Thiol Redox Regulation Research Articles

Hydrogen Peroxide-sensitive Cysteines in the Sty1 MAPK Regulate the Transcriptional Response to Oxidative Stress

MAPK are activated by and orchestrate responses to multiple, diverse stimuli. Although these responses involve the increased phosphorylation of substrate effector proteins, e.g. transcription factors, the mechanisms by which responses are tailored to particular stimuli are unclear. In the fission yeast Schizosaccharomyces pombe, the Sty1 MAPK is crucial for changes in gene expression that allow adaptation to many forms of environmental stress. Here, we have identified two cysteine residues in Sty1, Cys-153 and Cys-158, that are important for hydrogen peroxide-induced gene expression and oxidative stress resistance but not for other functions of Sty1. Many Sty1-dependent changes in gene expression are mediated by the Atf1 transcription factor. In response to stress, Sty1 increases Atf1 levels by (i) promoting increases in atf1 mRNA and by (ii) directly phosphorylating and stabilizing Atf1 protein. Although dispensable for phosphorylation and stabilization of Atf1 protein, we find that both Cys-153 and Cys-158 are required for increases in atf1 mRNA levels and Atf1-dependent gene expression in response to hydrogen peroxide but not osmotic stress. Indeed, our data indicate that oxidation of Sty1, by formation of a disulfide bond between Cys-153 and Cys-158, is important for maintaining atf1 mRNA stability at high concentrations of hydrogen peroxide. Together, these data reveal that redox regulation of cysteine thiols in Sty1 is involved in a stress-specific mechanism regulating transcriptional responses to oxidative stress. Intriguingly, the conservation of these cysteine residues in other MAPK raises the possibility that similar mechanisms may ensure appropriate responses to hydrogen peroxide in other eukaryotes.

Thiol-Redox Regulation of Mercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Thiol-Redox Regulation of Mercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Modulation of Cell Surface Protein Free Thiols; A Potential Novel Mechanism of Action of the Sesquiterpene Lactone Parthenolide in Non-Hodgkin's Lymphoma.

Abstract 3774Poster Board III-710Recent data supports the concept that redox regulation of cell surface protein thiols (i.e., exofacial thiols) effects critical cellular functions. We therefore hypothesized that redox-active anti-cancer therapeutics would modulate exofacial thiols, and that such modulation could be related to cell death mechanisms. To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Parthenolide treatment results in a dose dependent decrease in mantle cell (MCL) and diffuse large cell lymphoma cell viability. Indeed, parthenolide decreases the level of free (ie. reduced) exofacial thiols on Granta MCL cells as assessed by flow cytometry. Parthenolide specifically modifies free thiols of surface proteins having molecular weights of ∼12 kd and ∼22 kd, as determined using a biotinylated thiol reactive reagent N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), which binds to free thiol groups and is detected by streptavidin-peroxidase staining of western blots. We further show that the central redox regulator thioredoxin is one of the surface protein thiol targets modified by parthenolide; specifically; (a) thioredoxin is detected on the Granta cell surface; (b) parthenolide directly interacts with free thiol groups on purified human thioredoxin, and; (c) parthenolide directly modifies Granta membrane associated thioredoxin, as determined with BIAM and neutravidin pull-down. To examine the functional consequences of parthenolide induced surface protein thiol modification, Granta cells were pretreated with the cell impermeable thiol antioxidant glutathione (GSH). Pretreatment with GSH inhibits the parthenolide induced; (a) decrease in exofacial free thiols; (b) modification of surface thioredoxin and; (c) Granta cell death. Pretreatment of Granta cells with GSH also inhibits parthenolide-mediated activation of JNK and inhibition of NFkB, two activities previously associated with the drug's anti-cancer activity. Based on these data, we postulate that at least one component of parthenolide's anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Indeed, to our knowledge, our data is the first to suggest that cancer cell exofacial thiols may be novel and important targets for cancer therapy.Supported by an NCI SPORE grant in lymphoma 1P50 CA130805. Disclosures:Bernstein:millenium: Consultancy; genentech: Consultancy, Speakers Bureau; enzon: Consultancy.

Chloroplast NADPH-thioredoxin reductase interacts with photoperiodic development in Arabidopsis.

