Redox Proteomics Research Articles

OP35 - Mitochondrial oxidative stress in skeletal muscle and cardiac aging

Mitochondrial dysfunction plays an important role in many chronic diseases, including aging. However the role of oxidative stress in this dysfunction remains controversial. We tested whether mitochondrial oxidative stress underlies not only mitochondrial deficits, but also declines in performance of skeletal and cardiac muscle in aged mice. Direct manipulation of mitochondrial H 2 O 2 (mtH 2 O 2 ) production by lifelong overexpression of mitochondrial targeted catalase reduced mitochondrial deficits in aged skeletal muscle and protected against age-related declines in cardiac function. To test whether manipulating mitochondrial oxidative stress late in life could reverse deficits in aged skeletal muscle and heart we used a novel mitochondrial targeted peptide, SS-31, that interacts with cardiolipin on the inner mitochondrial membrane. SS-31 reduces mtH 2 O 2 production in dysfunctional aged mitochondria and improved glutathione redox status while not affecting young healthy mitochondria. Acute (1 hr) and 8 week treatment with SS-31 had similar effects on mitochondrial function and skeletal muscle performance. Both treatments reversed age-related declines in in vivo maximal mitochondrial ATP production and efficiency of oxidative phosphorylation (P/O) while increasing muscle fatigue resistance. Treatment for 8 weeks also reversed cardiac dysfunction, especially diastolic dysfunction. These improvements in skeletal muscle and cardiac function translated to improved treadmill endurance capacity in the aged mice. These results reveal a dynamic relationship between mitochondrial deficits and the redox environment of the cell that affects function in these tissues. To examine the mechanisms underlying this interaction we are examining the effect of manipulating mitochondrial oxidant production on the thiol redox proteome. Aging is associated with increased S-glutathionylation of the thiol proteome in aged skeletal muscle. Treatment with SS-31 partially reverses many of these age-related changes, including proteins involved in E-C coupling, muscle contraction, and protein quality control. These results demonstrate that mitochondrial oxidant production alters redox dependent post-translational modifications throughout the muscle thereby providing a potential mechanism linking mitochondrial oxidative stress to loss of muscle function with age. Furthermore, we demonstrate that reversing this mitochondrial oxidative stress can rapidly improve function highlighting the interaction between mitochondria and cellular redox environment as an attractive target to improve health in the elderly.

Oxidative stress in Alzheimer disease and mild cognitive impairment: evidence from human data provided by redox proteomics.

Alzheimer disease (AD) is a neurodegenerative disease with many known pathological features, yet there is still much debate into the exact cause and mechanisms for progression of this degenerative disorder. The amyloid-beta (Aβ)-induced oxidative stress hypothesis postulates that it is the oligomeric Aβ that inserts into membrane systems to initiate much of the oxidative stress observed in brain during the progression of the disease. In order to study the effects of oxidative stress on tissue from patients with AD and amnestic mild cognitive impairment (MCI), we have developed a method called redox proteomics that identifies specific brain proteins found to be selectively oxidized. Here, we discuss experimental findings of oxidatively modified proteins involved in three key cellular processes implicated in the pathogenesis of AD progression: energy metabolism, cell signaling and neurotransmission, as well as the proteasomal degradation pathways and antioxidant response systems. These proteomics studies conducted by our laboratory and others in the field shed light on the molecular changes imposed on the cells of AD and MCI brain, through the deregulated increase in oxidative/nitrosative stress inflicted by Aβ and mitochondrial dysfunction.

The Redox Code.

