Inactivation of human peroxiredoxin I during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid

By following peroxiredoxin I (Prx I)-dependent NADPH oxidation spectrophotometrically, we observed that Prx I activity decreased gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by two-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys(51)-SH to Cys(51)-SO(2)H. Thus, Cys(51)-SOH generated as an intermediate during catalysis appeared to undergo occasional further oxidation to Cys(51)-SO(2)H, which cannot be reversed by thioredoxin. The presence of H(2)O(2) alone was not sufficient to cause oxidation of Cys(51) to Cys(51)-SO(2)H. Rather, the presence of complete catalytic components (H(2)O(2), thioredoxin, thioredoxin reductase, and NADPH) was necessary, indicating that such hyperoxidation occurs only when Prx I is engaged in the catalytic cycle. Likewise, hyperoxidation of Cys(172)/Ser(172) mutant Prx I required not only H(2)O(2), but also a catalysis-supporting thiol (dithiothreitol). Kinetic analysis of Prx I inactivation in the presence of a low steady-state level (<1 microm) of H(2)O(2) indicated that Prx I was hyperoxidized at a rate of 0.072% per turnover at 30 degrees C. Hyperoxidation of Prx I was also detected in HeLa cells treated with H(2)O(2).     

Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro

Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliana, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cys(149). Catalytic Cys(149) is the only solvent-exposed cysteine of the protein and its thiol is relatively acidic (pK(a)=5.7). This property makes GapC1 sensitive to oxidation by H(2)O(2), which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO(2)(-) and -SO(3)(-)) via a labile sulfenate intermediate (-SO(-)). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSH-dependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic Cys(149).     

Differential susceptibilities of serine/threonine phosphatases to oxidative and nitrosative stress

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are signal-transducing molecules that regulate the activities of a variety of proteins. In the present investigation, we have compared the effects of superoxide (O2-), nitric oxide (NO), and hydrogen peroxide (H2O2) on the activities of three highly homologous serine/threonine phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), and calcineurin (protein phosphatase type 2B). Although superoxide, generated from xanthine/xanthine oxidase or paraquat, and NO, generated from (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide or sodium nitroprusside, potently inhibited the phosphatase activity of calcineurin in neuroblastoma cell lysates, they had relatively little effect on the activities of PP1 or PP2A. In contrast, H2O2 inhibited the activities of all three phosphatases in lysates but was not a potent inhibitor for any of the enzymes. Calcineurin inactivated by O2-, NO, and H2O2 could be partially reactivated by the reducing agent ascorbate or by the thiol-specific reagent dithiothreitol (DTT). Maximal reactivation was achieved by the addition of both reagents, which suggests that ROS and RNS inhibit calcineurin by oxidizing both a catalytic metal(s) and a critical thiol(s). Reactivation of H2O2-treated PP1 also required the combination of both ascorbate and DTT, whereas PP2A required only DTT for reactivation. These results suggest that, despite their highly homologous structures, calcineurin is the only major Ser/Thr phosphatase that is a sensitive target for inhibition by superoxide and nitric oxide and that none of the phosphatases are sensitive to inhibition by hydrogen peroxide.     

Abscisic Acid Activation of Plasma Membrane Ca2+ Channels in Guard Cells Requires Cytosolic NAD(P)H and Is Differentially Disrupted Upstream and Downstream of Reactive Oxygen Species Production in abi1-1 and abi2-1 Protein Phosphatase 2C Mutants

The hormone abscisic acid (ABA) regulates stress responses and developmental processes in plants. Calcium-permeable channels activated by reactive oxygen species (ROS) have been shown recently to function in the ABA signaling network in Arabidopsis guard cells. Here, we report that ABA activation of these I(Ca) Ca(2)+ channels requires the presence of NAD(P)H in the cytosol. The protein phosphatase 2C (PP2C) mutant abi1-1 disrupted ABA activation of I(Ca) channels. Moreover, in abi1-1, ABA did not induce ROS production. Consistent with these findings, in abi1-1, H(2)O(2) activation of I(Ca) channels and H(2)O(2)-induced stomatal closing were not disrupted, suggesting that abi1-1 impairs ABA signaling between ABA reception and ROS production. The abi2-1 mutation, which lies in a distinct PP2C gene, also disrupted ABA activation of I(Ca). However, in contrast to abi1-1, abi2-1 impaired both H(2)O(2) activation of I(Ca) and H(2)O(2)-induced stomatal closing. Furthermore, ABA elicited ROS production in abi2-1. These data suggest a model with the following sequence of events in early ABA signal transduction: ABA, abi1-1, NAD(P)H-dependent ROS production, abi2-1, I(Ca) Ca(2)+ channel activation followed by stomatal closing.     

