol, lanosterol, and squalene in various isoprenoid pathway mutants including
As regards ergosterol, the level in
Wild-type cells and
Wild-type cells and Δ
Wild-type cells and Δ
(A) Levels of ergosterol, lanosterol, and squalene in wild-type cells and Δ
As regards lanosterol, the levels in
As regards squalene, the level in
We also examined whether fission yeast cells can grow normally under the condition of lowest ergosterol and lanosterol such as in Δ
As described above, the
(A) Effect of pravastatin and FK506 on the growth of wild-type cells. Wild-type cells and
In order to investigate the genetic interaction between
Previously, it has been reported that various isoprenoid pathway mutants including
Interestingly, squalene is highly produced in Δ
Intriguingly, lanosterol levels in Δ
In conclusion, the intermediate and final products of the fission yeast isoprenoid pathway were quantified using LC-MS/MS in this study. The findings presented here suggest that the pleiotropic phenotypes caused by the
Pravastatin, terbinafine, and miconazole were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan), Novartis Pharma K.K. (Tokyo, Japan), Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan), respectively. Squalene, lanosterol, and ergosterol (85.0+% (HPLC)) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Farnesyl pyrophosphate ammonium salt (≥95% (TLC)), and geranylgeranyl pyrophosphate ammonium salt (≥95% (TLC)) were purchased from Sigma-Aldrich, St. Louis, MO, USA. All other reagents were from commercial sources.
Strain | Genotype | Reference |
HM123 |
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Our stock |
KP207 |
|
Our stock |
KP4127 |
|
|
KP4334 | NBRP (FY13124) | |
KP4248 |
|
|
KP4249 |
|
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KP4250 |
|
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KP4251 |
|
Extraction of squalene, lanosterol, and ergosterol from cells was performed essentially as previously described
Extraction of FPP and GGPP from cells was performed essentially as previously described
Squalene, lanosterol, and ergosterol were measured using LC-MS/MS under previously described conditions
No. | Compound | Precursor ion | Product ion | Collision energy |
1 | Squalene | 35% | ||
2 | Lanosterol | 35% | ||
3 | Ergosterol | 35% | ||
4 | Pyrene | - | - |
FPP and GGPP were measured using LC-MS/MS under previously described conditions
No. | Compound | Precursor ion | Product ion | Collision energy |
1 | FPP | 35% | ||
2 | GGPP | 35% |
The peak area ratios of the compounds squalene, lanosterol, and ergosterol to pyrene in mutants or knockout cells were compared to those in wild-type cells. The peak area values of FPP and GGPP in mutants or knockout cells were compared to those in wild-type cells. The level of the respective compounds in wild-type cells was taken as 100%, and the level of the corresponding compounds in mutants or knockout cells was calculated as a percentage of that of the wild-type cells.
The calibration curve of each standard was constructed to confirm that LC-MS/MS system used in this study gave correct quantitative data. Each standard solution to construct a calibration curve was prepared as follows. Squalene and lanosterol were dissolved in methanol. The concentrations of the standard solutions of squalene were 1, 5, 10, 50, 100, and 500 µmol/l. The concentrations of the standard solutions of lanosterol were 0.1, 0.2, 0.5, 1, 2, 5, and 10 µmol/l. Pyrene was used as an internal standard. The concentration of the standard solution of pyrene was 90 µmol/l.
FPP and GGPP were dissolved in water/methanol (1∶1, v/v). The concentrations of the standard solutions of both FPP and GGPP were 10, 20, 40, 100, 200, and 400 nmol/l. An internal standard for the quantification of FPP and GGPP was not used.
Ergosterol was quantified absolutely by constructing a calibration curve of Δ
LC-MS/MS analysis of isoprenoids. A) SRM chromatograms of squalene, lanosterol, ergosterol, and pyrene in the standard solution. B) SRM chromatograms of FPP and GGPP in the standard solution.
(TIF)
Calibration curves of squalene (
(TIF)
LC-MS/MS analysis of ergosterol in fission yeast extracts. SRM chromatograms of ergosterol (Left) and pyrene (Right) in the blank sample (A), the zero sample (B), and the LLOQ sample (C).
(TIF)
Calibration curve of ergosterol (
(TIF)
We thank Kaoru Takegawa for providing the strains and plasmids, and Susie O. Sio for critical reading of the manuscript. Original strain of Δ