Characterization of an inducible oxidative stress response in Vitreoscilla C1

Vitreoscilla becomes resistant to killing by hydrogen peroxide and heat shock when pretreated with nonlethal levels of hydrogen peroxide. The pretreated Vitreoscilla cells (60 microM hydrogen peroxide for 120 min) significantly increased survival of the lethal dose of 20 mM hydrogen peroxide or heat shock (22 degrees C --> 37 degrees C). This indicates the existence of an adaptive response to oxidative stress. However, cells pretreated with 60 microM hydrogen peroxide became nonresistant to a lethal dose of a menadione. This result shows that hydrogen peroxide does not induce cross-resistance to menadione in Vitreoscilla. Furthermore, Vitreoscilla treated with hydrogen peroxide, heat shock, and menadione showed a change in the protein composition, as monitored by a two-dimensional gel analysis. During adaptation to hydrogen peroxide, 12 proteins were induced. Also, 18 new proteins synthesized in response to heat shock were detected by a 2-D gel analysis. The redox-cycling agents also elicited the synthesis of 6 other proteins that were unseen with hydrogen peroxide.     

Proteomic analysis of antioxidant strategies of Staphylococcus aureus: diverse responses to different oxidants

The high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF MS and a recently developed fluorescence-based thiol modification assay were used to investigate the cellular response of Staphylococcus aureus to oxidative stress. Addition of hydrogen peroxide, diamide, and the superoxide generating agent paraquat to exponentially growing cells revealed complex changes in the protein expression pattern. In particular, proteins involved in detoxification, repair systems, and intermediary metabolism were found to be up-regulated. Interestingly, there is only a small overlap of proteins induced by all these stressors. Exposure to hydrogen peroxide mediated a significant increase of DNA repair enzymes, whereas treatment with diamide affected proteins involved in protein repair and degradation. The activity of proteins under oxidative stress conditions can be modulated by oxidation of thiol groups. In growing cells, protein thiols were found to be mainly present in the reduced state. Diamide mediated a strong increase of reversibly oxidized thiols in a variety of metabolic enzymes. By contrast, hydrogen peroxide resulted in the reversible oxidation especially of proteins with active site cysteines. Moreover, high levels of hydrogen peroxide influenced the pI of three proteins containing cysteines within their active sites (GapA1, AhpC, and HchA) indicating the generation of sulfinic or sulfonic acid by irreversible oxidation of thiols.     

The Saccharomyces cerevisiae Sln1p-Ssk1p two-component system mediates response to oxidative stress and in an oxidant-specific fashion

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, environmental gamma and UV radiation, as well as several chemicals also generate reactive oxygen species, which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Oxidative stress induces the expression of several genes in yeast Saccharomyces cerevisiae. However, the primary sensor(s) that trigger the response is unknown. This study demonstrates that primary sensors of osmotic stress, the Sln1p-Ssk1p two-component proteins, are involved in sensing oxidative stress specifically induced by hydrogen peroxide and diamide, but not by other oxidants used in the study. Wild type and sln1-ssk1 mutant were treated with hydrogen peroxide, diamide, menadione, UV, and gamma-radiation. Results show that sln1-ssk1 mutant is only sensitive to hydrogen peroxide and diamide but not to other oxidants. S. cerevisiae contains an additional cell surface osmosensor, Sho1p, that targets the osmotic signal to Hog1p. Data is presented that shows Sho1 and Hog1 proteins are also involved in signaling oxidant-specific cellular damage. Furthermore, it is demonstrated that expression of the mammalian homolog of Hog1p provides protection from oxidative stress induced by hydrogen peroxide. These results suggest that Sln1p-Ssk1p and Sho1p signal transduction pathways participate in oxidative stress response. However, this response to oxidative stress is limited to specific oxidants.     

Proteasome-dependent turnover of protein disulfide isomerase in oxidatively stressed cells.

