Effect of nitrosative stress on Schizosaccharomyces pombe: inactivation of glutathione reductase by peroxynitrite

Oxidative stress has been shown to alter cellular redox status in various cell types. Changes in expressions of several antioxidative and antistress-responsive genes along with activation or inactivation of various proteins were also reported during oxidative insult as well as during nitrosative stress. In the present study, we show the effect of nitrosative stress on cellular redox status of fission yeast Schizosaccharomyces pombe. This is the first report of S-nitrosoglutathione (GSNO) reductase activity in S. pombe and its inactivation by GSNO. We also show the inactivation of glutathione reductase (GR) and glutathione peroxidase in the presence of various reactive nitrogen species in vivo. In addition, we first observe the inactivation of GR by peroxynitrite in vivo using S. pombe cells and also similar observations under in vitro conditions. An immunoreactive band against monoclonal anti-3-nitrotyrosine antibody confirms the modification of GR under in vitro conditions. We also show the effect of nitrosative stress on Deltapap1 cells of S. pombe, which are more sensitive to nitrosative stress, indicating the involvement of Pap1 in the protection against nitrosative stress. Finally, exposure of S. pombe cells to reactive nitrogen species reveals an important role of cellular thiol pool in protection against nitrosative stress.     

Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.     

Genes involved in glucose repression and oxidative stress response in the fission yeast Schizosaccharomyces pombe

We looked for changes in gene expression and novel genes that could be involved in the interaction between glucose repression and oxidative stress response in the fission yeast, Schizosaccharomyces pombe, using a constitutive invertase mutant, ird11, which is resistant to glucose. BLAST analysis was made of the S. pombe genome database of cDNAs whose expression ratios differentially decreased or increased upon exposure to mild oxidative stress in this mutant compared to the wild type. Genes with this type of activity were identified as rpl302, encoding 60S ribosomal protein L3, and mpg1, encoding mannose-1-phosphate guanyltransferase; their expression patterns were measured using quantitative real-time PCR. We found that the expression levels of rpl302 and mpg1 genes in ird11 under unstressed conditions were increased compared to those of the wild type. Under stress conditions, the expression levels of the rpl302 gene were decreased in both strains, while mpg1 expression levels remained unchanged. These results suggest that these genes play a role in the response to oxidative stress in this mutant strain.     

Oxidative and nitrosative stress in Staphylococcus aureus biofilm

Diverse chemical and physical agents can alter cellular functions associated with oxidative metabolism, thus stimulating the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) in planktonic bacterial physiology. However, more research is necessary to determine the precise role of cellular stress in biofilm. The present study was designed to address the issues of Staphylococcus aureus biofilm formation with respect to the generation of oxidative and nitrosative stress. We studied three pathogenic S. aureus clinical strains and an ATCC strain exposed to a different range of culture conditions (time, temperature, pH, reduction and atmospheric conditions) using quantitative methods of biofilm detection. We observed that cellular stress could be produced inside biofilms, thereby affecting their growth, resulting in an increase of ROS and RNI production, and a decrease of the extracellular matrix under unfavorable conditions. These radical oxidizers could then accumulate in an extracellular medium and thus affect the matrix. These results contribute to a better understanding of the processes that enable adherent biofilms to grow on inert surfaces and lead to an improved knowledge of ROS and RNI regulation, which may help to clarify the relevance of biofilm formation in medical devices.     

Cellular functions and transcriptional regulation of a third thioredoxin from Schizosaccharomyces pombe

The structural gene encoding a third thioredoxin (Trx) homologue, TRX3, of the fission yeast Schizosaccharomyces pombe was characterized and its regulation was studied. The determined DNA sequence encoded a putative 290 amino acid sequence of Trx with a molecular mass of 31,889 Da. The TRX3 mRNA level was increased in S. pombe cells harboring plasmid pTRX3, suggesting that the cloned TRX3 gene was functional. Yeast cultures harbouring plasmid pTRX3 exhibited shorter generation times and higher survival on solid minimal media plates incorporating mercury chloride (0.01 mmol/L) or hydrogen peroxide (1 mmol/L) compared with control cultures. Yeast cells containing extra copies of TRX3, but not TRX1 and TRX2, gave rise to lower reactive oxygen species levels than control cells. Oxidative stress owing to hydrogen peroxide and menadione enhanced the synthesis of beta-galactosidase from the TRX3-lacZ fusion gene in Pap1-positive cells but not in Pap1-negative cells. The TRX3 mRNA level was increased by oxidative stress only in Pap1-positive cells. Basal expression of the TRX3 gene also depended on Pap1. We concluded that S. pombe TRX3 is linked with yeast growth and oxidative stress response, with its expression being regulated by oxidative stress in a Pap1-dependent manner.     

