Oxygen stress: a regulator of apoptosis in yeast.

Oxygen radicals are important components of metazoan apoptosis. We have found that apoptosis can be induced in the yeast Saccharomyces cerevisiae by depletion of glutathione or by low external doses of H2O2. Cycloheximide prevents apoptotic death revealing active participation of the cell. Yeast can also be triggered into apoptosis by a mutation in CDC48 or by expression of mammalian bax. In both cases, we show oxygen radicals to accumulate in the cell, whereas radical depletion or hypoxia prevents apoptosis. These results suggest that the generation of oxygen radicals is a key event in the ancestral apoptotic pathway and offer an explanation for the mechanism of bax-induced apoptosis in the absence of any established apoptotic gene in yeast.     

Effect of temperature on ascorbate peroxidase activity and flowering of Arabidopsis thaliana ecotypes under different light conditions

Four Arabidopsis thaliana ecotypes were grown at 14 degrees C and 22 degrees C under two light conditions (300 microE m-2 s-1 or 150 microE m-2 s-1) and the effect of temperature on their growth and flowering time was studied. Flowering occurred within 31 days (experimental period) at 22 degrees C, whereas a decrease in growth temperature resulted in a delay in flowering (63 days) under both light conditions. At 14 degrees C, membrane-bound APX (tAPX) activity decreased and total chlorophyll (Chl) content increased with growth under both light conditions. However, at 22 degrees C, the tAPX activity increased and total Chl content decreased with growth under both light conditions. These results suggest that at 22 degrees C oxidative stress was high under both light conditions and consequently Chl content decreased under stressful conditions or vice versa for all the four A. thaliana ecotypes studied. Under both the temperature and light conditions, soluble APX activity showed an irregular pattern of growth. The increase in tAPX activity, with growth only at 22 degrees C but not at 14 degrees C, suggests increased H2O2 formation in flowering plants at 22 degrees C for all the four A. thaliana ecotypes studied. Before flowering, the tAPX activity showed a significantly negative correlation with flowering time. Higher oxidative stress in the lower-latitude ecotypes might induce earlier flowering than the higher-latitude ecotypes. From these results, we propose a hypothesis that H2O2 is one of the possible factors in flower induction.     

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.     

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.     

Bax-induced cell death of Arabidopsisis meditated through reactive oxygen-dependent and -independent processes

An Arabidopsisprotoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsismesophyll protoplasts expressing murine BaxcDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivotargeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N-acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsisnucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of ArabidopsisBax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.     

Prevention of geranylgeranoic acid-induced apoptosis by phospholipid hydroperoxide glutathione peroxidase gene

Micromolar concentrations (0.5 approximately 5 microM) of all-trans geranylgeranoic acid (GGA) induced cell death in a guinea pig cell line, 104C1, whereas under the same conditions GGA was unable to kill 104C1/O4C, a clone established from 104C1 cells by transfection of them with the human phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene. GGA (5 microM) induced a loss of the mitochondrial inner membrane potential (DeltaPsim) in 104C1 cells in 2 h, and their apoptotic cell death became evident in 6 h. On the other hand, 104C1/O4C cells were resistant to loss of DeltaPsim and showed intact morphology until at least 24 h after addition of 10 microM GGA. Dihydroethidine, superoxide-sensitive probe, was immediately oxidized 15 min after addition of GGA in both 104C1 and 104C1/O4C cells. The peroxide-sensitive probe 2',7'-dichlorofluorescin diacetate (H2-DCF-DA) was strongly oxidized in 104C1 cells 4 h after the addition of 2.5 microM GGA, but not in 104C1/O4C cells even in the presence of 10 microM GGA. The present results suggest that GGA induced a hyper-production of superoxide and subsequently peroxides, which in turn may have led to dissipation of the DeltaPsim and final apoptotic cell death in 104C1 cells.     

Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells

The biological activities of C₆₀-bis(N,N-dimethylpyrrolidinium iodide), a water-soluble cationic fullerene derivative, on human promyeloleukaemia (HL-60) cells were investigated. The pyrrolidinium fullerene derivative showed cytotoxicity in HL-60 cells. The characteristics of apoptosis, such as DNA fragmentation and condensation of chromatin in HL-60 cells, were observed by exposure to the pyrrolidinium fullerene derivative. Caspase-3 and -8 were activated and cytochrome c was also released from mitochondria. The generation of reactive oxygen species (ROS) by the pyrrolidinium fullerene derivative was observed by DCFH-DA, a fluorescence probe for the detection of ROS. Pre-treatment with α-tocopherol suppressed cell death and intracellular oxidative stress caused by the pyrrolidinium fullerene derivative. The apoptotic cell death induced by the pyrrolidinium fullerene derivative was suggested to be mediated by ROS generated by the pyrrolidinium fullerene derivative.     

Role of reactive oxygen species (ROS) in apoptosis induction

Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS. Cytochrome c release from mitochondria, that triggers caspase activation, appears to be largely mediated by direct or indirect ROS action. On the other hand, ROS have also anti-apoptotic effects. This review focuses on the role of ROS in the regulation of apoptosis, especially in inflammatory cells.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Diethyldithiocarbamate inhibits in vivo Cu,Zn-superoxide dismutase and perturbs free radical processes in the yeast Saccharomyces cerevisiae cells

Copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese superoxide dismutase (Mn-SOD) in some model experiments in vitro demonstrated antioxidant as well as pro-oxidant properties. In the present study, yeast Saccharomyces cerevisiae lacking Mn-SOD were studied using Cu,Zn-SOD inhibitor N-N'-diethyldithiocarbamate (DDC) as a model system to study the physiological role of the yeast Cu,Zn-SOD. Yeast treatment by DDC caused dose-dependent inhibition of SOD in vivo, with 75% inhibition at 10mM DDC. The inhibition of SOD by DDC resulted in modification of carbonylprotein levels, indicated by a bell-shaped curve. The activity of glutathione reductase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase (enzymes associated with antioxidant) increased, demonstrating a compensatory effect in response to SOD inhibition by different concentrations of DDC. A strong positive correlation (R2=0.97) was found between SOD and catalase activities that may be explained by the protective role of SOD for catalase. All observed effects were absent in the isogenic SOD-deficient strain that excluded direct DDC influence. The results are discussed from the point of view that in vivo Cu,Zn-SOD of S. cerevisiae can demonstrate both anti- and pro-oxidant properties.     

Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species

CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis.     

Production of recombinant proteins by yeast cells

Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed.     

The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.