Significance: The redox code is a set of principles that defines the positioning of the nicotinamide adenine dinucleotide (NAD, NADP) and thiol/disulfide and other redox systems as well as the thiol redox proteome in space and time in biological systems. The code is richly elaborated in an oxygen-dependent life, where activation/deactivation cycles involving O2 and H2O2 contribute to spatiotemporal organization for differentiation, development, and adaptation to the environment. Disruption of this organizational structure during oxidative stress represents a fundamental mechanism in system failure and disease. Recent Advances: Methodology in assessing components of the redox code under physiological conditions has progressed, permitting insight into spatiotemporal organization and allowing for identification of redox partners in redox proteomics and redox metabolomics. Critical Issues: Complexity of redox networks and redox regulation is being revealed step by step, yet much still needs to be learned. Future Directions: Detailed knowledge of the molecular patterns generated from the principles of the redox code under defined physiological or pathological conditions in cells and organs will contribute to understanding the redox component in health and disease. Ultimately, there will be a scientific basis to a modern redox medicine. Antioxid. Redox Signal. 23, 734–746.

Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation, Influenza Virus Infection and Oxidative Stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.

Antioxidant enzymes as redox-based biomarkers: a brief review.

The field of redox proteomics focuses to a large extent on analyzing cysteine oxidation in proteins under different experimental conditions and states of diseases. The identification and localization of oxidized cysteines within the cellular milieu is critical for understanding the redox regulation of proteins under physiological and pathophysiological conditions, and it will in turn provide important information that are potentially useful for the development of novel strategies in the treatment and prevention of diseases associated with oxidative stress. Antioxidant enzymes that catalyze oxidation/reduction processes are able to serve as redox biomarkers in various human diseases, and they are key regulators controlling the redox state of functional proteins. Redox regulators with antioxidant properties related to active mediators, cellular organelles, and the surrounding environments are all connected within a network and are involved in diseases related to redox imbalance including cancer, ischemia/reperfusion injury, neurodegenerative diseases, as well as normal aging. In this review, we will briefly look at the selected aspects of oxidative thiol modification in antioxidant enzymes and thiol oxidation in proteins affected by redox control of antioxidant enzymes and their relation to disease. [BMB Reports 2015; 48(4): 200-208]

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle.

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid—a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

Redox proteomics identification of specifically carbonylated proteins in the hippocampi of triple transgenic Alzheimer's disease mice at its earliest pathological stage

Alzheimer's disease (AD) is the most common cause of dementia in the elderly population. Attempts to develop therapies for the treatment of the late stage AD have been unsuccessful. Increasing evidences indicate that oxidative stress is an early event of neurodegeneration, however the pathogenic mechanism of AD remains unclarified. In the present study, slot-blot analysis was used to determine the levels of protein carbonyls in the hippocampi of 3-month-old triple transgenic AD mice (3×Tg-AD). The increased levels of protein carbonyls were observed in the hippocampi of 3×Tg-AD mice as compared to the non-transgenic controls (non-Tg). Using a redox-proteomic approach, twelve proteins were found to be significantly altered in the levels of protein carbonyls in the hippocampus. These proteins are crucial in energy metabolism, protein folding, cell structure, signal transduction and excitotoxicity. Immunoprecipitation and Western blot were used to validate two proteins identified by the redox proteomics. In addition, increased expression level of carbonyl reductase 1 (CBR1) was observed in the hippocampi of 3×Tg-AD mice. These results demonstrate that significant protein carbonylation occurs early in the 3-month-old 3×Tg-AD mice, which support the viewpoint that oxidative stress is an early event in AD progression. Biological significanceIn this study, we have observed increased levels of protein carbonyls in the hippocampi of 3×Tg-AD mice before the appearance of Aβ plaques and neurofibrillary tangles (NFTs). By redox proteomics, twelve specifically carbonylated proteins were identified. Among them, alpha-enolase (ENO1) and glutamine synthetase (GS) were identified as the common targets of oxidation in the brains of 3×Tg-AD mice, mild cognitive impairment (MCI) sufferers and AD patients. For the first time, the oxidation of t-complex protein 1 subunit epsilon (CCT5) and protein disulfide-isomerase A3 (PDIA3) were reported to be associated with AD. These results indicated that the combination of monoclonal anti-DNP antibody with digital imaging system could enhance the specificity and accuracy of redox proteomics analysis. Those data support the viewpoint that oxidative stress occurs at the early pathological stage of AD. In addition, this paper provides new information for understanding the pathological process of AD and for developing more appropriate therapies to intervene AD progression.