Chloroplast NADPH-thioredoxin reductase (NTRC) belongs to the thioredoxin systems that control crucial metabolic and regulatory pathways in plants. Here, by characterization of T-DNA insertion lines of NTRC gene, we uncover a novel connection between chloroplast thiol redox regulation and the control of photoperiodic growth in Arabidopsis (Arabidopsis thaliana). Transcript and metabolite profiling revealed severe developmental and metabolic defects in ntrc plants grown under a short 8-h light period. Besides reduced chlorophyll and anthocyanin contents, ntrc plants showed alterations in the levels of amino acids and auxin. Furthermore, a low carbon assimilation rate of ntrc leaves was associated with enhanced transpiration and photorespiration. All of these characteristics of ntrc were less severe when plants were grown under a long 16-h photoperiod. Transcript profiling revealed that the mutant phenotypes of ntrc were accompanied by differential expression of genes involved in stomatal development, chlorophyll biosynthesis, chloroplast biogenesis, and circadian clock-linked light perception systems in ntrc plants. We propose that NTRC regulates several key processes, including chlorophyll biosynthesis and the shikimate pathway, in chloroplasts. In the absence of NTRC, imbalanced metabolic activities presumably modulate the chloroplast retrograde signals, leading to altered expression of nuclear genes and, ultimately, to the formation of the pleiotrophic phenotypes in ntrc mutant plants.

Glutathione and apoptosis

Apoptosis or programmed cell death represents a physiologically conserved mechanism of cell death that is pivotal in normal development and tissue homeostasis in all organisms. As a key modulator of cell functions, the most abundant non-protein thiol, glutathione (GSH), has important roles in cellular defense against oxidant aggression, redox regulation of proteins thiols and maintaining redox homeostasis that is critical for proper function of cellular processes, including apoptosis. Thus, a shift in the cellular GSH-to-GSSG redox balance in favour of the oxidized species, GSSG, constitutes an important signal that could decide the fate of a cell. The current review will focus on three main areas: (1) general description of cellular apoptotic pathways, (2) cellular compartmentation of GSH and the contribution of mitochondrial GSH and redox proteins to apoptotic signalling and (3) role of redox mechanisms in the initiation and execution phases of apoptosis.

Redox Regulation of Sperm Surface Thiols Modulates Adhesion to the Fallopian Tube Epithelium1

Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.

Thiol Redox and Immune Regulation in Corneal Transplantation

Penetrating keratoplasty (PKP) is the most common type of clinical grafting performed in humans. Although PKP has emerged as the most successful form of transplantation, PKP in "high-risk" eyes shows high incidence of allograft rejection. The incidence of epithelial rejection after limbal transplantation (LT) is extremely higher and swifter than PKP rejection, and even intensive systemic immunosuppressive therapy is often of no avail. Because failure of corneal grafts is an important cause of blindness, developing new strategies for suppressing graft rejection is a worthy goal for research. Corneal allograft rejection is mainly mediated by the TH1-type immune response, which leads to a delayed-type hypersensitivity reaction. Because the TH2-type immune response regulates the TH1-type immune response, we have successfully elicited allograft survival after both PKP and LT by inducing systemic TH2-type immune responses. Because intracellular thiol redox status of antigen-presenting cells (APC) reportedly regulates TH1/TH2 balance via distinctive cytokine production by APC, we also investigated the effect of modulating macrophage intracellular thiol redox status on corneal allograft survival. These strategies are quite effective in major histocompatibility complex (MHC) matching in mice, although it is believed that MHC matching has no effect on corneal allograft survival according to many rodent studies. Recently, many laboratories are reconsidering HLA matching for allograft survival in human corneal transplantation. It may be possible that MHC matching improves corneal allograft survival in the context of TH1 suppression. We propose that the suppression of the TH1-type immune response and MHC matching together may promote allograft survival in humans.

Effect of selenium‐supplement on the calcium signaling in human endothelial cells

Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP3-triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+-ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins.

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Redox regulation of surface protein thiols: identification of integrin alpha-4 as a molecular target by using redox proteomics.

Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1-2 h) of cells with 0.1-5 mM N-acetyl-L-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiol N-acetyl-D-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin alpha-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and beta-actin. NAC pretreatment augmented integrin alpha-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes.

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.

Thiol Metabolism and Redox Regulation of Cellular Functions. Series I: Life and Behavioural Sciences

Thiol Metabolism and Redox Regulation of Cellular Functions. Series I: Life and Behavioural Sciences

Oxidative signaling and glutathione synthesis.

