Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide

Molecular Biology - The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a...     

Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Δ reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h⁻¹ in anaerobic batch cultures containing lactic acid but only 0.16 h⁻¹ in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.Lactic acid is an organic acid with a wide range of applications. In the food industry, lactic acid has traditionally been used as an antimicrobial as well as a flavor enhancer. Besides having applications in textile, cosmetic, and pharmaceutical industries (5), lactic acid has been applied for the manufacture of lactic acid polymers (11, 40). These polymers have properties that are similar to those of petroleum-derived plastics. Skyrocketing oil prices caused by dwindling fossil fuel reserves coupled with pressures to tackle environmental issues are creating increased demand for bioderived, and often biodegradable, polymers, such as poly-lactic acid.Current industrial lactic acid fermentations are based on different species of lactic acid bacteria. These bacteria have complex nutrient requirements due to their limited ability to synthesize B vitamins and amino acids (8) and are intolerant to acidic conditions with a pH between 5.5 and 6.5 required for growth (40). Acidification of the growth medium during lactic acid fermentation is typically counteracted by the addition of neutralizing agents (e.g., CaCO₃), resulting in the formation of large quantities of insoluble salts, such as gypsum, during downstream processing.Saccharomyces cerevisiae has received attention as a possible alternative biocatalyst. This organism is relatively tolerant to low pH and has simple nutrient requirements. The production of lactic acid with metabolically engineered S. cerevisiae was achieved by introducing a NAD⁺-dependent lactate dehydrogenase, leading to the simultaneous formation of both ethanol and lactate (1a, 12, 31, 32, 36). Further improvements were made by constructing a pyruvate decarboxylase-negative (Pdc⁻) S. cerevisiae strain (1a, 31, 44) that converted glucose to lactic acid as the sole fermentation product.Although the redox balance and ATP generation in lactic acid fermentation are analogous to those in alcoholic fermentation, engineered homolactic S. cerevisiae strains could not sustain anaerobic growth (44). In addition, the lactate formation rate under anaerobic conditions in the presence of excess glucose was significantly lower than the specific ethanol production rate of the wild-type strain. Moreover, exposure of the anaerobic cell suspension to oxygen immediately led to a 2.5-fold increase in the lactate formation rate. The stimulatory effect of oxygen on lactic acid fermentation may reflect an energetic constraint in lactate fermentation, probably as a consequence of energy-dependent product export (42, 44). In agreement with this hypothesis, intracellular ATP concentrations and the related energy charge decrease rapidly during anaerobic homolactic fermentation by S. cerevisiae (1). Consequently, industrial production of lactic acid with S. cerevisiae may require (micro)aerobic conditions to allow for the generation of sufficient ATP to enable cell growth and, even under nongrowing conditions, maintenance.The formation of reactive oxygen species (ROS) during cellular respiration is an unavoidable side effect of aerobic life relying on oxygen as the final electron acceptor. At least 2% of oxygen consumed in mitochondrial respiration undergoes only one electron reduction, mainly by the semiquinone form of coenzyme Q, generating superoxide radicals (O₂⁻) (26). In addition, the prooxidant effects of organic acids have been demonstrated using sod mutants (30). An in vitro study by Ali et al. (3) also linked ROS formation to weak organic acids and showed enhanced hydroxy radical (OH) generation in the presence of lactic acid.Among different ROS, the hydroxy radical that originates from H₂O₂ in the metal-mediated Fenton/Haber-Weiss reactions is especially reactive. It indiscriminately oxidizes intracellular proteins, nucleic acids, and lipids in the cell membranes (4, 38). Lactate interacts with the ferric ion (Fe³⁺) to form a stable complex of Fe³⁺-lactate at a molar ratio of 1:2. This complex then reacts with H₂O₂ to enhance the OH generation via the Fenton reaction (2, 3). Although a similar in vivo mechanism has not yet been proven, previous research indicates that lactic acid and other weak organic acids enhance oxidative stress of aerobic yeast cultures.Like other eukaryotic organisms, S. cerevisiae possesses enzymatic defense mechanisms, including several crucial antioxidant enzymes, such as catalase and superoxide dismutase (SOD). SOD removes O₂⁻ by converting it to H₂O₂, which, in turn, can be disproportionated to water by catalase or glutathione peroxidase. Cytosolic catalase, Ctt1p, is thought to play a general role, as CTT1 expression is regulated by various stresses, including oxidative stress, osmotic stress, and starvation (15, 23, 33). More recently, catalase has also been implicated in response to acetic acid tolerance and acetic acid-induced programmed cell death (17, 47).The goals of the present study were to assess the in vivo relevance of lactate-mediated oxidative stress in S. cerevisiae and to investigate whether its effects could be ameliorated by enhanced expression of catalase.     

Polyhydroxyfullerene Binds Cadmium Ions and Alleviates Metal-Induced Oxidative Stress in Saccharomyces cerevisiae

The water-soluble polyhydroxyfullerene (PHF) is a functionalized carbon nanomaterial with several industrial and commercial applications. There have been controversial reports on the toxicity and/or antioxidant properties of fullerenes and their derivatives. Conversely, metals have been recognized as toxic mainly due to their ability to induce oxidative stress in living organisms. We investigated the interactive effects of PHF and cadmium ions (Cd) on the model yeast Saccharomyces cerevisiae by exposing cells to Cd (≤5 mg liter⁻¹) in the absence or presence of PHF (≤500 mg liter⁻¹) at different pHs (5.8 to 6.8). In the absence of Cd, PHF stimulated yeast growth up to 10.4%. Cd inhibited growth up to 79.7%, induced intracellular accumulation of reactive oxygen species (ROS), and promoted plasma membrane disruption in a dose- and pH-dependent manner. The negative effects of Cd on growth were attenuated by the presence of PHF, and maximum growth recovery (53.8%) was obtained at the highest PHF concentration and pH. The coexposure to Cd and PHF decreased ROS accumulation up to 36.7% and membrane disruption up to 30.7% in a dose- and pH-dependent manner. Two mechanisms helped to explain the role of PHF in alleviating Cd toxicity to yeasts: PHF decreased Cd-induced oxidative stress and bound significant amounts of Cd in the extracellular medium, reducing its bioavailability to the cells.     