Effects of anthracene on filtration rates, antioxidant defense system, and redox proteomics in the Mediterranean clam Ruditapes decussatus (Mollusca: Bivalvia).

This study aimed at analyzing the impact of a toxic polyaromatic hydrocarbon (PAH), anthracene (ANT), on Ruditapes decussatus collected from a Tunisian coastal lagoon (Bizerte Lagoon). Filtration rates, several antioxidant enzymes--superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione transferase (GST)--as well as indices of protein oxidation status were determined in various tissues of this bivalve. Specimens were exposed to 100 μg/L of ANT for 2 days. ANT levels were evaluated using HPLC and were detected in the gill and digestive gland at different amounts. ANT exposure altered the behavior of bivalves by changing the siphon movement and decreasing filtration rate significantly. The enzymatic results indicated that ANT exposure affected the oxidative stress status of the gills of R. decussatus. In addition, modification of proteins was detected in the gills using redox proteomics after ANT treatment. Three protein spots were successfully identified by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS). These proteins can be roughly related to muscle contraction function. In contrast, no significant modification of enzymatic and protein responses was detected in the digestive gland after ANT treatment. These data demonstrate that combined behavioral and biochemical analyses are a powerful tool to provide valuable insights into possible mechanisms of toxicity of anthracene in R. decussatus. Additionally, the results highlight the potential of the gill as a valuable candidate for investigating PAH toxicity.

Application of redox proteomics to the study of oxidative degradation products in archaeological wool

Abstract Most archaeological and historical textiles (clothing, tapestries, blankets, carpets, etc.) present traces of UV-induced damage when exposed to light during their lifetime. Yellowing of the fibres, fading of the dyes and loss of physical properties, such as tensile strength are the typical indicators of photodegradation. Natural fibres made of proteins, such as wool and silk are particularly sensitive to UV damage. Photo-oxidative damage is caused by the accumulation of chemical modification at the amino acid residue level that lead to a range of oxidation products, including chromophores responsible for changes in coloration, as well as to the breaking of peptide bonds in the protein backbone. Amino acid residues with aromatic side-chain groups are particularly sensitive to photo-oxidation and breakthroughs have been made in recent years in the field of protein science to identify the photoproducts and locate them within proteins. This study explores new methodologies using redox proteomics-based strategies to assess the extent of photodamage in ancient wool textiles, by identifying modifications occurring at the molecular level. Using a scoring system to determine the level of oxidation in amino acids with aromatic side-chains (tryptophan, tyrosine, histidine and phenylalanine), we compare the effects of dyes and mordants on fibres after UV ageing, and assess the extent of oxidation on the different proteins composing the wool fibres. We determine that dyes and mordants have the capability of slowing down photo-oxidation during ageing. We also assess the effect of UV irradiation on deamidation, a modification targeting glutamine and asparagine, as it is a common marker of ageing in ancient proteins.

Quantifying reversible oxidation of protein thiols in photosynthetic organisms.

Photosynthetic organisms use dynamic post-translational modifications to survive and adapt, which include reversible oxidative modifications of protein thiols that regulate protein structure, function, and activity. Efforts to quantify thiol modifications on a global scale have relied upon peptide derivatization, typically using isobaric tags such as TMT, ICAT, or iTRAQ that are more expensive, less accurate, and provide less proteome coverage than label-free approaches--suggesting the need for improved experimental designs for studies requiring maximal coverage and precision. Herein, we present the coverage and precision of resin-assisted thiol enrichment coupled to label-free quantitation for the characterization of reversible oxidative modifications on protein thiols. Using C. reinhardtii and Arabidopsis as model systems for algae and plants, we quantified 3662 and 1641 unique cysteinyl peptides, respectively, with median coefficient of variation (CV) of 13% and 16%. Further, our method is extendable for the detection of protein abundance changes and stoichiometries of cysteine oxidation. Finally, we demonstrate proof-of-principle for our method, and reveal that exogenous hydrogen peroxide treatment regulates the C. reinhardtii redox proteome by increasing or decreasing the level of oxidation of 501 or 67 peptides, respectively. As protein activity and function is controlled by oxidative modifications on protein thiols, resin-assisted thiol enrichment coupled to label-free quantitation can reveal how intracellular and environmental stimuli affect plant survival and fitness through oxidative stress.