Reversible inhibition of the protein phosphatase 1 by hydrogen peroxide. Potential regulation of eIF2 alpha phosphorylation in differentiated PC12 cells

Oxidative inactivation of protein tyrosine phosphatases and calcineurin is a well established mechanism; however, little information with regard to the effect of oxidants on PP1 and PP2A activity is available. Herein, we show that PP1 activity is inhibited by H(2)O(2) treatment in differentiated PC12 cells both in vitro and in vivo experiments. Thiol-antioxidant N-acetyl-cysteine (NAC) and reduced glutathione (GSH), when added in vitro to lysates from H(2)O(2)-treated cells, reversed PP1 inhibition. H(2)O(2) treatment increased eIF2 alpha phosphorylated levels (eIF2 alpha P) in a time- and dose-dependent fashion and promoted protein synthesis inhibition. Interestingly, NAC pretreatment protected cells from H(2)O(2)-induced PP1 inactivation and, consequently, it abolished increased H(2)O(2)-induced eIF2 alpha phosphorylation and protein synthesis inhibition. In addition, PP1 inhibitor tautomycin prevented both NAC-induced PP1 reactivation and eIF2 alpha P dephosphorylation in H(2)O(2)-treated cells. Taken together, our findings support a role for PP1 in eIF2 alpha phosphorylation and oxidative stress-triggered translation down regulation.     

Activation of insulin-like growth factor type-1 receptor is required for H2O2-induced PKB phosphorylation in vascular smooth muscle cells

Evidence accumulated in recent years has revealed a potential role for reactive oxygen species (ROS) in the pathophysiology of cardiovascular diseases. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully established. Previous work from our laboratory has indicated that exogenous hydrogen peroxide (H2O2) activates several signaling protein kinases, such as extracellular signal-regulated kinase 1 and 2 (ERK1/2) and protein kinase B (PKB) in A10 vascular smooth muscle cells (VSMC). However, the upstream elements responsible for this activation remain unclear. Although a role for epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK) in H2O2-induced ERK1/2 signaling has been suggested, the contribution of this PTK or other receptor or nonreceptor PTKs to PKB activation is not well defined in VSMC. In this study, we used pharmacological inhibitors to investigate the role of receptor and Src-family-PTKs in H2O2-induced PKB phosphorylation. AG1478, a specific inhibitor of EGFR, failed to attenuate the H2O2-induced increase in PKB Ser473 phosphorylation, whereas AG1024, an inhibitor of insulin-like growth factor type1 receptor (IGF-1R)-PTK, almost completely blocked this response. H2O2 treatment also enhanced tyrosine phosphorylation of the IGF-1Rbeta subunit, which was significantly inhibited by AG1024 pretreatment of cells. Furthermore, pharmacological inhibition of Src by PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole(3,4-d) pyrimidine) decreased PKB phosphorylation. Moreover, H2O2-induced PKB phosphorylation was associated with increased tyrosine phosphorylation of c-Src and Pyk2 in an AG1024- and PP2-inhibitable manner. In conclusion, these data provide evidence of the contribution of IGF-1R-PTK in initiating H2O2-evoked PKB phosphorylation in A10 VSMC, with an intermediary role for c-Src and Pyk2 in this process.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.     

