Zinc and copper rescue statin toxicity in a yeast-based screen
To maximize the effect that exogenously added metabolites would have on statin-treated yeast, we used yeast bearing a PDR5
encodes an ABC cassette efflux pump responsible for the removal of a variety of small molecules, including xenobiotics and drugs.11
In the presence of lovastatin, the knockout strain reached cell densities approximately 40% that of the PDR+
strain (Supplementary Fig. 1
). The assay was performed in synthetic complete media to minimize the availability of metabolites in the media and to avoid undefined components in autolyzed yeast extract.
Yeast cultures were pretreated overnight with lovastatin to exhaust the intracellular ergosterol reserve and then diluted into fresh media containing metabolites from the library. Growth was measured every 30 minutes for 24 hours (). We chose a lovastatin concentration that inhibited yeast growth by approximately 50%, enabling the identification of metabolites that enhanced or rescued statin toxicity (, CuSO4 is shown).
Figure 1 A growth-based assay of drug-metabolite interactions reveals divalent metal ion-mediated rescue of statin growth inhibition. (a) Yeast cultures were pretreated with statin to deplete ergosterol reserves. After pretreatment, the yeast were diluted into (more ...)
Statin treatment in yeast at high concentrations and for sustained periods can cause degradation of mitochondrial DNA and the consequent appearance of the petite phenotype.9, 12, 13
We established that at the concentration of statin and for the period of treatment we employed, petite formation did not occur (Supplementary Fig. 1
We screened a small library designed to cover a spectrum of metabolite classes, including enzyme cofactors and metals as well as nutritional and nutriceutical compounds (). Metabolites were added at high concentration (typically mM) unless toxicity was observed, in which case lower concentrations were chosen (Supplementary Table 1
). We generated growth curves from triplicate cultures that were averaged and transformed into a fitness measurement. We examined the fitness impact of each metabolite on the background of statin treatment by calculating chemical epistasis values (Epistasis = Fitnessstatin+metabolite
, Supplementary Table 1
The epistasis values comprised a distribution with a mean score = 0.073 +/− 0.022 (). Statistical analysis of the distribution of epistasis scores revealed that the divalent metal ions zinc, copper and manganese rescued statin toxicity (). We included in the screen two positive controls: mevalonic acid, the product of HMG-CoA reductase; and ergosterol, the endpoint of the pathway. Mevalonic acid produced a strong rescue effect, in accord with previous studies.9, 10
Ergosterol did not rescue, likely because it is sparingly soluble in water and therefore not bioavailable to yeast after dilution into the culture. Indeed, reports of the effectiveness of ergosterol in the rescue of yeast treated with sterol synthesis inhibitors are conflicting, likely for this reason.9
Given that the three hits from the screen were metals, we tested a variety of additional metals in our assay. Other divalent metals, including iron, nickel and calcium, did not rescue statin growth inhibition. Additionally, neither chromium nor lithium rescued statin growth inhibition. By testing both chloride and sulfate salts of copper and zinc, we established that the rescue is independent of the counterion present. Furthermore, the copper and zinc rescue effects were dose-dependent, occurring maximally over a narrow range of concentrations (1–2 mM) just below the point at which toxicity dominated (Supplementary Fig. 1
). From these data, we conclude that the rescue effects observed for copper, zinc and manganese are due to the metals themselves, and cannot be generalized to other metals.
We performed experiments to rule out possible confounding features of our assay. Testing in mating type α yeast revealed that the rescue effect was not specific to the mating type a
strain used in the screen (Supplementary Fig. 1
). Copper is highly oxidatively active, and zinc has been reported to induce oxidative stress responses in yeast at high concentrations.15, 16
Therefore, we examined whether hydrogen peroxide, a strong oxidant, could rescue statin growth inhibition and found that it did not. Thus, the rescue effects are not strain-or mating type-specific nor do they arise from the induction of a general oxidative stress response.