BioFactorsVolume 17, Issue 1-4 p. 1-12 Article Oxidative signaling and glutathione synthesis† Henry Jay Forman, Corresponding Author Henry Jay Forman Department of Environmental Health Sciences, School of Public Health, and, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USADr. Henry Jay Forman, Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 1530 3RD Avenue S, RPHB-534, Birmingham, AL 35294–0022, USA. Tel.: +1 205 975 8949; Fax: +1 205 975 6341; E-mail: hforman@uab.eduSearch for more papers by this authorDale A. Dickinson, Dale A. Dickinson Department of Environmental Health Sciences, School of Public Health, and, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USASearch for more papers by this author Henry Jay Forman, Corresponding Author Henry Jay Forman Department of Environmental Health Sciences, School of Public Health, and, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USADr. Henry Jay Forman, Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 1530 3RD Avenue S, RPHB-534, Birmingham, AL 35294–0022, USA. Tel.: +1 205 975 8949; Fax: +1 205 975 6341; E-mail: hforman@uab.eduSearch for more papers by this authorDale A. Dickinson, Dale A. Dickinson Department of Environmental Health Sciences, School of Public Health, and, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USASearch for more papers by this author First published: 16 December 2008 https://doi.org/10.1002/biof.5520170101Citations: 49 † This article also appeared in Thiol Metabolism and Redox Regulation of Cellular Functions, A. Pompella, G. Bánhegyi and M. Wellman-Rousseau, eds, IOS Press, Amsterdam. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume17, Issue1-42003Pages 1-12 RelatedInformation

Redox-regulation of DNA repair.

BioFactorsVolume 17, Issue 1-4 p. 315-324 Article Redox-regulation of DNA repair† Joseph Lunec, Joseph Lunec Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this authorK. Holloway, K. Holloway Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this authorMarcus Cooke, Marcus Cooke Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this authorMark Evans, Mark Evans Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this author Joseph Lunec, Joseph Lunec Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this authorK. Holloway, K. Holloway Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this authorMarcus Cooke, Marcus Cooke Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this authorMark Evans, Mark Evans Oxidative Stress Group; Department of Clinical Biochemistry, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary NHS Trust, PO Box 65, Leicester LE2 7LX, UK. Tel.: +44 0 116 252 5890; Fax: +44 0 116 252 5887; E-mail: [email protected]; URL: www.le.ac.uk/cb/Search for more papers by this author First published: 16 December 2008 https://doi.org/10.1002/biof.5520170131Citations: 2 † This article also appeared in Thiol Metabolism and Redox Regulation of Cellular Functions, A. Pompella, G. Bánhegyi and M. Wellman-Rousseau, eds, IOS Press, Amsterdam. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume17, Issue1-42003Pages 315-324 RelatedInformation

Oxidative stress and acclimation mechanisms in plants.

One of the most crucial functions of plant cells is their ability to respond to fluctuations in their environment. Understanding the connections between a plant's initial responses and the downstream events that constitute successful adjustment to its altered environment is one of the next grand challenges of plant biology. Oxidative stress from environmental sources and developmental transitions such as seed maturation involves the formation of reactive oxygen species (ROS) in plant cells. The redox-modulated changes that follow are central events in cellular responses. Thiol redox regulation (Figure 1) partially mediated through the redox state of the glutathione pool (GSH/GSSG), regulation of the glutathione biosynthetic pathway, and ROS themselves are each thought to have important roles as environmental sensors and/or modulators of global patterns of gene expression in development and defense. Exposure of green tissue to potentially damaging light intensities involves redox sensing molecular events throughout the plant, originating at the plastoquinone (PQ) pool in the thylakoid membrane. Major defense genes whose expression is affected by the redox state of the PQ pool include both cytosolic and chloroplast ascorbate peroxidases (APX) ( Karpinska et al. 2000 ). The superoxide dismutase (SOD) gene families appear to be specialized in function with respect to subcellular location and other as yet unknown factors (Alscher et al. 2002). In the case of peroxisomes, the imposition of oxidative stress gives rise to organelle proliferation, thus adding another layer of complexity to stress responses (Lopez-Huertas et al. 2000). Defense mechanisms involving molecular chaperones and methionine sulfoxide reductase are becoming recognized as important players in resistance to oxidative stress throughout the cell. Figure 1. Thiol redox control and stress defense Intracellular origins of ROS and their multiple damaging effects Any circumstance in which cellular redox homeostasis is disrupted can lead to oxidative stress or the generation of ROS (Asada 1994). Production of ROS during environmental stress is one of the main causes for decreases in productivity, injury, and death that accompany these stresses in plants. ROS are produced in both unstressed and stressed cells, and in various locations (Halliwell and Gutteridge 1989) (Figure 2). They are generated endogenously during certain developmental transitions such as seed maturation and as a result of normal, unstressed, photosynthetic and respiratory metabolism. An initial oxyradical product, the superoxide radical (O2-.), upon further reaction within the cell, can form more ROS such as hydroxyl radicals and singlet oxygen. Superoxide is a charged molecule and cannot cross biological membranes. Subcellular compartmentation of defense mechanisms is, therefore, crucial for efficient removal of superoxide anions at their sites of generation throughout the cell. Hydrogen peroxide, on the other hand, which is formed as a result of SOD action, is capable of diffusing across membranes and is thought to fulfil a signaling function in defense responses (Mullineaux et al. 2000). Figure 2. Reactive oxygen species (ROS) arise throughout the cell. ROS play an important role in endonuclease activation and consequent DNA damage (Hagar et al. 1996). In the presence of metal ions such as Fe or Cu(II), hydroxyl radicals are formed very rapidly. Hydroxyl radicals can cause damage to all classes of biologically important macromolecules, especially nucleic acids. Hydroxyl radicals can also modify proteins so as to make them more susceptible to proteolytic attack. There is evidently considerable specificity associated with this degradative process since proteins have widely differing susceptibilities to attack by ROS (Davies 1987). Once damaged, proteins can be broken down further by specific endopeptidases such as the one found bound to the thylakoid membrane (Casano et al. 1994). A multicatalytic proteinase complex has been demonstrated in plant systems, with the capacity to selectively break down oxidatively damaged proteins (Van Nocker et al. 1996)