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an “oxidative environment” by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis.

Oxidative Stress Regulates Titin Elasticity by Affecting Ig-Domain Stability

Background: The elasticity of titin is regulated through several mechanisms, including isoform switching and phosphorylation of unique spring elements (N2-Bus, PEVK). However, the titin springs consist mainly of immunoglobulin-like (Ig) domains, which are centrally involved in the molecular mechanism of titin elasticity. Passive force-regulating mechanisms targeting the Ig-domains of titin have not been described.Methods and Results: We have elucidated a novel oxidative stress-related mechanism regulating muscle elasticity by altering the stability of titin-Ig domains (Alegre-Cebollada et al., Cell. 2014;156:1235-46). Using single-molecule AFM force spectroscopy, force measurements of isolated skinned human myocytes, and redox proteomics, we show that I-band Ig-domains of titin are weakened by oxidative modification of cryptic cysteines. We demonstrate that mechanical unfolding of these Ig domains exposes hidden cysteines, which now become accessible to disulfide bonding or S-glutathionylation in the presence of millimolar concentrations of oxidized glutathione (GSSG). In the AFM experiments, the cysteines of unfolded titin-Ig domains preferentially formed mixed disulfides with glutathione, which prevented the refolding of these domains. Oxidation by GSSG substantially reduced the passive tension of stretched human myocytes, and the effect was fully reversible with the incubation of reduced glutathione. Exposing perfused mouse hearts to oxidative stress (0.1 mM H2O2) revealed that Ig-domains from I-band titin are preferential targets of oxidation, as monitored using ICAT labeling/mass spectrometry.Conclusions: Titin elasticity in striated muscle is modulated by oxidative stress through reversible weakening of Ig-domain stability via S-glutathionylation of buried cysteines. These titin Ig domains could also represent individual mechanosensors, whose mechanical properties determine mechano-chemical signaling processes in stressed myocytes.

Thiol-based redox proteomics in cancer research.

Cancer cells maintain their intracellular ROS concentrations at required levels for their survival. Changes in ROS concentrations can regulate biochemical signaling mechanisms that control cell function. It has been demonstrated that ROS regulate the cellular events through redox regulation of redox-sensitive proteins (redox sensors). Upon oxidative stress, redox sensors undergo redox modifications that cause the allosteric changes of these proteins and endow them with different functions. Understanding the altered functions of redox sensors and the underlying mechanisms is critical for the development of novel cancer therapeutics. Recently, a series of high-throughput proteomics approaches have been developed for screening redox processes. In this manuscript, we review these methodologies and discuss the important redox sensors recently identified that are related to cancer.

Redox proteomics and physiological responses in Cistus albidus shrubs subjected to long-term summer drought followed by recovery.

