Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress

The aim of this study was to determine whether exposure to heat stress would lead to oxidative stress and whether this effect varied with different exposure periods. We kept 1-, 6- and 12-month-old male Wistar rats at an ambient temperature of either 22 degrees C or 40 degrees C for 3 and 7 days and measured glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST) activities and levels of thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH) and oxidized glutathione (GSSG) in erythrocytes and determined GSH/GSSG ratio, total glutathione and the redox index. G-6-PD and CAT activities were found to be significantly increased in 1- and 6-month-old rats after 3 and 7 days of heat stress, but G-6-PD activities decreased in 12-month-old rats. Cu, Zn-SOD activity decreased in 1-month-old rats after heat stress, whereas it increased in 6- and 12-month-old rats. GST activity increased in all groups. GSH and total GSH levels and GSH/GSSG ratios decreased in 1- and 6-month-old rats but they increased in 12-month-old rats after heat stress. GSSG levels increased in 1- and 6-month-old rats but decreased in 12-month-old rats after heat stress. TBARS levels increased in all groups. Seven days of stress is more effective in altering enzyme activities and levels of GSH, GSSG and TBARS. When the effects of both heat stress and aging were examined together, it was interesting to note that they mostly influenced G-6-PD activity.     

Stimulation of colonic mucosal growth associated with oxidized redox status in rats

Limited data in animal models suggest that colonic mucosa undergoes adaptive growth following massive small bowel resection (SBR). In vitro data suggest that intestinal cell growth is regulated by reactive oxygen species and redox couples [e.g., glutathione (GSH)/glutathione disulfide (GSSG) and cysteine (Cys)/cystine (CySS) redox]. We investigated the effects of SBR and alterations in redox on colonic growth indexes in rats after either small bowel transection (TX) or 80% midjejunoileal resection (RX). Rats were pair fed +/- blockade of endogenous GSH synthesis with buthionine sulfoximine (BSO). Indexes of colonic growth, proliferation, and apoptosis and GSH/GSSG and Cys/CySS redox potentials (E(h)) were determined. RX significantly increased colonic crypt depth, number of cells per crypt, and epithelial cell proliferation [crypt cell bromodeoxyuridine (BrdU) incorporation]. Administration of BSO markedly decreased colonic mucosal GSH, GSSG, and Cys concentrations in both TX and RX groups, with a resultant oxidation of GSH/GSSG and Cys/CySS E(h). BSO did not alter colonic crypt cell apoptosis but significantly increased all colonic mucosal growth indexes (crypt depth, cells/crypt, and BrdU incorporation) in both TX and RX groups in a time- and dose-dependent manner. BSO significantly decreased plasma GSH and GSSG, oxidized GSH/GSSG E(h), and increased plasma Cys and CySS concentrations. Collectively, these data provide in vivo evidence indicating that oxidized colonic mucosal redox status stimulates colonic mucosal growth in rats. The data also suggest that GSH is required to maintain normal colonic and plasma Cys/CySS homeostasis in these animal models.     

Effects of buthionine sulfoximine treatment on diaphragm contractility and SR Ca2+ pump function in rats.

The purpose of this study was to examine the effects of glutathione (GSH) depletion and cellular oxidation on rat diaphragm contractility and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in vitro under basal conditions and following fatiguing stimulation. Buthionine sulfoximine (BSO) treatment (n = 10) for 10 days (20 mM in drinking water) reduced (P < 0.05) diaphragm GSH content (nmol/mg protein) and the ratio of GSH to glutathione disulfide (GSH/GSSG) by 91% and 71%, respectively, compared with controls (CTL) (n = 10). Western blotting showed that Hsp70 expression in diaphragm was not increased (P > 0.05) with BSO treatment. As hypothesized, basal peak twitch force (g/mm(2)) was increased (P < 0.05), and fatigability in response to repetitive stimulation (350-ms trains at 100 Hz once every 1 s for 5 min) was also increased (P < 0.05) in BSO compared with CTL. Both Ca(2+) uptake and maximal SERCA activity (mumol.g protein(-1).min(-1)) measured in diaphragm homogenates that were prepared at rest were increased (P < 0.05) with BSO treatment, an effect that could be partly explained by a twofold increase (P < 0.05) in SERCA2a expression with BSO. In response to the 5-min stimulation protocol, both Ca(2+) uptake and maximal SERCA activity were increased (P < 0.05) in CTL but not (P > 0.05) in BSO diaphragm. We conclude that 1) cellular redox state is more optimal for contractile function and fatigability is increased in rat diaphragm following BSO treatment, 2) SERCA2a expression is modulated by redox signaling, and 3) regulation of SERCA function in working diaphragm is altered following BSO treatment.     