Sensitized photooxygenation and peroxidase-catalyzed inactivation of xanthine oxidase--evidence of cysteine damage by singlet oxygen

Xanthine oxidase (XO) has been investigated for its decreased activity in several cancerous tissues and constitutive generation of reactive oxygen species (ROS) in vivo seems to contribute significantly to its inactivation. Singlet oxygen (1O2) production has been suggested to be relevant when considering folic acid metabolism by cancer cells. Thus, the susceptibility of XO to inactivation by 1O2 generated either by the bioenergized systems folic acid/peroxidase/GSH/Mn2+/O2 and malonaldehyde/peroxidase/Mn2+/O2 or by methylene blue (MB) or eosin-sensitized photooxygenation was studied. Our results showed that other ROS were also responsible for XO inactivation when MB was used. In contrast, eosin produced almost exclusively 1O2. Kinetic studies of XO oxidation in the malonaldehyde/peroxidase system showed that histidine (His) is a competitive inhibitor with respect to XO. A similar result was observed in the eosin-photosensitized process, suggesting the involvement of 1O2 in both processes. In addition, an efficient quenching of XO oxidation by guanosine in the folic acid/peroxidase system was observed. Amino acid analysis revealed that cysteine (Cys) is more affected than other XO amino acids also prone to oxidation such as tyrosine (Tyr), methionine (Met) and His. These results indicate that 1O2 may cause oxidative damage to the Cys residues of XO, with loss of enzyme activity. Alteration of the flavin prosthetic site is hypothesized.     

Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR

In Bacillus subtilis most peroxide-inducible oxidative stress genes are regulated by a metal-dependent repressor, PerR. PerR is a dimeric, Zn2+-containing metalloprotein with a regulatory metal-binding site that binds Fe2+ (PerR:Zn,Fe) or Mn2+ (PerR: Zn,Mn). Reaction of PerR:Zn,Fe with low levels of hydrogen peroxide (H2O2) leads to oxidation of two His residues thereby leading to derepression. When bound to Mn2+, the resulting PerR:Zn,Mn is much less sensitive to oxidative inactivation. Here we demonstrate that the structural Zn2+ is coordinated in a highly stable, intrasubunit Cys4:Zn2+ site. Oxidation of this Cys4:Zn2+ site by H2O2 leads to the formation of intrasubunit disulfide bonds. The rate of oxidation is too slow to account for induction of the peroxide stress response by micromolar levels of H2O2 but could contribute to induction under severe oxidative stress conditions. In vivo studies demonstrated that inactivation of PerR:Zn,Mn required 10 mM H2O2, a level at least 1000 times greater than that needed for inactivation of PerR:Zn,Fe. Surprisingly even under these severe oxidation conditions there was little if any detectable oxidation of cysteine residues in vivo: derepression was correlated with oxidation of the regulatory site. Because oxidation at this site required bound Fe2+ in vitro, we suggest that treatment of cells with 10 mM H2O2 released sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR: Zn,Fe form. This model is supported by metal ion affinity measurements demonstrating that PerR bound Fe2+ with higher affinity than Mn2+.     

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.     

HMG盒转录因子1(HBP1)参与H2O2诱导的细胞衰老过程

为探讨HMG盒转录因子1 (HBP1)在过氧化氢（H2O2）诱导的细胞衰老中所起的作用,通过慢病毒感染得到稳定表达HBP1的MDA-MB-231细胞,以H2O2处理细胞．采用Western免疫印迹杂交试验和实时PCR检测HBP1、p16和细胞周期蛋白D1（cyclinD1）表达水平的变化．用荧光免疫试验检测H2O2对HBP1表达的影响,以及HBP1在H2O2的诱导下对于p16和细胞周期蛋白D1启动子的影响．用细胞增殖试验检测H2O2对于细胞增殖的影响． 用基因敲减实验和衰老相关β半乳糖苷酶(SA-β-Gal)染色检测在H2O2诱导的细胞衰老中HBP1所起的作用．Western和免疫荧光实验结果显示,细胞经H2O2处理后,HBP1表达增高的同时促进了p16的表达,降低了细胞周期蛋白D1的表达．细胞增殖实验结果显示,H2O2显著抑制了细胞的增殖．基因敲减实验和SA-β-Gal染色实验说明,H2O2可诱导HBP1表达正常的MDA-MB-231细胞衰老,而HBP1的敲减则抑制了H2O2诱导的细胞衰老过程．本研究结果提示,在H2O2诱导的衰老中,HBP1的表达显著增加,并通过促进衰老相关基因p16的表达和抑制生长因子cyclinD1的表达来阻碍细胞增殖,促进细胞衰老．HBP1在H2O2诱导的细胞衰老过程中起着重要作用,H2O2诱导的细胞衰老必须在HBP1存在的情况下才能发生．     