Generalized increases in protein oxidation and protein degradation in response to mild oxidative stress have been widely reported, but only a few individual proteins have actually been shown to undergo selective, oxidation-induced proteolysis. Our goal was to find such proteins in Clone 9 liver cells exposed to hydrogen peroxide. Using metabolic radiolabeling of intracellular proteins with [35S]cysteine/methionine, and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), we found at least three labeled proteins ("A," "B," and "C") whose levels were decreased significantly more than the generalized protein loss after mild oxidative stress. "Protein C" was excised from 2-D PAGE and subjected to N-terminal amino acid microsequencing. "Protein C" was identified as Protein Disulfide Isomerase or PDI (E.C. 5.3.4.1), and this identity was reconfirmed by Western blotting with a C-terminal anti-PDI monoclonal antibody. A combination of quantitative radiometry and Western blotting in 2-D PAGE revealed that PDI was selectively degraded and then new PDI was synthesized, following H2O2 exposure. PDI degradation was blocked by inhibitors of the proteasome, and by cell treatment with proteasome C2 subunit antisense oligonucleotides, indicating that the proteasome was largely responsible for oxidation-induced PDI degradation.     

Oxidative stress response in an anaerobe, Bacteroides fragilis: a role for catalase in protection against hydrogen peroxide.

Survival of Bacteroides fragilis in the presence of oxygen was dependent on the ability of bacteria to synthesize new proteins, as determined by the inhibition of protein synthesis after oxygen exposure. The B. fragilis protein profile was significantly altered after either a shift from anaerobic to aerobic conditions with or without paraquat or the addition of exogenous hydrogen peroxide. As determined by autoradiography after two-dimensional gel electrophoresis, approximately 28 newly synthesized proteins were detected in response to oxidative conditions. These proteins were found to have a broad range of pI values (from 5.1 to 7.2) and molecular weights (from 12,000 to 79,000). The hydrogen peroxide- and paraquat-inducible responses were similar but not identical to that induced by oxygen as seen by two-dimensional gel protein profile. Eleven of the oxidative response proteins were closely related, with pI values and molecular weights from 5.1 to 5.8 and from 17,000 to 23,000, respectively. As a first step to understanding the resistance to oxygen, a catalase-deficient mutant was constructed by allelic gene exchange. The katB mutant was found to be more sensitive to the lethal effects of hydrogen peroxide than was the parent strain when the ferrous iron chelator bipyridyl was added to culture media. This suggests that the presence of ferrous iron in anaerobic culture media exacerbates the toxicity of hydrogen peroxide and that the presence of a functional catalase is important for survival in the presence of hydrogen peroxide. Further, the treatment of cultures with a sublethal concentration of hydrogen peroxide was necessary to induce resistance to higher concentrations of hydrogen peroxide in the parent strain, suggesting that this was an inducible response. This was confirmed when the bacterial culture, treated with chloramphenicol before the cells were exposed to a sublethal concentration of peroxide, completely lost viability. In contrast, cell viability was greatly preserved when protein synthesis inhibition occurred after peroxide induction. Complementation of catalase activity in the mutant restored the ability of the mutant strain to survive in the presence of hydrogen peroxide, showing that the catalase (KatB) may play a role in oxidative stress resistance in aerotolerant anaerobic bacteria.     

Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts

We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H2O2, and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H2O2, the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein.     

Pkc1 and the upstream elements of the cell integrity pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, are required for cellular responses to oxidative stress

In this study we analyze the participation of the PKC1-MAPK cell integrity pathway in cellular responses to oxidative stress in Saccharomyces cerevisiae. Evidence is presented demonstrating that only Pkc1 and the upstream elements of the cell integrity pathway are essential for cell survival upon treatment with two oxidizing agents, diamide and hydrogen peroxide. Mtl1 is characterized for the first time as a cell-wall sensor of oxidative stress. We also show that the actin cytoskeleton is a cellular target for oxidative stress. Both diamide and hydrogen peroxide provoke a marked depolarization of the actin cytoskeleton, being Mtl1, Rom2 and Pkc1 functions all required to restore the correct actin organization. Diamide induces the formation of disulfide bonds in newly secreted cell-wall proteins. This mainly provokes structural changes in the cell outer layer, which activate the PKC1-MAPK pathway and hence the protein kinase Slt2. Our results led us to the conclusion that Pkc1 activity is required to overcome the effects of oxidative stress by: (i) enhancing the machinery required to repair the altered cell wall and (ii) restoring actin cytoskeleton polarity by promoting actin cable formation.     

Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis

Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. In this study, we developed a fluorescence-based thiol modification assay and combined it with two-dimensional gel electrophoresis and mass spectrometry to monitor the in vivo thiol state of cytoplasmic proteins. For the Gram-positive model organism Bacillus subtilis our results show that protein thiols of growing cells are mainly present in the reduced state. Only a few proteins were found to be thiol-modified, e.g. enzymes that include oxidized thiols in their catalytic cycle. To detect proteins that are particularly sensitive to oxidative stress we exposed growing B. subtilis cells to diamide, hydrogen peroxide or to the superoxide generating agent paraquat. Diamide mediated a significant increase of oxidized thiols in a variety of metabolic enzymes, whereas treatment with paraquat affected only a few proteins. Exposure to hydrogen peroxide forced the oxidation especially of proteins with active site cysteines, e.g. of cysteine-based peroxidases and glutamine amidotransferase-like proteins. Moreover, high levels of hydrogen peroxide were observed to influence the isoelectric point of proteins of this group indicating the generation of irreversibly oxidated thiols. From the overlapping set of oxidatively modified proteins, also enzymes necessary for methionine biosynthesis were identified, e.g. cobalamin-independent methionine synthase MetE. Growth experiments revealed a methionine limitation after diamide and hydrogen peroxide stress, which suggests a thiol-oxidation-dependent inactivation of MetE. Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis.     

Oligomerization of water soluble proteins of rabbit crystalline lens under the action of diamide

The study has examined the effects of the SH-oxidizing agent diamide (Diazane dicarboxylic acid bis-(N,N-dimethyl-amide)) on the water-soluble portion of proteins from rabbit lenses. The dialyzed protein extracts were incubated for 1-1.5 hrs with various concentrations of diamide. Treatments were monitored for alterations in sulphydryl contents, gel filtration and gel electrophoresis profiles of proteins. The response to 2 mM diamide treatment for 1 hr consists of rapid oxidation (up to 40%) of protein-bound sulphydryl groups accompanied by an appearance of polypeptides with apparent molecular weights. The protein with molecular weight of 29 kilodaltons was shown to be involved in cross-linking. The linkages in the dialyzed water-soluble lens polypeptide fraction induced by diamide may be reduced by GSH (10 mM) treatment of protein extract. The main target of oxidative insult induced by diamide in the water-soluble proteins of the lens is probably the superficially localized sulphydryl groups of crystallins. Our observations suggest that the described oxidative system of proteins may be a useful tool for cataract research.     

Identification of a novel response regulator, Crr1, that is required for hydrogen peroxide resistance in Candida albicans

Candida albicans colonises numerous niches within humans and thus its success as a pathogen is dependent on its ability to adapt to diverse growth environments within the host. Two component signal transduction is a common mechanism by which bacteria respond to environmental stimuli and, although less common, two component-related pathways have also been characterised in fungi. Here we report the identification and characterisation of a novel two component response regulator protein in C. albicans which we have named CRR1 (Candida Response Regulator 1). Crr1 contains a receiver domain characteristic of response regulator proteins, including the conserved aspartate that receives phosphate from an upstream histidine kinase. Significantly, orthologues of CRR1 are present only in fungi belonging to the Candida CTG clade. Deletion of the C. albicans CRR1 gene, or mutation of the predicted phospho-aspartate, causes increased sensitivity of cells to the oxidising agent hydrogen peroxide. Crr1 is present in both the cytoplasm and nucleus, and this localisation is unaffected by oxidative stress or mutation of the predicted phospho-aspartate. Furthermore, unlike the Ssk1 response regulator, Crr1 is not required for the hydrogen peroxide-induced activation of the Hog1 stress-activated protein kinase pathway, or for the virulence of C. albicans in a mouse model of systemic disease. Taken together, our data suggest that Crr1, a novel response regulator restricted to the Candida CTG clade, regulates the response of C. albicans cells to hydrogen peroxide in a Hog1-independent manner that requires the function of the conserved phospho-aspartate.     

Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus

The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.     

The whcA gene plays a negative role in oxidative stress response of Corynebacterium glutamicum

In this study, we analyzed the whcA gene from Corynebacterium glutamicum , which codes for a homologue of the WhiB-family of proteins. Deletion of the gene did not affect the growth of the mutant cells, indicating that the whcA gene was not essential under ordinary growth conditions. However, cells overexpressing the protein not only showed retarded growth as compared with the wild-type or the Δ whcA mutant cells but also showed increased sensitivity to a variety of oxidants, such as diamide, menadione, and hydrogen peroxide. Thioredoxin reductase activity was repressed in the whcA -overexpressing cells, whereas its activity in the Δ whcA mutant strain was derepressed regardless of the presence of oxidative stress. The whcA gene was constitutively expressed throughout the growth phase and its expression level was not affected by oxidative stress. A set of proteins under the control of whcA were identified by two-dimensional polyacrylamide gel electrophoresis and they were annotated as NADH oxidase, alcohol dehydrogenase, quinone reductase, and cysteine desulfurase. The corresponding genes encoding the identified proteins were not transcribed in Δ sigH mutant cells. Collectively, these data suggest that the whcA gene of C. glutamicum plays a negative role in the sigH -mediated stress response pathway.     

Patterns of protein carbonylation following oxidative stress in wild-type and<Emphasis Type="Italic"> sigB Bacillus subtilis</Emphasis> cells

Oxidative stress causes damage to nucleic acids, membrane lipids and proteins. One striking effect is the metal-catalyzed, site-specific carbonylation of proteins. In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the ^B-dependent general stress response act together to make cells more resistant to oxidative stress. In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B. subtilis. Furthermore, we asked whether the ^B-dependent response to oxidative stress also confers protection against protein carbonylation. To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis. The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide. Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated. Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation. Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells. We could not detect any differences between wild-type and sigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress. However, artificial preloading with proteins that are normally induced by ^B-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.Communicated by W. Goebel     

Differential correlations between changes to glutathione redox state,protein ubiquitination,and stress-inducible HSPA chaperone expression after different types of oxidative stress

In primary bovine fibroblasts with an hspa1b/luciferase transgene, we examined the intensity of heat-shock response (HSR) following four types of oxidative stress or heat stress (HS), and its putative relationship with changes to different cell parameters, including reactive oxygen species (ROS), the redox status of the key molecules glutathione (GSH), NADP(H) NAD(H), and the post-translational protein modifications carbonylation, S-glutathionylation, and ubiquitination. We determined the sub-lethal condition generating the maximal luciferase activity and inducible HSPA protein level for treatments with hydrogen peroxide (H₂O₂), UVA-induced oxygen photo-activation, the superoxide-generating agent menadione (MN), and diamide (DA), an electrophilic and sulfhydryl reagent. The level of HSR induced by oxidative stress was the highest after DA and MN, followed by UVA and H₂O₂ treatments, and was not correlated to the level of ROS production nor to the extent of protein S-glutathionylation or carbonylation observed immediately after stress. We found a correlation following oxidative treatments between HSR and the level of GSH/GSSG immediately after stress, and the increase in protein ubiquitination during the recovery period. Conversely, HS treatment, which led to the highest HSR level, did not generate ROS nor modified or depended on GSH redox state. Furthermore, the level of protein ubiquitination was maximum immediately after HS and lower than after MN and DA treatments thereafter. In these cells, heat-induced HSR was therefore clearly different from oxidative stress-induced HSR, in which conversely early redox changes of the major cellular thiol predicted the level of HSR and polyubiquinated proteins.