Redox Proteomics Research Articles

Changes in Milk Protein Interactions and Associated Molecular Modification Resulting from Thermal Treatments and Storage

We investigated protein modifications that occur during short- and long-term storage of raw, pasteurized, and ultra-high-temperature processed (UHT) milks using RE-HPLC and redox proteomics. The RE-HPLC results show that casein dissociation and whey protein/κ-casein association occurred in both pasteurized and UHT milk. The extent of protein interactions was more pronounced in UHT milk after storage. The redox proteomics analyses show that primary structural level protein modifications were not correlated to processing type on the of day processing but did occur and increase during storage. Methionine oxidation was the most significant type of oxidative modification in all samples, particularly in the caseins. Methionine oxidation increased in the UHT-treated milk samples with longer storage times, especially in the micelle-phase proteins, likely due to the increasing exposure of these proteins as they migrated to the serum phase. Glycated and lactosylated early-stage Maillard reaction products were also found after heat treatment, particularly in UHT-treated milk, with the levels of these products maintained and generally increased with increasing storage time. PRACTICAL APPLICATION: Understanding changes in protein modification during heat processing and storage of liquid milk products may help develop a model to predict the quality and shelf-life stability of heat treated milk products.

Regulation of thiol metabolism as a factor that influences the development and storage capacity of beech seeds

Seeds are the basis of propagation for the common beech (Fagus sylvatica L.), but the seed set of the beech is unsteady, with 5–10 years between abundant crops. Beech seeds are very difficult to store and lose their viability quickly even in optimum storage conditions. To date, it has not been possible to determine factors indicative of the aging process and the loss of viability of beech seeds during storage. To address this important economic challenge and interesting scientific problem, we analyzed the adjustment of the redox state during the development and storage of seeds. Many metabolic processes are based on reduction and oxidation reactions. Thiol proteins control and react to the redox state in the cells. The level of thiol proteins increased during seed maturation and decreased during storage. Gel-based redox proteomics identified 17 proteins in beech seeds during development. The proteins could be assigned to processes like metabolism and antioxidant functions. During storage, the number of proteins decreased to only six, i.e., oxidoreductases, peptidases, hydrolases and isomerases. The occurrence of peroxiredoxins (PRX) as thiol peroxidases and redox regulators indicates an important role of cytosolic 1CysPRX and PRXIIC, mitochondrial PRXIIF, and plastidic PRXIIE, 2CysPRX, and PRXQ in beech seeds during development and storage. Particularly, 2CysPRX was present in beech seeds during development and storage and may perform an important function in regulation of the redox state during both seed development and storage. The role of thiol proteins in the regulation of the redox state during the development and storage of beech seeds is discussed.

Uncovering sexual dimorphism in type 2 diabetes by redox proteomics.

Uncovering sexual dimorphism in type 2 diabetes by redox proteomics.

Redox and global interconnected proteome changes in mice exposed to complex environmental hazards surrounding Doñana National Park

Natural environments are receiving an increasing number of contaminants. Therefore, the evaluation and identification of early responses to pollution in these complex habitats is an urgent and challenging task. Doñana National Park (DNP, SW Spain) has been widely used as a model area for environmental studies because, despite its strictly protected core, it is surrounded by numerous threat sources from agricultural, mining and industrial activities. Since many pollutants often induce oxidative stress, redox proteomics was used to detect redox-based variations within the proteome of Mus spretus mice captured in DNP and the surrounding areas. Functional analysis showed that most differentially oxidized proteins are involved in the maintenance of homeostasis, by eliciting mechanisms to respond to toxic substances and oxidative stress, such as antioxidant and biotransformation processes, immune and inflammatory responses, and blood coagulation. Furthermore, changes in the overall protein abundance were also analysed by label-free quantitative proteomics. The upregulation of phase I and II biotransformation enzymes in mice from Lucio del Palacio may be an alert for organic pollution in the area located at the heart of DNP. Metabolic processes involved in protein turnover (proteolysis, amino acid catabolism, new protein biosynthesis and folding) were activated in response to oxidative damage to these biomolecules. Consequently, aerobic respiratory metabolism increased to address the greater ATP demands. Alterations of cholesterol metabolism that could cause hepatic steatosis were also detected. The proteomic detection of globally altered metabolic and physiological processes offers a complete view of the main biological changes caused by environmental pollution in complex habitats.