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.     

Direct activation of the fission yeast PAK Shk1 by the novel SH3 domain protein, Skb5

The p21-activated kinase (PAK) homolog Shk1 is essential for cell viability in the fission yeast Schizosaccharomyces pombe. Roles have been established for Shk1 in the regulation of cell morphology, sexual differentiation, and mitosis in S. pombe. In this report, we describe the genetic and molecular characterization of a novel SH3 domain protein, Skb5, identified as a result of a two-hybrid screen for Shk1 interacting proteins. S. pombe cells carrying a deletion of the skb5 gene exhibit no discernible phenotypic defects under normal growth conditions, but when subjected to hypertonic stress, become spheroidal in shape and growth impaired. Both of these defects can be suppressed by overexpression of the Shk1 modulator, Skb1. The growth inhibition that results from overexpression of Shk1 in S. pombe cells is markedly suppressed by a null mutation in the skb5 gene, suggesting that Skb5 contributes positively to the function of Shk1 in vivo. Consistent with this notion, we show that Skb5 stimulates Shk1 catalytic function in S. pombe cells. Furthermore, and perhaps most significantly, we show that bacterially expressed recombinant Skb5 protein directly stimulates the catalytic activity of recombinant Shk1 kinase in vitro. These and additional data described herein demonstrate that Skb5 is a direct activator of Shk1 in fission yeast.     

Saccharomyces cerevisiae Hsp31p, a stress response protein conferring protection against reactive oxygen species

The Saccharomyces cerevisiae HSP31 (YDR533c) gene encodes a protein that belongs to the DJ-1/PfpI family and its function is unknown. Homologs to Hsp31p polypeptide can be found in organisms from all systematic groups of eukaryotes and prokaryotes, and the functions of the vast majority of them are also hypothetical. One of the homologs is human protein DJ-1. Various amino acid substitutions within this protein correlate with early onset hereditary Parkinson's disease. The deletion of the HSP31 gene displays no apparent phenotype under standard growth conditions, but a thorough functional analysis of S. cerevisiae revealed that its absence makes the cells sensitive to a subset of reactive oxygen species (ROS) generators. HSP31 is induced under conditions of oxidative stress in a YAP1-dependent manner. Similar to other stress response genes, it is also induced in the postdiauxic phase of growth and this induction is YAP1-independent. The patterns of sensitivities to various ROS generators of the hsp31Delta strain and the strain with the deletion of SOD1, another gene defending the cell against ROS, are different. We postulate that Hsp31p protects the cell against oxidative stress and complements other stress protection systems within the cell.     

eIF4E isoform 2 in Schizosaccharomyces pombe is a novel stress-response factor

Cap-binding proteins of the elF4E family are generally involved in mediating ribosome recruitment to capped mRNA via an interaction with the initiation factor elF4G. However, Schizosaccharomyces pombe has two elF4E isoforms, one of which (elF4E2, encoded by tif452) has a relatively low affinity for elF4G. We show that tif452 is required for specific stress responses. An S. pombe, tif452delta mutant manifests slow growth under conditions of nutrient, temperature and salt stress. elF4E2 shows a distinct subcellular distribution to elF4E1, the cap-binding factor that is required for mainstream translation. In response to salt stress, the cellular level of elF4E2 increases, whereas the amount of intact elF4G decreases, leaving elF4E2 as the predominant elF4E isoform in a cell deficient in ElF4G. The presence of elF4E2 modifies the competence of S. pombe ribosomes to translate mRNAs with structured leaders in vivo. The tif452 promoter has putative stress-response (T-rich) motifs, whereas elF4E2 seems to be a new type of stress-response factor.     

The role of protein quality control in mitochondrial protein homeostasis under oxidative stress

Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS‐dependent proteolysis. Enzymes containing oxidation‐sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress‐dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP‐dependent protease Pim1/LON as a major factor in the degradation of ROS‐modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.