Main conclusion The interaction between enzymatic and non-enzymatic antioxidants, endogenous levels of ABA and ABA-GE, the rapid recuperation of photosynthetic proteins under re-watering as well the high level of antioxidant proteins in previously drought-stressed plants under re-watering conditions, will contribute to drought resistance in plants subjected to a long-term drought stress under Mediterranean field conditions. This work provides an overview of the mechanisms of Cistus albidus acclimation to long-term summer drought followed by re-watering in Mediterranean field conditions. To better understand the molecular mechanisms of drought resistance in these plants, a proteomic study using 2-DE and MALDI-TOF/TOF MS/MS was performed on leaves from these shrubs. The analysis identified 57 differentially expressed proteins in water-stressed plants when contrasted to well watered. Water-stressed plants showed an increase, both qualitatively and quantitatively, in HSPs, and downregulation of photosynthesis and carbon metabolism enzymes. Under drought conditions, there was considerable upregulation of enzymes related to redox homeostasis, DHA reductase, Glyoxalase, SOD and isoflavone reductase. However, upregulation of catalase was not observed until after re-watering was carried out. Drought treatment caused an enhancement in antioxidant defense responses that can be modulated by ABA, and its catabolites, ABA-GE, as well as JA. Furthermore, quantification of protein carbonylation was shown to be a useful marker of the relationship between water and oxidative stress, and showed that there was only moderate oxidative stress in C. albidus plants subjected to water stress. After re-watering plants recovered although the levels of ABA-GE and antioxidant enzymes still remain higher than in well-watered plants. We expect that our results will provide new data on summer acclimation to drought stress in Mediterranean shrubs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2221-0) contains supplementary material, which is available to authorized users.

Redox proteomics reveal stress responsive proteins linking peroxiredoxin-1 status in glioma to chemosensitivity and oxidative stress

The combined deletion of chromosomal arms 1p and 19q has been described as a prognostic marker for oligodendroglial tumors. These tumors show a better response to chemotherapy and radiotherapy. Recently, we found a lower abundance of peroxiredoxin 1 (PRDX1) in oligodendroglial tumors with 1p/19q deletion, suggesting a potential role of this enzyme in the clearance of therapy induced reactive oxygen species (ROS). Here, we confirmed the importance of PRDX1 in tumor cell survival by PRDX1 knockdown and overexpression in A-172 cells treated with the alkylating agent bis-chloroethyl nitrosourea (BCNU). Overexpression of PRDX1 resulted in a higher resistance of cells to BCNU treatment. In addition, BCNU challenged cells showed higher levels of ROS in PRDX1 knockdown cells. We applied a modified version of the redox two dimensional difference gel electrophoresis approach to analyze ROS mediated effects on protein thiols after BCNU treatment by labeling protein thiols with fluorescent dyes. Altogether eleven proteins were identified showing PRDX1 dependent altered labeling, many of them have been previously linked to stress response processes. Furthermore, 30 additional potentially redox active proteins were identified. The majority of them is involved in therapy associated processes like cellular stress response, DNA damage and regulation of cell death and therewith suggests that tumor cells maintain a network of redox sensitive proteins to escape chemotherapy. This article is part of a Special Issue entitled: Medical Proteomics.

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

BackgroundIn the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts.ResultsHere we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme.ConclusionsThe data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1064) contains supplementary material, which is available to authorized users.

Proteome-wide Light/Dark Modulation of Thiol Oxidation in Cyanobacteria Revealed by Quantitative Site-specific Redox Proteomics

Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of ∼2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for ∼1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover, the redox sensitivity along with the stoichiometric data enabled prediction of potential functional Cys-sites for proteins of interest. The functional significance of redox-sensitive Cys-sites in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin (AhpC/TSA family protein Sll1621), and glucose 6-phosphate dehydrogenase was further confirmed with site-specific mutagenesis and biochemical studies. Together, our findings provide significant insights into the broad redox regulation of photosynthetic organisms.

Redox Modulation Matters: Emerging Functions for Glutaredoxins in Plant Development and Stress Responses.