Bioinspired Small-Molecule Tools for the Imaging of Redox Biology.

The availability of electrons to biological systems underpins the mitochondrial electron transport chain (ETC) that powers living cells. It is little wonder, therefore, that the sufficiency of electron supply is critical to cellular health. Considering mitochondrial redox activity alone, a lack of oxygen (hypoxia) leads to impaired production of adenosine triphosphate (ATP), the major energy currency of the cell, whereas excess oxygen (hyperoxia) is associated with elevated production of reactive oxygen species (ROS) from the interaction of oxygen with electrons that have leaked from the ETC. Furthermore, the redox proteome, which describes the reversible and irreversible redox modifications of proteins, controls many aspects of biological structure and function. Indeed, many major diseases, including cancer and diabetes, are now termed "redox diseases", spurring much interest in the measurement and monitoring of redox states and redox-active species within biological systems. In this Account, we describe recent efforts to develop magnetic resonance (MR) and fluorescence imaging probes for studying redox biology. These two classes of molecular imaging tools have proved to be invaluable in supplementing the structural information that is traditionally provided by MRI and fluorescence microscopy, respectively, with highly sensitive chemical information. Importantly, the study of biological redox processes requires sensors that operate at biologically relevant reduction potentials, which can be achieved by the use of bioinspired redox-sensitive groups. Since oxidation-reduction reactions are so crucial to modulating cellular function and yet also have the potential to damage cellular structures, biological systems have developed highly sophisticated ways to regulate and sense redox changes. There is therefore a plethora of diverse chemical structures in cells with biologically relevant reduction potentials, from transition metals to organic molecules to proteins. These chemical groups can be harnessed in the development of exogenous molecular imaging agents that are well-tuned to biological redox events. To date, small-molecule redox-sensitive tools for oxidative stress and hypoxia have been inspired from four classes of cellular regulators. The redox-sensitive groups found in redox cofactors, such as flavins and nicotinamides, can be used as reversible switches in both fluorescent and MR probes. Enzyme substrates that undergo redox processing within the cell can be modified to provide fluorescence or MR readout while maintaining their selectivity. Redox-active first-row transition metals are central to biological homeostasis, and their marked electronic and magnetic changes upon oxidation/reduction have been used to develop MR sensors. Finally, redox-sensitive amino acids, particularly cysteine, can be utilized in both fluorescent and MR sensors.

OxSWATH: An integrative method for a comprehensive redox-centered analysis combined with a generic differential proteomics screening

Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation without considering the variation in the total levels of the proteins. However, protein synthesis and protein degradation also belong to the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in PTMs-focused analyses, such as cysteine redox characterization. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using a combination of commonly available alkylating reagents (oxSWATH) is presented, by which it is possible to integrate the information regarding relative cysteine oxidation with the analysis of the total protein levels in a cost-effective high-throughput approach.The proposed method was tested using a redox-regulated protein and further applied to a comparative analysis of secretomes obtained from cells cultured under control or oxidative stress conditions to strengthen the importance of considering the overall proteome changes. Using the OxSWATH method it was possible to determine both the relative proportion of reduced and reversible oxidized oxoforms, as well as the total levels of each oxoform by taking into consideration the total levels of the protein. Therefore, using OxSWATH the comparative analyses can be performed at two different levels by considering the relative proportion or the total levels at both peptide and protein level. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can also be performed, allowing a truly comprehensive evaluation of proteomics changes upon the oxidative stimulus.Data are available via ProteomeXchange and SWATHAtlas with the identifiers PXD006802, PXD006802, and PASS01210.