Lovastatin inhibits ergosterol biosynthesis by preventing the synthesis of mevalonic acid, an early intermediate in the pathway. Mevalonic acid is also used by the cell to generate heme and coenzyme Q10
as well as to prenylate proteins. Statin-mediated toxicity in yeast arises from a lack of protein prenylation9
as well as ergosterol starvation. To investigate whether these non-ergosterol products of mevalonic acid play a role in the metal rescue effect, we tested ketoconazole, an antifungal agent that inhibits Erg11, an enzyme that acts after the pathway is committed to ergosterol.17
We asked whether copper and zinc rescue ketoconazole-mediated growth inhibition in yeast. Both metals produced a rescue effect similar to that seen when they are combined with lovastatin (Supplementary Fig. 1
). These data show that the metals act by restoring ergosterol biosynthesis rather than by modulating other products of mevalonic acid.
Zinc and copper increase the level of ergosterol and its precursors in lovastatin-treated yeast
We reasoned that metal rescue of lovastatin growth inhibition was likely mediated by an increase in ergosterol levels. We tested this hypothesis by measuring the ergosterol content of yeast cultures by spectroscopic analysis. We found that lovastatin treatment reduced ergosterol to one quarter the level found in untreated cells (). Treatment with zinc or copper alone resulted in an approximately twofold increase in ergosterol level relative to untreated cells. In the statin-treated cultures, the addition of zinc or copper also led to a twofold increase in ergosterol relative to statin treatment alone. The addition of mevalonic acid alone suppressed ergosterol biosynthesis, likely the result of the very high mevalonic acid concentrations used. The mechanism by which mevalonic acid supplementation produces this result is unclear; however, the sterol biosynthesis pathway is subject to feedback inhibition.18
In the lovastatin-treated culture, mevalonic acid produced an increase in ergosterol levels equivalent to that observed with lovastatin plus zinc or copper.
Figure 2 Copper and zinc increase levels of ergosterol and ergosterol biosynthetic intermediates. (a) Ergosterol was measured by spectrophotometric quantitation after extraction with n-heptane and by GCxGC-MS (inset). Comparison of ergosterol levels in untreated (more ...)
We explored ergosterol biosynthesis in more detail by profiling levels of intermediates in this pathway using two-dimensional gas chromatography and mass spectrometry (GCxGC-MS). A heptane-based extraction isolated these highly hydrophobic intermediates. Using standards and chemical library information,19
we identified eight ergosterol pathway intermediates as well as ergosterol itself (). We quantified each of these intermediates after treatment with combinations of lovastatin, zinc, copper and mevalonic acid. Copper, zinc or mevalonic acid alone induced increases in intermediate levels (PCu
= 4e-11, PZn
= 0.0001, Pmevalonic acid
= 3e-5, t-test) (). In contrast, statin treatment alone led to a striking depletion of intermediates, with most being reduced to immeasurably low concentrations (P = 1.4e-12, t-test). Treatment with copper, zinc or mevalonic acid along with lovastatin increased the levels of these intermediates relative to their low levels in the presence of the statin alone (PCu
= 0.003, PZn
= 0.02, Pmevalonic acid
= 5e-16, t-test).
As a complement to our spectroscopic approach, we used GCxGC-MS to examine ergosterol levels in yeast treated with combinations of copper, zinc and lovastatin (, inset). Copper alone produced a large and significant increase in the level of ergosterol (P = 2e-6, t-test). Zinc alone produced a small increase that is not significant. Mevalonic acid alone reduced ergosterol levels (P = 0.001, t-test). Treatment with lovastatin alone reduced ergosterol by 70%. In the presence of statin, both copper and zinc increased the diminished ergosterol level due to the statin, although this increase was small and statistically significant only for copper (P = 0.046, t-test). As expected, mevalonic acid treatment resulted in an increase in ergosterol level. These observations are in general agreement with the ergosterol levels determined by spectrophotometric measurement ().
These metabolite profiling data reveal a detailed picture of the effect of copper, zinc and lovastatin on yeast ergosterol biosynthesis. Lovastatin treatment eliminated ergosterol precursors and reduced ergosterol levels by approximately 70%. Copper and zinc stimulated ergosterol production, increasing levels of ergosterol precursors as well as ergosterol itself. This metal-mediated enhancement of ergosterol biosynthesis occurred in the context of lovastatin-treated yeast. Thus, metal enhancement of ergosterol biosynthesis likely mediates the observed rescue of lovastatin-mediated growth inhibition.
