Protein disulfide bond formation in the cytoplasm during oxidative stress

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both beta4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.     

Preparation of protein conjugates via intermolecular disulfide bond formation

Conjugates of two unlike proteins can be prepared via the intermolecular disulfide interchange reaction, namely, protein A containing thiol groups reacts with protein B containing 4-dithiopyridyl groups to yield a conjugate with the release of 4-thiopyridone. Thiol groups can be introduced into proteins upon amidination with methyl 3-mercaptopropionimidate ester or 2-iminothiolane, and 4-dithiopyridyl groups can be introduced into proteins with these same reagents in the presence of 4,4'-dithiodipyridine. 2-Iminothiolane is stable on storage in contrast to the known lability of imidate esters; therefore 2-iminothiolane is a more convenient reagent for the modification of protein than are the imidate esters. All the reactions can be carried out easily under mild conditions in good yields. Conjugates of bovine plasma albumin with itself, ribonuclease, or a copolymer of D-glutamic acid and D-lysine and of sheep antibody and horseradish peroxidase were prepared with modified proteins containing an average of 1 to 5 thiol or dithiopyridyl groups per mol. These conjugates formed mainly dimers, trimers, and tetramers. The peroxidase labeled antibody retained more than 80% of its enzymatic and antigenic binding activities.     

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lagphase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.     

The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.     

Identification of intra- and intermolecular disulphide bonding in the plant mitochondrial proteome by diagonal gel electrophoresis

Redox active proteins in plant mitochondria were examined using 2-D oxidant/reductant diagonal-SDS-PAGE to separate and identify proteins with intermolecular or intramolecular disulphide bonds using diamide in the first dimension and DTT in the second dimension. Eighteen proteins spots were resolved either above or below the diagonal and these were in-gel digested and identified by MS/MS. This analysis revealed intermolecular disulphide bonds in alternative oxidase, O-acetylserine (thiol) lyase, citrate synthase and between subunits of the ATP synthase. Intramolecular disulphide bonds were observed in a range of mitochondrial dehydrogenases, elongation factor Tu, adenylate kinase and the phosphate translocator. Many of the soluble proteins found were known glutaredoxin/thioredoxin targets in other plants, but the membrane proteins were not found by these methods nor were the nature of the disulphides able to be investigated. The accessibility of thiols involved in disulphide bonds to modification by a lipid derived aldehyde gave an insight into the potential impact of Cys modification on redox-functions in mitochondria during lipid peroxidation. Comparison of the protein sequences of the identified proteins with homologs from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins.     

Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress

t-Butyl hydroperoxide was utilized to alter the thiol homeostasis in rat brain mitochondria. Following exposure to t-butyl hydroperoxide (50-500 microM), intramitochondrial GSH content decreased rapidly and irreversibly with a major portion of the depleted GSH being accounted for as protein-SS-Glutathione mixed disulfide. Formation of GSSG was not observed nor was efflux of GSSG or GSH from the mitochondria detected in the incubation medium. The loss of intramitochondrial GSH was accompanied by loss of protein thiols. Unlike liver mitochondria, which can reverse t-butyl hydroperoxide induced formation of GSSG, addition of 50 microM t-butyl hydroperoxide resulted in irreversible loss; indicating greater susceptibility of brain mitochondria to oxidative stress than liver mitochondria.     

Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis

Phosphocellulose-purified tubulin has been shown to form a characteristic "ladder" of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120-129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of alpha- and beta-subunits of tubulin. Omission of beta-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, alpha- or beta-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of beta-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included greater than 75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in alpha beta-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide-crosslinked tubulin aggregates.     

Subchronic nandrolone administration reduces cardiac oxidative markers during restraint stress by modulating protein expression patterns

lncRNAs are an emerging class of regulators involved in multiple biological processes. MEG3, an lncRNA, acts as a tumor suppressor, has been reported to be linked with osteogenic differentiation of MSCs. However, limited knowledge is available concerning the roles of MEG3 in the multilineage differentiation of hASCs. The current study demonstrated that MEG3 was downregulated during adipogenesis and upregulated during osteogenesis of hASCs. Further functional analysis showed that knockdown of MEG3 promoted adipogenic differentiation, whereas inhibited osteogenic differentiation of hASCs. Mechanically, MEG3 may execute its role via regulating miR-140-5p. Moreover, miR-140-5p was upregulated during adipogenesis and downregulated during osteogenesis in hASCs, which was negatively correlated with MEG3. In conclusion, MEG3 participated in the balance of adipogenic and osteogenic differentiation of hASCs, and the mechanism may be through regulating miR-140-5p.     