Effect of 1-methyl-4-phenylpyridinium on glutathione in rat pheochromocytoma PC 12 cells

We investigated the effect of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on glutathione redox status and the generation of reactive oxygen intermediates (ROI) in rat pheochromocytoma PC 12 cells in vitro. Treatment with MPP+ (250 microM) led to a 63% increase of reduced glutathione (GSH) after 24 h, while a 10-fold higher concentration of MPP+ (2.5 mM) depleted cellular GSH to 12.5% of control levels within that time. Similarly, the complex I-inhibitor rotenone induced a time-dependent loss of GSH at 1 and 10 microM, whereas treatment with lower concentrations of rotenone (0.1, 0.01 microM) increased cellular GSH. Both MPP+ and rotenone increased cellular levels of oxidised glutathione (GSSG) and the higher concentrations of both compounds led to an elevated ratio of oxidised glutathione (GSSG) vs total glutathione (GSH + GSSG) indicating a shift in cellular redox balance. MPP+ or rotenone did not induce the generation of ROI or significant elevation of intracellular levels of thiobabituric acid reactive substances (TBARS) for up to 48 h. Our data suggest that MPP+ has differential effects on glutathione homeostasis depending on the degree of complex I-inhibition and that inhibition of complex I is not sufficient to generate ROI in this paradigm.     

Diamide induced shift in protein and glutathione thiol: disulfide status delays DNA rejoining after X-irradiation of human cancer cells

By treating a human tumor cell line with various concentrations of diamide, we explored the relationship between extent and duration of protein and nonprotein thiol oxidation, initiation of DNA double-strand break rejoining after X-rays, and the degree of radiosensitization. We also examined the relationship between protein thiol status and the non-protein thiol, glutathione (GSH). A549 cells were irradiated and incubated postirradiation with 0, 100, 300 or 500 microM diamide for 1 h. The dose of radiation required to give 10% survival decreased from 4.8 Gy to 3.2 Gy with 300 microM and to 2.7 Gy with 500 microM diamide (enhancement ratios of 1.5 and 1.8, respectively) but was not significantly affected by 100 microM diamide. The time of initiation of double-stranded DNA rejoining after X-irradiation (DNA repair) was delayed by 300 and 500 microM diamide. Furthermore, DNA rejoining began only after total cellular protein thiol content recovered to 55% of pretreatment levels for both concentrations. Intracellular GSH/GSSG ratios decreased immediately after diamide addition to less than 1. Large decreases in GSH/GSSG ratio preceded significant loss of protein thiols, but protein-glutathione mixed disulfides accounted for a minor percentage of the total protein thiol oxidized (up to 20%). We believe that diamide-induced protein thiol loss, and not GSH oxidation, is related to the cessation of DNA strand rejoining after X-irradiation, thereby affecting survival.     

Hepatic response to the oxidative stress induced by E. coli endotoxin: Glutathione as an index of the acute phase during the endotoxic shock

Reactive oxygen species are important mediators of cellular damage during endotoxic shock. In order to investigate the hepatic response to the oxidative stress induced by endotoxin, hepatic and plasma glutathione (total, GSH and GSSG), GSSG/GSH ratio as well as Mn-superoxide dismutase and catalase activities were determined during the acute and recovery phases of reversible endotoxic shock in the rat. A significant increase in liver and plasma total glutathione content was observed 5 h after endotoxin treatment (acute phase), followed by a diminution of these parameters below control values at 48 h (recovery phase). The significant increases of GSSG levels and GSSG/GSH ratio are indicative of oxidative stress occurring during the acute phase. Liver Mn-SOD activity showed a similar time dependency as the GSSG/GSH ratio; however, a marked decrease in the liver catalase activity was observed during the process. These results indicate the participation of liver glutathione in the response to endotoxin and the possible use of plasma glutathione levels and GSSG/GSH ratio as indicators of the acute phase during the endotoxic process. (Mol Cell Biochem 159: 115-121, 1996)     

Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-cysteine synthetase) and, particularly, GLR1 (glutathione reductase) are required for survival on DPS. DPS is uniquely thiol-specific, and we found that the cellular mechanisms for DPS detoxification differ substantially from that of the commonly used thiol oxidant diamide. In contrast to this oxidant, the full antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS sensitivity. DPS seems to induce a specific disulfide stress, where an increase in the cytoplasmic/nuclear GSSG/GSH ratio results in putative DPS target(s) becoming sensitive to DPS.     

Lipid hydroperoxide-induced apoptosis in human colonic CaCo-2 cells is associated with an early loss of cellular redox balance.

Apoptosis plays a critical role in maintaining homeostasis of the intestinal epithelium. Dietary oxidants like peroxidized lipids could perturb cellular redox status and disrupt mucosal turnover. The objective of this study was to delineate the role of lipid hydroperoxide (LOOH) -induced redox shifts in intestinal apoptosis using the human colonic CaCo-2 cell. We found that subtoxic concentrations of LOOH increased CaCo-2 cell apoptosis. This LOOH-induced apoptosis was associated with a significant decrease in the ratio of reduced glutathione-to-oxidized glutathione (GSH/GSSG), which preceded DNA fragmentation by 12 to 14 h, suggesting a temporal relationship between the two events. Oxidation of GSH with the thiol oxidant diamide caused significant decreases in cellular GSH and GSH/GSSG at 15 min that correlated with the activation of caspase 3 (60 min) and cleavage of PARP (120 min), confirming a temporal link between induction of cellular redox imbalance and initiation of apoptotic cell death. These kinetic studies further reveal that oxidant-mediated early redox change (within 1 h) was a primary inciting event of the apoptotic cascade. Once initiated, the recovery of redox balance did not prevent the progression of CaCo-2 cell apoptosis to its biological end point at 24 h. Collectively, the study shows that subtoxic levels of LOOH disrupt intestinal redox homeostasis, which contributes to apoptosis. These results provide insights into the mechanism of hydroperoxide-induced mucosal turnover that have important implications for understanding oxidant-mediated genesis of gut pathology.     

摄入异烟肼对家蚕幼虫体内谷胱甘肽氧化还原循环和谷胱甘肽S-转移酶活性的影响

为了建立家蚕Bombyx mori的药物筛选和毒性评价模型, 以剂量为2 000 mg/kg的抗结核模药异烟肼饲喂家蚕5龄第3天幼虫后检测其中肠和脂肪体的抗氧化解毒相关代谢的变化。结果表明： 雌蚕中肠组织中, 总谷胱甘肽(GSH+2GSSG)、 还原型谷胱甘肽(reduced glutathione, GSH)和氧化型谷胱甘肽(oxidized glutathione, GSSG)含量均呈现迅速上升再缓慢下降趋势; 谷胱甘肽S 转移酶(glutathione S-transferase, GST)活性升高到较大值后逐渐降低; GSH/GSSG的比值下降表明, 在72 min后中肠组织向氧化态转移。脂肪体组织中, 总谷胱甘肽、 GSH和GSSG含量变化均呈现迅速下降再迅速上升的趋势; GST活性达到最大值后逐渐降低后趋于平稳; GSH/GSSG比值升高表明, 在72 min后脂肪体组织向还原态转移。无论雌蚕还是雄蚕, 总谷胱甘肽、 GSH和GSSG含量以及GST活性均是脂肪体高于中肠。雌蚕的总谷胱甘肽含量、 GSH和GSSG含量高于雄蚕, 但雄蚕的GST活性高于雌性。结果说明, 摄入异烟肼引起了家蚕幼虫体内谷胱甘肽氧化还原状态的改变和酶活性的变化, 在这个过程中脂肪体起主要解毒代谢作用。     

Novel functions of the alpha-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H(2)O(2) and to probe the mechanism underlying these changes. Since the response to H(2)O(2) is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H(2)O(2) decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H(2)O(2) inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H(2)O(2) converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H(2)O(2) diminished glutathione to a similar level after 24 h. However, H(2)O(2), but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H(2)O(2) together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.     