The JmjC domain histone demethylase Ndy1 regulates redox homeostasis and protects cells from oxidative stress

The histone H3 demethylase Ndy1/KDM2B protects cells from replicative senescence. Changes in the metabolism of reactive oxygen species (ROS) are important for establishing senescence, suggesting that Ndy1 may play a role in redox regulation. Here we show that Ndy1 protects from H(2)O(2)-induced apoptosis and G(2)/M arrest and inhibits ROS-mediated signaling and DNA damage, while knockdown of Ndy1 has the opposite effects. Consistent with these observations, whereas Ndy1 overexpression promotes H(2)O(2) detoxification, Ndy1 knockdown inhibits it. Ndy1 promotes the expression of genes encoding the antioxidant enzymes aminoadipic semialdehyde synthase (Aass), NAD(P)H quinone oxidoreductase-1 (Nqo1), peroxiredoxin-4 (Prdx4), and serine peptidase inhibitor b1b (Serpinb1b) and represses the expression of interleukin-19. At least two of these genes (Nqo1 and Prdx4) are regulated directly by Ndy1, which binds to specific sites within their promoters and demethylates promoter-associated histone H3 dimethylated at K36 and histone H3 trimethylated at K4. Simultaneous knockdown of Aass, Nqo1, Prdx4, and Serpinb1b in Ndy1-expressing cells to levels equivalent to those detected in control cells was sufficient to suppress the Ndy1 redox phenotype.     

Inhibitory role of RhoA on senescence-like growth arrest by a mechanism involving modulation of phosphatase activity

Recently, negative effects of phosphatase in tumorigenesis and metastasis have been suggested in various tumor types. In this study, we showed that RhoA activation modulated phosphatase during senescence-like arrest in human prostate cancer cells. Under senescence-inducing condition, decreased Erk phosphorylation was detected in caRhoA-transfected cells and inactivation of Erk, but not p38, prevented doxorubicin-induced cell senescence. Cells were induced to senescence by inhibition of phosphatase activity (VHR, MKP3, or PP2A) without additional cellular stress. Of interest, caRhoA prevented doxorubicin-induced decrease of phosphatase. Thus, we postulate that RhoA signaling may protect cells against cellular senescence by maintaining phosphatase activity and Erk dephosphorylation.     

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal-regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H(2)O(2) as well as in PC12 cells stimulated by nerve growth factor and H(2)O(2). Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.     

转录因子HBP1调控H2O2诱导的骨肉瘤细胞生长阻滞过程

为探讨氧化应激对人骨肉瘤细胞增殖的影响及其作用机理,首先用H2O2处理U2OS细 胞,采用Western印迹和real-time PCR检测HMG盒转录因子1 (HBP1)及其下游靶基因 DNMT1和p16表达水平的变化. 用荧光素酶报告基因实验检测在H2O2诱导下, HBP1对于DNMT1 和p16启动子的影响. 用细胞增殖试验（BrdU掺入,细胞生长曲线）检测 H2O2对细胞增殖的影响以及HBP1的作用. 用衰老相关β半乳糖苷酶(SA-β-Gal)染色 检测在H2O2诱导的细胞衰老中HBP1所起的作用. Western 印迹, real-time PCR及荧光素酶报告基因实验结果显示,细胞经H2O2处理后,明显增高HBP1表达水平,转录抑制DNMT1的表达, 促进p16蛋白的表达. 细胞增殖实验结果显示, H2O2显著抑制了细胞的增殖,HBP1 knockdown可部分逆转这种抑制作用. SA-β-Gal染色实验说明, H2O2可诱导HBP1表达正常的U2OS细胞衰老,而HBP1 knockdown使这种促衰老作用减弱. 研究结果说明, H2O2可抑制人骨肉瘤细胞增殖,诱导细胞衰老. 其作用机制是通过上调转录因子HBP1的表达,转录抑制或促进其下游靶基因DNMT1或p16的表达来抑制细胞增殖,促进细胞衰老.