Large‐Scale Analysis of Redox‐Sensitive Conditionally Disordered Protein Regions Reveals Their Widespread Nature and Key Roles in High‐Level Eukaryotic Processes

Recently developed quantitative redox proteomic studies enable the direct identification of redox-sensing cysteine residues that regulate the functional behavior of target proteins in response to changing levels of reactive oxygen species. At the molecular level, redox regulation can directly modify the active sites of enzymes, although a growing number of examples indicate the importance of an additional underlying mechanism that involves conditionally disordered proteins. These proteins alter their functional behavior by undergoing a disorder-to-order transition in response to changing redox conditions. However, the extent to which this mechanism is used in various proteomes is currently unknown. Here, a recently developed sequence-based prediction tool incorporated into the IUPred2A web server is used to estimate redox-sensitive conditionally disordered regions at a large scale. It is shown that redox-sensitive conditional disorder is fairly widespread in various proteomes and that its presence strongly correlates with the expansion of specific domains in multicellular organisms that largely rely on extra stability provided by disulfide bonds or zinc ion binding. The analyses of yeast redox proteomes and human disease data further underlie the significance of this phenomenon in the regulation of a wide range of biological processes, as well as its biomedical importance.

Protein carbonylation in human bronchial epithelial cells exposed to cigarette smoke extract.

Cigarette smoke is a well-established exogenous risk factor containing toxic reactive molecules able to induce oxidative stress, which in turn contributes to smoking-related diseases, including cardiovascular, pulmonary, and oral cavity diseases. We investigated the effects of cigarette smoke extract on human bronchial epithelial cells. Cells were exposed to various concentrations (2.5-5-10-20%) of cigarette smoke extract for 1, 3, and 24h. Carbonylation was assessed by 2,4-dinitrophenylhydrazine using both immunocytochemical and Western immunoblotting assays. Cigarette smoke induced increasing protein carbonylation in a concentration-dependent manner. The main carbonylated proteins were identified by means of two-dimensional electrophoresis coupled to MALDI-TOF mass spectrometry analysis and database search (redox proteomics). We demonstrated that exposure of bronchial cells to cigarette smoke extract induces carbonylation of a large number of proteins distributed throughout the cell. Proteins undergoing carbonylation are involved in primary metabolic processes, such as protein and lipid metabolism and metabolite and energy production as well as in fundamental cellular processes, such as cell cycle and chromosome segregation, thus confirming that reactive carbonyl species contained in cigarette smoke markedly alter cell homeostasis and functions.

Detection of S-Nitrosation and S-Glutathionylation of the Human Branched-Chain Aminotransferase Proteins.

The human branched-chain aminotransferase (hBCAT) enzymes play an integral role in brain glutamate and branched-chain amino acid (BCAA) metabolism. Optimal hBCAT activity is dependent on the oxidation state of their redox reactive thiols, where post-translational modification by nitric oxide (NO) and glutathione results in reversible inhibition. Incubation of the cytosolic isoform (hBCATc) with S-nitrosating agents was found to inhibit in both a time and dose dependent manner through formation of a mixture of products including cysteine-nitric oxide (SNO) and S-glutathionylation. Mechanistic details of these redox interactions were studied using labeling with fluorescein-5-maleimide and confirmed via mass spectrometry and Western blot analysis. Though the mitochondrial isoform (hBCATm) was inhibited by nitrosating agents adduct formation could only be observed by DTNB titration as neither SNO, S-glutathionylation or disulfide bond formation could be detected. These studies revealed that the two isoforms of hBCAT, namely hBCATc and hBCATm, were differently regulated by S-nitrosation or S-glutathionylation pointing to distinct functional/mechanistic responses to GSNO modification. Detection of these adducts is essential for studies into the effect of NO on cells and the redox proteome which can offer insight into several pathological states and normal functioning of the cell.

Protein carbonylation as a possible way to modulate breast cancer cell proliferation