Gene expression analysis of metal-mediated rescue of lovastatin-treated yeast
To understand the basis for metal modulation of ergosterol biosynthesis, we analyzed gene expression. Expression data were collected for yeast treated with lovastatin, with copper or zinc, or with both the statin and a metal. We searched for genes whose expression was significantly affected by both lovastatin and metal treatment using a linear model-based analysis. We found 321 such genes at a false discovery rate of 5%. Clustering of these genes identified six major groups; of these, four were primarily responsive to statin and two were primarily responsive to metal (Supplementary Fig. 2
). Gene Ontology (GO) analysis of these groups revealed that statin treatment in general modulated the expression of sterol, lipid and cell wall biosynthetic genes, whereas metal treatment modulated the expression of mitochondrial and metal binding/transport genes (Supplementary Table 2
The expression level of many genes in yeast is dependent on growth rate.20
Given that statin treatment resulted in decreased growth rate (doubling time for untreated = 5.5 hours, statin = 10.75 hours), we checked whether the transcriptional changes observed were due to such growth rate-dependent expression effects. Of the 321 genes found to be significantly up- or downregulated, only 17 had growth rate-dependent slopes of similar sign in the Brauer et al. data set.20
Thus, the transcriptional changes do not originate primarily from changes in growth rate.
The yeast transcriptional response to zinc and, in particular, to copper has been extensively studied;15, 16, 21–24
our results generally agree with previous observations. The two clusters that responded primarily to metal (metal responsive, MR) contain many genes known to respond to changes in metal availability, including metalloproteins and metal transporters (Supplementary Table 2
). MR cluster #1 contains genes whose expression was upregulated in response to zinc or copper (). MR cluster #1 genes are representative of metal-stress responses and include the metallothioneins CUP1
. A large number of mitochondria-associated genes are also found in this cluster; mitochondrial metabolism is sensitive to metal and oxidoreductive perturbations.25
Figure 3 Gene expression clusters primarily responsive to metal treatment. Measurement of gene expression following treatment of yeast cultures with statin, zinc/copper or both resulted in clusters of genes with particular expression patterns. Each panel represents (more ...)
MR cluster #2 contains genes that were downregulated in response to metals (). Included in this cluster are genes that characterize the primary yeast response to high zinc concentrations, which is mediated by Zap1 repression of genes carrying zinc responsive elements.23, 24, 26
Genes from this regulon that were downregulated by zinc include ZPS1
, ZRT1, ZRT2
. Other genes that were downregulated include FET5
, all of which play a role in the uptake or transport of copper. Many of the genes found in both MR clusters relate to iron metabolism, which is also disrupted by high copper and zinc concentrations.16, 21
Treatment with statins or other antifungal agents, along with the study of a variety of genetic modifications that interfere with ergosterol metabolism, has revealed that yeast respond to ergosterol deprivation primarily through the transcription factors Ecm22 and Upc2, which act on Sterol Responsive Elements (SREs) to upregulate many ergosterol biosynthetic genes as well as the DAN/TIR
cell wall biosynthesis gene families.10, 17, 27, 28 UPC2
expression is expected to increase during ergosterol deprivation.29
We observed a sevenfold increase in UPC2
expression upon lovastatin treatment. However, this expression change was not considered significant in our model, which was designed to identify genes whose expression changed in response to both metal and statin treatment. Since UPC2
expression did not respond to metal treatment, the metal-mediated rescue of statin growth inhibition is likely not effected by Upc2. ECM22
expression is reported to decrease during ergosterol deprivation,29
but we did not observe a significant change upon statin treatment.
Four clusters of primarily statin-responsive (SR) genes showed an expression pattern that was also modulated by metal treatment. SR cluster #1 contains 72 genes whose expression was increased greatly by statin alone, was unaffected by metal alone, and was diminished by statin plus metal relative to statin alone (, see also Supplementary Fig. 2
). Based on GO analysis, this cluster is enriched for sterol and lipid biosynthetic as well as cell wall genes (Supplementary Table 2
). These genes characterize the stereotypic SRE-driven response which upregulates ergosterol and cell wall biosynthesis.