Light-harvesting complex II kinase and chloroplast redox signaling

Winter rye plants were grown under contrasting environmental conditions, or just transiently shifted to varying light/temperature regimes, to elucidate the acclimative processes of the light-harvesting complex II. Photosystem II (PSII) excitation pressure, reflecting the plastoquinone redox state, and the steady-state phosphorylation level of the thylakoid light-harvesting proteins (LHCII and CP29), together with accumulation of lhcb mRNA were monitored in vivo. Regulatory mechanisms of the phosphorylation of LHCII and CP29 were additionally explored in vitro. Obtained results allow us to make the following conclusions. (i) LHCII protein phosphorylation is a sensitive tool to monitor redox changes in chloroplasts. (ii) LHCII protein phosphorylation and lhcb mRNA accumulation are co-regulated, possibly via an activation state of the LHCII kinase. (iii) Excitation pressure of photosystem II (reduction state of the plastoquinone pool) is not directly involved in the regulation of lhcb mRNA accumulation. Instead, (iv) the redox status of the electron acceptors of photosystem I in the stromal compartment seems to be highly regulated and crucial for regulation of both the LHCII protein phosphorylation and lhcb gene expression in the nucleus. (v) Phosphorylation of CP29 is induced under plastoquinol-reducing conditions, and does not involve thiol redox-regulation

G6PD Deficient Cells and the Bioreduction of Disulfides: Effects of DHEA, GSH Depletion and Phenylarsine Oxide

We used Glucose 6 phosphate dehydrogenase (G6PD) minus cells (89 cells) and G6PD containing cells (K1) to understand the mechanisms of bioreduction of disulfide and the redox regulation of protein and non protein thiols in mammalian cells. The 89 cells reduce hydroxyethyldisulfide (HEDS) to mercaptoethanol (ME) at a slower rate than K1 cells. HEDS reduction results in loss of nonprotein thiols (NPSH) and a decrease in protein thiols (PSH) in 89 cells. The effects are less dramatic with K1 cells. However, the loss of NPSH and PSH in K1 cells are increased in the absence of glucose. Glutathione-depletion with L-BSO partially blocks HEDS reduction in K1 and 89 cells. Treatment with the vicinal thiol reagent phenyl arsenic oxide (PAO) blocks reduction of HEDS in both cells. Surprisingly, dehydroepiandrosterone (DHEA), a known inhibitor of G6PD, inhibits the growth and blocks the reduction of HEDS both in 89 and K1 cells suggesting that its mechanism for inhibition of growth is not G6PD related.

Nitric oxide and thiol redox regulation of Janus kinase activity.

The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2 in vitro, presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggered in vivo activation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagents in vitro and to nitric oxide donors in vivo. We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.