Analysis of outer membrane protein complexes and heat-modifiable proteins in Neisseria strains using two-dimensional diagonal electrophoresis

Two-dimensional diagonal SDS-PAGE was used to resolve membrane complexes and identify proteins with temperature-dependent mobility in Neisseria meningitidis and N. lactamica. The main membrane complexes were composed of porins and were formed by heteromers of PorA, PorB and RmpM in N. meningitidis, and by PorB and RmpM in N. lactamica. Also, other proteins, including Opa, with temperature-dependent mobility were clearly demonstrated. The method allows improved detection of the components of membrane complexes and proteins with temperature-dependent mobility which is difficult to resolve with other analytical approaches.     

Balancing oxidative protein folding: The influences of reducing pathways on disulfide bond formation

Oxidative protein folding is confined to few compartments, including the endoplasmic reticulum, the mitochondrial intermembrane space and the bacterial periplasm. Conversely, in compartments in which proteins are translated such as the cytosol, the mitochondrial matrix and the chloroplast stroma proteins are kept reduced by the thioredoxin and glutaredoxin systems that functionally overlap. The highly reducing NADPH pool thereby serves as electron donor that enables glutathione reductase and thioredoxin reductase to keep glutathione pools and thioredoxins in their reduced redox state, respectively. Notably, also compartments containing oxidizing machineries are linked to these reducing pathways. Reducing pathways aid in proofreading of disulfide bond formation by isomerization or they provide reducing equivalents for the reduction of disulfides prior to degradation. In addition, they contribute to the thiol-dependent regulation of protein activities, and they help to counteract oxidative stress. The existence of oxidizing and reducing pathways in the same compartment poses a potential problem as the cell has to avoid futile cycles of oxidation and subsequent reduction reactions. Thus, compartments that contain oxidizing machineries have developed sophisticated ways to spatiotemporally balance and regulate oxidation and reduction. In this review, we discuss oxidizing and reducing pathways in the endoplasmic reticulum, the periplasm and the mitochondrial intermembrane space and highlight the role of glutathione especially in the endoplasmic reticulum and the intermembrane space. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Endogenous formation of protein adducts with carcinogenic aldehydes: implications for oxidative stress

In the present study, we characterize the covalent modification of a protein by crotonaldehyde, a representative carcinogenic aldehyde, and describe the endogenous production of this aldehyde in vivo. The crotonaldehyde preferentially reacted with the lysine and histidine residues of bovine serum albumin and generated a protein-linked carbonyl derivative. Upon incubation with the histidine and lysine derivatives, crotonaldehyde predominantly generated beta-substituted butanal adducts of histidine and lysine and N(epsilon)-(2,5-dimethyl-3-formyl-3,4-dehydropiperidino)lysine (dimethyl-FDP-lysine) as the putative carbonyl derivatives generated in the crotonaldehyde-modified protein. To verify the endogenous formation of crotonaldehyde in vivo, we raised the monoclonal antibody (mAb82D3) against the crotonaldehyde-modified protein and found that it cross-reacted with the protein-bound 2-alkenals, such as crotonaldehyde, 2-pentenal, and 2-hexenal. The anti-2-alkenal antibody recognized multiple crotonaldehyde-lysine adducts, including dimethyl-FDP-lysine and an unknown product, which showed the greatest immunoreactivity with the antibody. On the basis of the chemical and spectroscopic evidence, the major antigenic product was determined to be a novel Schiff base-derived crotonaldehyde-lysine adduct, N(epsilon)-(5-ethyl-2-methylpyridinium)lysine (EMP-lysine). It was found that the lysine residues that had disappeared in the protein treated with crotonaldehyde were partially recovered by EMP-lysine. The presence of immunoreactive materials with mAb82D3 in vivo was demonstrated in the kidney of rats exposed to the renal carcinogen, ferric nitrilotriacetate. In addition, the observations that the metal-catalyzed oxidation of polyunsaturated fatty acids in the presence of proteins resulted in an increase in the antigenicity of the protein indicated that lipid peroxidation represents a potential pathway for the formation of crotonaldehyde/2-alkenals in vivo. These data suggest that the formation of carcinogenic aldehydes during lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.     