Glutaredoxins (GRXs) are small ubiquitous glutathione (GSH)-dependent oxidoreductases that catalyze the reversible reduction of protein disulfide bridges or protein-GSH mixed disulfide bonds via a dithiol or monothiol mechanism, respectively. Three major classes of GRXs, with the CPYC-type, the CGFS-type or the CC-type active site, have been identified in many plant species. In spite of the well-characterized roles for GRXs in Escherichia coli, yeast and humans, the biological functions of plant GRXs have been largely enigmatic. The CPYC-type and CGFS-type GRXs exist in all organisms, from prokaryotes to eukaryotes, whereas the CC-type class has thus far been solely identified in land plants. Only the number of the CC-type GRXs has enlarged dramatically during the evolution of land plants, suggesting their participation in the formation of more complex plants adapted to life on land. A growing body of evidence indicates that plant GRXs are involved in numerous cellular pathways. In this review, emphasis is placed on the recently emerging functions for GRXs in floral organ development and disease resistance. Notably, CC-type GRXs have been recruited to participate in these two seemingly unrelated processes. Besides, the current knowledge of plant GRXs in the assembly and delivery of iron-sulfur clusters, oxidative stress responses and arsenic resistance is also presented. As GRXs require GSH as an electron donor to reduce their target proteins, GSH-related developmental processes, including the control of flowering time and the development of postembryonic roots and shoots, are further discussed. Profiling the thiol redox proteome using high-throughput proteomic approaches and measuring cellular redox changes with fluorescent redox biosensors will help to further unravel the redox-regulated physiological processes in plants.

Proteomics of protein post-translational modifications implicated in neurodegeneration.

Mass spectrometry (MS)-based proteomics has developed into a battery of approaches that is exceedingly adept at identifying with high mass accuracy and precision any of the following: oxidative damage to proteins (redox proteomics), phosphorylation (phosphoproteomics), ubiquitination (diglycine remnant proteomics), protein fragmentation (degradomics), and other posttranslational modifications (PTMs). Many studies have linked these PTMs to pathogenic mechanisms of neurodegeneration. To date, identifying PTMs on specific pathology-associated proteins has proven to be a valuable step in the evaluation of functional alteration of proteins and also elucidates biochemical and structural explanations for possible pathophysiological mechanisms of neurodegenerative diseases. This review provides an overview of methods applicable to the identification and quantification of PTMs on proteins and enumerates historic, recent, and potential future research endeavours in the field of proteomics furthering the understanding of PTM roles in the pathogenesis of neurodegeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/2047-9158-3-23) contains supplementary material, which is available to authorized users.

Disturbed flow enhances inflammatory signaling and atherogenesis by increasing thioredoxin-1 level in endothelial cell nuclei.

BackgroundOxidative stress occurs with disturbed blood flow, inflammation and cardiovascular disease (CVD), yet free-radical scavenging antioxidants have shown limited benefit in human CVD. Thioredoxin-1 (Trx1) is a thiol antioxidant protecting against non-radical oxidants by controlling protein thiol/disulfide status; Trx1 translocates from cytoplasm to cell nuclei due to stress signaling, facilitates DNA binding of transcription factors, e.g., NF-κB, and potentiates inflammatory signaling. Whether increased nuclear Trx1 contributes to proatherogenic signaling is unknown.Methodology/Principal Findings In vitro and in vivo atherogenic models were used to test for nuclear translocation of Trx1 and associated proinflammatory signaling. Disturbed flow by oscillatory shear stress stimulated Trx1 nuclear translocation in endothelial cells. Elevation of nuclear Trx1 in endothelial cells and transgenic (Tg) mice potentiated disturbed flow-stimulated proinflammatory signaling including NF-κB activation and increased expression of cell adhesion molecules and cytokines. Tg mice with increased nuclear Trx1 had increased carotid wall thickening due to disturbed flow but no significant differences in serum lipids or weight gain compared to wild type mice. Redox proteomics data of carotid arteries showed that disturbed flow stimulated protein thiol oxidation, and oxidation was higher in Tg mice than wild type mice.Conclusions/SignificanceTranslocation of Trx1 from cytoplasm to cell nuclei plays an important role in disturbed flow-stimulated proatherogenesis with greater cytoplasmic protein oxidation and an enhanced nuclear transcription factor activity. The results suggest that pharmacologic interventions to inhibit nuclear translocation of Trx1 may provide a new approach to prevent inflammatory diseases or progression.