Mitochondrial Respiratory Electron Carriers Are Involved in Oxidative Stress during Heat Stress in Saccharomyces cerevisiae

In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50 degrees C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H(2)O(2) at 42 as at 30 degrees C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H(2)O(2). We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.     

Evidence for a Nonendosomal Function of the Saccharomyces cerevisiae ESCRT-III-Like Protein Chm7

Endosomal sorting complex required for transport (ESCRT) proteins are involved in a number of cellular processes, such as endosomal protein sorting, HIV budding, cytokinesis, plasma membrane repair, and resealing of the nuclear envelope during mitosis. Here we explored the function of a noncanonical member of the ESCRT-III protein family, the Saccharomyces cerevisiae ortholog of human CHMP7. Very little is known about this protein. In silico analysis predicted that Chm7 (yeast ORF YJL049w) is a fusion of an ESCRT-II and ESCRT-III-like domain, which would suggest a role in endosomal protein sorting. However, our data argue against a role of Chm7 in endosomal protein sorting. The turnover of the endocytic cargo protein Ste6 and the vacuolar protein sorting of carboxypeptidase S (CPS) were not affected by CHM7 deletion, and Chm7 also responded very differently to a loss in Vps4 function compared to a canonical ESCRT-III protein. Our data indicate that the Chm7 function could be connected to the endoplasmic reticulum (ER). In line with a function at the ER, we observed a strong negative genetic interaction between the deletion of a gene function (APQ12) implicated in nuclear pore complex assembly and messenger RNA (mRNA) export and the CHM7 deletion. The patterns of genetic interactions between the APQ12 deletion and deletions of ESCRT-III genes, two-hybrid interactions, and the specific localization of mCherry fusion proteins are consistent with the notion that Chm7 performs a novel function at the ER as part of an alternative ESCRT-III complex.     

Stress tolerance of pressure-shocked Saccharomyces cerevisiae

Pressure shock treatment induced synthesis of heat shock protein (hsp104) and tolerance against various stresses such as high temperature, high pressure and high concentration of ethanol in Saccharomyces cerevisiae. The optimum pressures that induced maximal tolerance against these stresses were in the range of 50–75 MPa and depended on the type of stress. However, pressure shock did not stimulate trehalose production in the cells. © Rapid Science Ltd. 1998     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7

H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target. Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2). In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase. The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death. Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity. The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p. The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation. In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins. However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent.     

Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H₂O₂, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.     

Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae

We have analyzed the proteins that are oxidatively damaged when Saccharomyces cerevisiae cells are exposed to stressing conditions. Carbonyl groups generated by hydrogen peroxide or menadione on proteins of aerobically respiring cells were detected by Western blotting, purified, and identified. Mitochondrial proteins such as E2 subunits of both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, aconitase, heat-shock protein 60, and the cytosolic fatty acid synthase (alpha subunit) and glyceraldehyde-3-phosphate dehydrogenase were the major targets. In addition we also report the in vivo modification of lipoamide present in the above-mentioned E2 subunits under the stressing conditions tested and that this also occurs with the homologous enzymes present in Escherichia coli cells that were used for comparative analysis. Under fermentative conditions, the main protein targets in S. cerevisiae cells treated with hydrogen peroxide or menadione were pyruvate decarboxylase, enolase, fatty acid synthase, and glyceraldehyde-3-phosphate dehydrogenase. Under the stress conditions tested, fermenting cells exhibit a lower viability than aerobically respiring cells and, consistently, increased peroxide generation as well as higher content of protein carbonyls and lipid peroxides. Our results strongly suggest that the oxidative stress in prokaryotic and eukaryotic cells shares common features.     

酿酒酵母NAD(H)激酶Pos5p在细胞抵抗氧化胁迫中的作用

酿酒酵母线粒体NAD(H)激酶Pos5p显示出重要功能,其缺失将导致细胞抗氧化性能出现障碍。为了了解Pos5p的抗氧化作用机制及其与调节辅酶NAD(H)和NADP(H)之间的关系,比较了在不同类型的氧化胁迫试剂作用下,野生型BY4742、POS5基因缺失体pos5-及其回补体pos5-/POS5-YEp的生长表型,同时采用高效液相色谱测定细胞内辅酶含量。结果表明,在超氧生成试剂甲萘醌(VK3)、过氧化氢(H2O2)和GSH消耗试剂马来酸二乙酯(DEM)存在时,pos5-都表现出明显的生长缺陷,而各抗氧化基因缺失体只在其相应胁迫下表现出生长缺陷。在正常生长条件下,pos5-的NADPH含量降低,pos5-/POS5-YEp则提高,表明Pos5p对胞内NADPH的供应有重要作用。在VK3、H2O2和DEM胁迫下,BY4742、pos5-及pos5-/POS5-YEp的NADP(H)含量均有不同程度的下降,其中pos5-的NADP(H)/NAD(H)比率下降最为严重,而pos5-/POS5-YEp较pos5-有明显提高,这与其氧化胁迫表型相一致。因此,在细胞面临不同类型的氧化胁迫时,Pos5p都能有效行使其NAD(H)激酶活性,补充NADP(H)的损耗,从而对细胞起到抗氧化保护作用。