Figure 4 Gene expression clusters primarily responsive to statin treatment. Measurement of gene expression following treatment of yeast cultures with statin, metal (copper or zinc) or both resulted in clusters of genes with particular expression patterns. Each (more ...)
SR cluster #2 contains 29 genes that were upregulated in response to statin alone, downregulated in response to metal alone, and upregulated more weakly or not at all in response to statin plus metal relative to statin alone (). No GO terms are enriched in this cluster, which contains a diverse array of seemingly unrelated genes. SR cluster #3 contains 94 genes that were downregulated in response to statin alone, unaffected by metal alone, and downregulated more weakly in response to statin plus metal relative to statin alone (). GO analysis reveals that this large cluster is enriched for amino acid and nucleotide biosynthetic genes.
SR cluster #4 contains 27 genes whose expression was upregulated weakly by statin alone, was upregulated weakly by metal alone, and was enhanced by statin plus metal relative to statin alone (). Thus, in this cluster, metal and statin treatments synergize to increase gene expression levels, which suggests that these genes could account for the observed metal-mediated rescue of statin growth inhibition. This hypothesis is strengthened by GO analysis, which revealed that this cluster is enriched for sterol biosynthetic genes (Bonferroni-corrected P = 2.66e-7). In fact, SR cluster #2 contains a large fraction (5 of 14) of the sterol biosynthesis genes identified by our model as having a significant response to the combination of lovastatin and metal treatment.
We performed a search for transcription factors that could be responsible for the observed expression changes. A search of YEASTRACT30
reveals consensus binding sites for the transcription factors Sfp1, Hap1 and Yap1 in the promoter regions of at least four of these five genes. Given that Sfp1 is a stress-sensitive regulator of ribosomal protein expression,31
that Yap1 activates a large transcriptional program in response to oxidative stress32
and that Hap1 responds to changes in oxygen availability,33
any of these transcription factors could be responsible for the metal effect. However, few of the other known targets of these factors were differentially expressed (7% for Yap1, 6% for Sfp1 and 13% for Hap1), making any of these factors unlikely to be responsible.
Intriguingly, the five ergosterol biosynthetic genes in SR cluster #4 (DAP1, ERG11, ERG5 , CYB5 and ERG1) are all involved in oxidoreductive reactions. Metal-induced oxidative stress might account for the differential regulation of these genes. However, we did not observe rescue of lovastatin-mediated growth inhibition upon treatment with hydrogen peroxide, a powerful inducer of oxidative stress. Nevertheless, perturbation of metal ion homeostasis induced by excess zinc and copper treatment could cause the observed upregulation of these genes after metal treatment.
Zinc and copper rescue growth inhibition in cultured HeLa cells
Many facets of sterol synthesis and regulation are conserved between yeast and humans, including most of the biosynthetic machinery.18
However, mammalian sterol homeostasis is achieved mostly by the Sterol Response Element Binding Proteins (SREBPs) whereas S. cerevisiae
regulates sterol homeostasis primarily through the Upc2 and Ecm22 transcription factors, which are not homologous to SREBPs. Given these differences, we asked whether zinc and copper could rescue statin growth inhibition in a mammalian cell culture system.
We treated HeLa cells with lovastatin to obtain an approximately 50% reduction in cell density. Treatment with zinc or copper produced a significant rescue of this statin growth inhibition (copper 40.4% increase, zinc 35% increase) (). Lithium, which failed to rescue statin growth inhibition in the yeast assay, did not result in a significant increase in HeLa cell growth. Mevalonic acid, the positive control in the yeast assay, produced a complete rescue of statin growth inhibition in HeLa cells. These results, showing consistent patterns in the activity of the two metals in lovastatin-treated yeast and mammalian cells, suggest that copper and zinc intake may be of relevance to human patients taking statins.
Figure 6 Zinc and copper rescue statin growth inhibition in cultured mammalian cells. HeLa cells were treated with statin in combination with lithium (negative control), copper, zinc and mevalonic acid (positive control). Cell growth was measured by calcein dye (more ...)