Introduction.High rates of cancer incidence and mortality worldwide dictate the necessity of developing new methodological approaches in understanding the molecular mechanisms of cancer progression associated with intracellular redox regulation imbalance.The objectiveof the study was to evaluate the role of protein carbonylation in regulating breast cancer cell proliferation under redox status modulation.Materials and Methods. In the intact breast cancer cells and in the cells cultured under redox status modulation using 5mM N-ethylmaleimide (an - SH group blocker) and 5 Mm 1,4-dithioerythritol (a thiol group protector), the concentration of thioredoxin and its carbonylated form was measured using Western blot analysis. The activity of thioredoxin reductase and the level of protein carbonyl derivatives were determined using spectrophotometry. Cell cycle phase distribution was evaluated by flow cytometry.Results and Discussion. Under the effect of N-ethylmaleimide, cell cycle arrest in the S-phase was confirmed by oxidative modification of proteins, including thioredoxin carbonylation. When culturing MCF-7 cells in the presence of 1,4-dithioerythritol, cell cycle arrest in the G0/G1 phases was associated with a rise in the concentrations of reduced thioredoxin and glutathione forms.Conclusion.The thioredoxin system and oxidative modification of proteins are involved in redox-dependent modulation of breast cancer cell proliferation. Studies in the area of redox proteomics offer great potential to seek molecular targets of malignant transformation of breast cells.

Site-Specific Proteomic Mapping of Modified Cysteine Residues.

The wide reactivity of the thiol group enables the formation of a number of chemically and biologically distinct posttranslational modifications. Proteins within nearly all major families undergo some form of cysteine modification and the modifications are associated with regulatory functions across many biological processes. However, the susceptibility of thiols to redox shifts, as well as the labile nature of most thiol modifications, renders detection difficult. Analysis difficulties are compounded further in complex protein mixtures due to the typical low abundance of cysteine modifications under normal physiological conditions. Here we describe methods for the analysis of three cysteine modifications: nitrosylation, glutathionylation, and S-acylation. The three methods use the same organic mercury-conjugated agarose resin as an enrichment platform. To date, over 2154 sites on 1446 proteins have been identified between the three modifications using this method. Using equivalent processing, enrichment, and analytical methods has enabled a more comprehensive picture of the redox proteome landscape.

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Quantifying changes in the bacterial thiol redox proteome during host-pathogen interaction

Phagocyte-derived production of a complex mixture of different oxidants is a major mechanism of the host defense against microbial intruders. On the protein level, a major target of these oxidants is the thiol group of the amino acid cysteine in proteins. Oxidation of thiol groups is a widespread regulatory post-translational protein modification. It is used by bacteria to respond to and to overcome oxidative stress. Numerous redox proteomic studies have shown that protein thiols in bacteria, such as Escherichia coli react towards a number of oxidants in specific ways. However, our knowledge about protein thiols in bacteria exposed to the complex mixture of oxidants encountered in the phagolysosome is still limited. In this study, we used a quantitative redox proteomic method (OxICAT) to assess the in vivo thiol oxidation status of phagocytized E. coli. The majority (65.5%) of identified proteins harbored thiols that were significantly oxidized (> 30%) upon phagocytosis. A substantial number of these proteins are from major metabolic pathways or are involved in cell detoxification and stress response, suggesting a systemic breakdown of the bacterial cysteine proteome in phagocytized bacteria. 16 of the oxidized proteins provide E. coli with a significant growth advantage in the presence of H2O2, when compared to deletion mutants lacking these proteins, and 11 were shown to be essential under these conditions.

Low-dose cadmium disrupts mitochondrial citric acid cycle and lipid metabolism in mouse lung

Cadmium (Cd) causes acute and chronic lung toxicities at occupational exposure levels, yet the impacts of Cd exposure at low levels through dietary intake remain largely uncharacterized. Health concerns arise because humans do not have an effective Cd elimination mechanism, resulting in a long (10- to 35-y) biological half-life. Previous studies showed increased mitochondrial oxidative stress and cell death by Cd yet the details of mitochondrial alterations by low levels of Cd remain unexplored. In the current study, we examined the impacts of Cd burden at a low environmental level on lung metabolome, redox proteome, and inflammation in mice given Cd at low levels by drinking water (0, 0.2, 0.6 and 2.0 mg Cd/L) for 16 weeks. The results showed that mice accumulated lung Cd comparable to non-smoking humans and showed inflammation in lung by histopathology at 2 mg Cd/L. The results of high resolution metabolomics combined with bioinformatics showed that mice treated with 2 mg Cd/L increased levels of lipids in the lung, accompanied by disruption in mitochondrial energy metabolism. In addition, targeted metabolomic analysis showed that these mice had increased accumulation of mitochondrial carnitine and citric acid cycle intermediates. The results of redox proteomics showed that Cd at 2 mg/L stimulated oxidation of isocitrate dehydrogenase, malate dehydrogenase and ATP synthase. Taken together, the results showed impaired mitochondrial function and accumulation of lipids in the lung with a Cd dose response relevant to non-smokers without occupational exposures. These findings suggest that dietary Cd intake could be an important variable contributing to human pulmonary disorders.