Glucose-induced oxidative stress leads to in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells

BackgroundDementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes.MethodsWe used SH-SY5Y cells cultured in high-glucose media.ResultsS-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation.ConclusionGlucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation.General significanceThe results may be useful for designing curative treatment strategies for dementia.     

Regulation of proteasome-mediated protein degradation during oxidative stress and aging

Protein degradation is a physiological process required to maintain cellular functions. There are distinct proteolytic systems for different physiological tasks under changing environmental and pathophysiological conditions. The proteasome is responsible for the removal of oxidatively damaged proteins in the cytosol and nucleus. It has been demonstrated that proteasomal degradation increases due to mild oxidation, whereas at higher oxidant levels proteasomal degradation decreases. Moreover, the proteasome itself is affected by oxidative stress to varying degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be resistant. Non-degradable protein aggregates and cross-linked proteins are able to bind to the proteasome, which makes the degradation of other misfolded and damaged proteins less efficient. Consequently, inhibition of the proteasome has dramatic effects on cellular aging processes and cell viability. It seems likely that during oxidative stress cells are able to keep the nuclear protein pool free of damage, while cytosolic proteins may accumulate. This is because of the high proteasome content in the nucleus, which protects the nucleus from the formation and accumulation of non-degradable proteins. In this review we highlight the regulation of the proteasome during oxidative stress and aging.     

Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach

Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes

Aggresomes are dynamic structures formed when the ubiquitin–proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with γ-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.     

Specific mixed disulfide formation with purified bovine cardiac glycogen synthase I and glutathione

Bovine cardiac glycogen-free glycogen synthase I reacts with oxidized glutathione at low temperature to partially inactivate the enzyme. Evidence is presented that a mixed disulfide between glutathione and the enzyme is formed in this reaction. A short incubation of the GSSG-treated enzyme with dithiothreitol restores full enzyme activity. The reaction with GSSG is pH dependent and the product is quite stable at neutral pH. Oxidation of one sulfhydryl group in glycogen synthase is associated with a loss of 60-70% of the enzyme activity. Further modification of protein sulfhydryls has less effect on the enzyme activity. Other low molecular weight disulfides also inactivate glycogen synthase and treatment with [35S]cystine to produce a 40% loss of enzyme activity gave rise to a single major radioactive peptide after cyanogen bromide digestion. Thus the GSSG-mediated inactivation of glycogen synthase apparently occurs through a single reactive sulfhydryl group that forms a mixed disulfide with low molecular weight disulfide molecules. Uridine 5'-diphosphate glucose and glycogen prevent the inactivation of glycogen-free glycogen synthase with GSSG, and glucose 6-phosphate retards the rate of inactivation. Reduction and reactivation of the GSSG-oxidized glycogen synthase is not affected by glycogen and it occurs readily at neutral pH with dithiothreitol, mercaptoethanol, or cysteamine. Oxidation of the reactive sulfhydryl group with GSSG has no effect on the rate of glycogen synthase phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.     

Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Process conditions with higher acidic peaks corresponded to elevated supernatant peroxide concentration, intracellular reactive oxygen species (ROS) levels, or both. Changes in glycation level were the primary cause of the charge heterogeneity, and for the first time, supernatant peroxide was found to positively correlate with glycation levels. Based on these findings, a novel mathematical model was developed to demonstrate that the rate of acidic species formation was exponentially proportional to the concentrations of supernatant peroxide and protein product. This work provides critical insights into charge variant formation during the cell culture process and highlights the importance of modulating of cell culture oxidative stress for charge variant control.