Low phosphate mitigates cadmium-induced oxidative stress in Saccharomyces cerevisiae by enhancing endogenous antioxidant defence system.

Cadmium is a highly toxic heavy metal that causes many harmful effects on human health and ecosystems. Metal chelation-based techniques have become a common approach for the treatment of metal poisoning and also for the remediation of metal contamination. Phosphate, an essential nutrient required for key cellular functions, has been supposed to be effective in reducing cadmium bioavailability, possibly through its chelating potential. In this study, we explored the effects of phosphate on cadmium toxicity and cellular response to cadmium stress in the eukaryotic model Saccharomyces cerevisiae. Our results reveal that cadmium toxicity is unexpectedly enhanced during phosphate repletion and optimal phosphate levels for yeast growth under cadmium stress conditions decline with increasing cadmium concentrations. The profound cadmium toxicity during phosphate repletion is unlikely to result from either elevated cadmium accumulation or dysregulated homeostasis of essential metals, but rather due to increased production of intracellular reactive oxygen species. We show that, under phosphate-depleted conditions, the activities of antioxidant enzymes, especially Mn-superoxide dismutase and catalase, are significantly promoted through transcriptional upregulation. Our findings highlight the important role of cellular response to phosphate limitation in mitigating cadmium toxicity and endogenous oxidative stress through the enhancement of antioxidant enzyme activity.

Effects of Moringa oleifera leaf extracts and its bioactive compound gallic acid on reducing toxicities of heavy metals and metalloid in Saccharomyces cerevisiae.

Moringa oleifera leaf extract is rich in antioxidants and has high potential for use to alleviate metal toxicity. Previously, we have reported the roles of aqueous M. oleifera leaf extract in mitigating intracellular cadmium (Cd) accumulation and Cd-induced oxidative stress. In this study, we investigated the protective role of aqueous and/or ethanolic M. oleifera leaf extracts (AMOLE and/or EMOLE) against other metal(loid)s in the eukaryotic model Saccharomyces cerevisiae. Our results show that only the AMOLE remarkably promoted the growth of yeast cells grown in the presence of arsenite (As(III)), Cd, nickel (Ni), and lead (Pb). Although the AMOLE contained lower amount of total phenolic and flavonoid contents and displayed lower DPPH scavenging capacity than the EMOLE, both AMOLE and EMOLE had the same capacity for reducing intracellular ROS levels in yeast cells exposed to As(III), Cd, Ni, and Pb. Moreover, the AMOLE was more effective than the EMOLE in inhibiting intracellular accumulation of these toxic metal(loid)s. In addition, we found that gallic acid, one of important phenolic constituents present in both extracts, could protect yeast cells against As(III) toxicity, likely through its role in decreasing As(III) accumulation and As(III)-induced ROS production. Furthermore, the hydroxyl and carboxyl groups of gallic acid appear to play a critical role in chelating As(III). The present study suggests the promising applications of the AMOLE (and also gallic acid) as protective agents against hazardous metal(loid)s.

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth.

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2(E301K)) suppressed the ubi4Δ growth defect. The rpt2(E301K) mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2(E301K) mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells.

Soluble Moringa oleifera leaf extract reduces intracellular cadmium accumulation and oxidative stress in Saccharomyces cerevisiae.

Moringa oleifera leaves are a well-known source of antioxidants and traditionally used for medicinal applications. In the present study, the protective action of soluble M. oleifera leaf extract (MOLE) against cadmium toxicity was investigated in the model eukaryote Saccharomyces cerevisiae. The results showed that this extract exhibited a protective effect against oxidative stress induced by cadmium and H2O2 through the reduction of intracellular reactive oxygen species. Interestingly, not only the co-exposure of soluble MOLE with cadmium but also pretreatment of this extract prior to cadmium exposure significantly reduced the cadmium uptake through an inhibition of Fet4p, a low-affinity iron(II) transporter. In addition, the supplementation of soluble MOLE significantly reduced intracellular iron accumulation in a Fet4p-independent manner. Our findings suggest the potential use of soluble extract from M. oleifera leaves as a dietary supplement for protection against cadmium accumulation and oxidative stress.

Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlor in Saccharomyces cerevisiae.

Alachlor is a widely used pre-emergent chloroacetanilide herbicide which has been shown to have many harmful ecological and environmental effects. However, the mechanism of alachlor-induced oxidative stress is poorly understood. We found that, in Saccharomyces cerevisiae, the intracellular levels of reactive oxygen species (ROS) including superoxide anions were increased only after long-term exposure to alachlor, suggesting that alachlor is not a pro-oxidant. It is likely that alachlor-induced oxidative stress may result from protein denaturation because alachlor rapidly induced an increased protein aggregation, leading to upregulation of SSA4 and HSP82 genes encoding heat shock proteins (Hsp) of Hsp70 and Hsp90 family, respectively. Although only SOD1 encoding Cu/Zn-superoxide dismutase (SOD), but not SOD2 encoding Mn-SOD, is essential for alachlor tolerance, both SODs play a crucial role in reducing alachlor-induced ROS. We found that, after alachlor exposure, glutathione production was inhibited while its utilization was increased, suggesting the role of glutathione in protecting cells against alachlor, which becomes more important when lacking Cu/Zn-SOD. Based on our results, it seems that alachlor primarily causes damages to cellular macromolecules such as proteins, leading to an induction of endogenous oxidative stress, of which intracellular antioxidant defense systems are required for elimination.

Peroxiredoxin chaperone activity is critical for protein homeostasis in zinc-deficient yeast.

Zinc is required for the folding and function of many proteins. In Saccharomyces cerevisiae, homeostatic and adaptive responses to zinc deficiency are regulated by the Zap1 transcription factor. One Zap1 target gene encodes the Tsa1 peroxiredoxin, a protein with both peroxidase and protein chaperone activities. Consistent with its regulation, Tsa1 is critical for growth under low zinc conditions. We previously showed that Tsa1's peroxidase function decreases the oxidative stress that occurs in zinc deficiency. In this report, we show that Tsa1 chaperone, and not peroxidase, activity is the more critical function in zinc-deficient cells. Mutations restoring growth to zinc-deficient tsa1 cells inactivated TRR1, encoding thioredoxin reductase. Because Trr1 is required for oxidative stress tolerance, this result implicated the Tsa1 chaperone function in tolerance to zinc deficiency. Consistent with this hypothesis, the tsa1Δ zinc requirement was complemented by a Tsa1 mutant allele that retained only chaperone function. Additionally, growth of tsa1Δ was also restored by overexpression of holdase chaperones Hsp26 and Hsp42, which lack peroxidase activity, and the Tsa1 paralog Tsa2 contributed to suppression by trr1Δ, even though trr1Δ inactivates Tsa2 peroxidase activity. The essentiality of the Tsa1 chaperone suggested that zinc-deficient cells experience a crisis of disrupted protein folding. Consistent with this model, assays of protein homeostasis suggested that zinc-limited tsa1Δ mutants accumulated unfolded proteins and induced a corresponding stress response. These observations demonstrate a clear physiological role for a peroxiredoxin chaperone and reveal a novel and unexpected role for protein homeostasis in tolerating metal deficiency.