Abstract
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 hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.
Highlights
The isoprenoid pathway is essential for all organisms
The correlation coefficient of the calibration curve of ergosterol is 0.9967 (Figure S4). These results suggest that the LCMS/MS system used in this study enabled absolute quantification of ergosterol and relative quantification of squalene, lanosterol, farnesyl pyrophosphate (FPP), and GGPP
It has been reported that various isoprenoid pathway mutants including hmg1-1 mutant, Dspo9 cells, Derg6 cells, Derg31Derg32 cells, Derg5 cells, and Dsts1 cells exhibited distinct phenotypes such as sensitivities to temperature, FK506, pravastatin, or cycloheximide [6,9]
Summary
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 [1]. The biosynthesis of isoprenoids occurs through the mevalonate pathway which starts with the biosynthesis of acetoacetyl coenzyme A and the subsequent reactions lead to the biosynthesis of mevalonate. Mevalonate is transformed into farnesyl pyrophosphate (FPP), a branch-point of the pathway that serves as a substrate for enzymes that synthesize sterol and nonsterol products (i.e. dolichols, ubiquinones and heme A) as well as prenyl groups for post-translational modification of proteins [2]. Ubiquinone involves electron transfer system that affects energy metabolisms [3] and dolichol involves glycosylation of proteins [2]. 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
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