外源GSH对NaCl胁迫下番茄幼苗光合特性及碳同化关键酶基因表达的影响

对NaCl胁迫下番茄幼苗叶片分别喷施还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)和谷胱甘肽合成抑制剂(BSO)构建不同氧化还原水平的番茄植株,研究外源GSH介导的氧化还原状态对NaCl胁迫下番茄幼苗光合作用的影响.结果表明: 外源喷施GSH诱导NaCl胁迫下番茄幼苗叶片的还原力水平提高,叶片净光合速率(P_n)、气孔导度(g_s)、蒸腾速率(T_r)及最大光化学效率(F_v/F_m)、实际光化学效率(Ф_PSⅡ)、光化学猝灭系数(q_P)、非光化学猝灭系数(NPQ)值均提高,核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)活性及大亚基(RbcL)、小亚基(RbcS)和Rubisco活化酶(RCA)的基因表达水平上调,从而有效保护了光合系统,促进了(光系统Ⅱ)PSⅡ光化学反应活性、降低了NaCl胁迫对光合暗反应的抑制,缓解了NaCl胁迫对番茄植株的危害.喷施GSSG显著降低了NaCl胁迫下番茄幼苗叶片还原力水平,造成叶片光损伤和光抑制加剧,但RbcS和RbcL的基因表达水平上调可能是导致NaCl+GSSG处理下叶片P_n未下降的原因.喷施BSO对NaCl胁迫下番茄幼苗叶片氧化还原状态、CO₂传导能力和PSⅡ反应中心无显著影响,但BSO上调碳同化关键酶Rubisco初始活性、总活性及RCA和RbcS表达水平是导致P_n提高的原因.     

Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of Tatar buckwheat calli

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10–20 times higher and the glutathione reductase activity was 2–2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6–8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation of proembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.     

Improved development of microspore-derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism

Glutathione has been shown to play an important role during embryo development in both plant and animal systems. The effects of altered glutathione metabolism during microspore-derived embryos (MDEs) of Brassica napus were investigated following exogenous application of reduced glutathione (GSH), its oxidized form (GSSG) and buthionine sulfoximine (BSO), an inhibitor of glutathione de novo synthesis. Applications of BSO which lowered the cellular glutathione redox status, i.e. GSH/(GSH + GSSG), enhanced significantly the quality of the embryos and their ability to convert into viable plants. Histological analyses revealed that inclusions of BSO in the culture medium altered the pattern of storage product accumulation in the embryos and improved the architecture of the shoot apical meristems (SAMs). Compared with their control counterparts which showed severe signs of SAM deterioration, such as the formation of intercellular spaces and differentiation of the meristematic cells, BSO-treated embryos had well-organized SAMs. The improved SAM organization observed in the presence of BSO also correlated with the proper localization pattern of WUSCHEL , a SAM molecular marker gene which was miss-expressed in control embryos. The beneficial effects of BSO on embryo development and conversion were ascribed to the increasing levels of ABA. The concentration of this growth regulator in BSO-treated embryos was always higher than that of control embryos during the second half of the maturation period. Furthermore, many structural alterations induced by BSO could be reproduced in embryos cultured in the presence of ABA. Taken together, these results suggest that a lowering of the glutathione redox status during embryo development may represent a metabolic switch needed for increasing the endogenous levels of ABA, which is required for successful completion of the developmental program.     

Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake

The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.     

Reduced and oxidized glutathione in brain and convulsions

Concentration changes of reduced glutathione (GSH) and oxidized glutathione (GSSG) were studied by fluorometric assay witho-phthalaldehyde to clarify the relationship between seizure mechanism and the glutathione redox state. In cerebellum the GSH/GSSG ratio was significantly decreased in the interictal stage of E1 mice (stimulated group), but in ddY mice this ratio was decreased before convulsions induced by pentylenetetrazol and during submaximal ECS. No change was found in the GSH/GSSG ratio of the cerebellum during and after convulsions induced by pentylenetetrazol and maximal ECS. GSH levels in cerebrum in the interictal stage of E1 mice (stimulated group) were lower compared to control E1 mice. In ddY mice submaximal ECS increased GSSG levels in cerebrum so that the GSH/GSSG ratio was decreased.