Differential regulation of cysteine oxidative post-translational modifications in high and low aerobic capacity

Given the association between high aerobic capacity and the prevention of metabolic diseases, elucidating the mechanisms by which high aerobic capacity regulates whole-body metabolic homeostasis is a major research challenge. Oxidative post-translational modifications (Ox-PTMs) of proteins can regulate cellular homeostasis in skeletal and cardiac muscles, but the relationship between Ox-PTMs and intrinsic components of oxidative energy metabolism is still unclear. Here, we evaluated the Ox-PTM profile in cardiac and skeletal muscles of rats bred for low (LCR) and high (HCR) intrinsic aerobic capacity. Redox proteomics screening revealed different cysteine (Cys) Ox-PTM profile between HCR and LCR rats. HCR showed a higher number of oxidized Cys residues in skeletal muscle compared to LCR, while the opposite was observed in the heart. Most proteins with differentially oxidized Cys residues in the skeletal muscle are important regulators of oxidative metabolism. The most oxidized protein in the skeletal muscle of HCR rats was malate dehydrogenase (MDH1). HCR showed higher MDH1 activity compared to LCR in skeletal, but not cardiac muscle. These novel findings indicate a clear association between Cys Ox-PTMs and aerobic capacity, leading to novel insights into the role of Ox-PTMs as an essential signal to maintain metabolic homeostasis.

Redox proteomics and amyloid β-peptide: insights into Alzheimer disease.

Alzheimer disease (AD) is a progressive neurodegenerative disorder associated with aging and characterized pathologically by the presence of senile plaques, neurofibrillary tangles, and neurite and synapse loss. Amyloid beta-peptide (1-42) [Aβ(1-42)], a major component of senile plaques, is neurotoxic and induces oxidative stress invitro and invivo. Redox proteomics has been used to identify proteins oxidatively modified by Aβ(1-42) invitro and invivo. In this review, we discuss these proteins in the context of those identified to be oxidatively modified in animal models of AD, and human studies including familial AD, pre-clinical AD (PCAD), mild cognitive impairment (MCI), early AD, late AD, Down syndrome (DS), and DS with AD (DS/AD). These redox proteomics studies indicate that Aβ(1-42)-mediated oxidative stress occurs early in AD pathogenesis and results in altered antioxidant and cellular detoxification defenses, decreased energy yielding metabolism and mitochondrial dysfunction, excitotoxicity, loss of synaptic plasticity and cell structure, neuroinflammation, impaired protein folding and degradation, and altered signal transduction. Improved access to biomarker imaging and the identification of lifestyle interventions or treatments to reduce Aβ production could be beneficial in preventing or delaying the progression of AD. This article is part of the special issue "Proteomics".

The physical and chemical disruption of human hair after bleaching - studies by transmission electron microscopy and redox proteomics.

To understand the structural and chemical effects of cosmetic peroxide bleaching on human hair. Human hair was progressively bleached using alkaline peroxide-persulphate treatment. Proteins lost through leaching were examined using amino acid analysis and mass spectrometric sequencing. Fibre damage was assessed using transmission electron microscopy, amino acid analysis and redox proteomics. Protein loss through leaching increased with bleaching severity. Leached proteins were not limited to the cuticle, but also included cortical intermediate filaments and matrix keratin-associated proteins. The leached proteins were progressively oxidized as bleaching severity increased. Bleached fibres demonstrated substantial damage to the cuticle layers and to the cortex. Extensive melanin granule degradation was present after the mildest bleach treatment. Protein oxidation in bleached fibres was principally in cortical intermediate filaments - the most abundant hair proteins - and targeted the sulphur-containing amino acids, particularly the conversion of cystine disulphide bonds to cysteic acid. Peroxide chemical treatments quickly access the cortex, causing untargeted oxidative damage across the fibre in addition to the desired loss of melanin. Peroxide ingress is likely facilitated by the considerable structural degradation caused to the cuticle layers of hair fibres. The consequences of the peroxide action within the cuticle and cortex are oxidation of the proteins, and subsequent protein loss from the fibre that correlates to bleaching severity.

Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

65 - Glutathione-S-transferase π promotes interleukin-1β induced pulmonary inflammation in association with S-glutathionylation of the glycolysis regulator, pyruvate kinase M2

Changes in lung metabolism and associated changes in redox homeostasis, including protein S-glutathionylation (PSSG), have been shown to be an important feature of chronic lung diseases. PSSG is a post-translational modification that can be catalyzed by Glutathione-S-transferase π (GSTP), and regulates protein function. A recent study by our laboratory demonstrated that Interleukin-1 beta (IL-1β) increased glycolysis in a house dust mite (HDM) mouse model of allergic airways disease resulted in enhanced inflammation. A number of enzymes in the glycolysis pathway have been shown to be redox regulated, and a redox proteomics study indicated increases in PSSG of the glycolysis regulator PKM2 (PKM2-SSG) in HDM-exposed mouse lungs. The goal of the present study was to determine whether GSTP-linked PSSG regulates glycolysis and controls allergic airways disease. We observed that absence of Gstp decreased HDM-mediated elevations in airway remodeling, airways hyperresponsiveness and lactate. HDM-dependent increases in PSSG were attenuated in Gstp-/- mice when compared to wildtype (WT) mice. While HDM led to comparable increases in PKM2 expression in lungs from WT and Gstp-/- mice, PKM2-SSG was strongly decreased in HDM-exposed Gstp-/- mice when compared to WT mice. Moreover, IL-1β induced pro-inflammatory signals in mouse tracheal epithelial cells (MTECs) and PKM2-SSG were also blunted when Gstp was ablated or inhibited, using TLK199, a clinically relevant inhibitor of GSTP. Treatment with the PKM2 activator, TEPP46, also attenuated IL-1β-mediated pro-inflammatory signals. Lastly, exposure of recombinant PKM2 to GSSG, a condition that induces PSSG, resulted in a loss of PKM2 enzymatic activity. Collectively, these results suggest the importance of GSTP-controlled S-glutathionylation of PKM2 in the promotion of pro-inflammatory signaling in epithelial cells that are known to be relevant to allergic airway remodeling.

87 - Characterization of unique products in reactions of GSNO with H2S

The interplay between NO and H2S signaling has attracted much interest in redox proteomics of nitrosative and oxidative stress. Both small molecules appear to target similar pathways in organisms, by reacting with protein free thiols or metalloenzymes to alter or enhance function. We have previously investigated the N-based product speciation in the reaction of GSNO with H2S, specifically noting that the N-based gas product distributions are analogous to those of reactions of GSNO with reductants (e.g. ascorbic acid). We have also identified a plethora of unique glutathione-based products by LC-HRMS analysis. Single Ion Chromatograms (SICs) confirm that these species are present in the reaction mixture and are separated on the LC column prior to ionization. A number of small reactive thiols can be trapped with iodoacetamide (IA), including polysulfides (SnH2), glutathione polysulfides (GSSnH) and trithionitrates, GSN(SnH)2. Unusual trithionitrates are also observed as acetamide adducts, GSN(SnA)2, but only at low ratios of H2S to GSNO. Competition experiments suggest one initial product, the sulfenic acid GSOH, acts as precursor to persulfide and oxidized glutathione-based products. Addition of a non-electrophilic radical trap, vinyl cyclopropane, has little effect on the product distributions, suggesting radical intermediates are short-lived. Other unique dimedone adducts were observed that are attributable to the sulfinyl (GS(O)H) tautomer. We also observe small S-oxoacids, HSOH and S(OH)2, as the derivatized ISnD and DSnD adducts. As with GSOH, unique thioacetals are observed which derive from the sulfenyl tautomers of both HS(O)H and GS(O)H. These unique mixed-valence sulfides have little precedent in the literature but remain observable in reaction mixtures after many hours on the bench.