Free Protein Thiols Research Articles

Thiol-Mediated Enhancement of Nε-Acetyllysine Formation in Lens Proteins.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Reactivity of amino acid residues towards 4-methylbenzoquinone: Effect on the site-specificity of quinone-protein reaction

The reactivity of amino acid residues in proteins towards quinones, along with the concentration of quinones, may play a crucial role in determining the specific sites of reaction between quinones and proteins. The reaction efficiencies of 20 different α-carbon amine group blocked amino acids with 4-methylbenzoquinone (4MBQ) were investigated at pH 7.0 by cyclic voltammetry. Among these, Nα-acetyl-l-lysine, Nα-Boc-l-histidine, Nα-acetyl-l-arginine, and Nα-acetyl-l-cysteine exhibited reactivity towards 4MBQ, with the reactivity sequence being: Nα-acetyl-l-cysteine > Nα-acetyl-l-lysine > Nα-acetyl-l-arginine > Nα-Boc-l-histidine. In addition, the reactivity of Nα-acetyl-l-cysteine towards 4MBQ was over 50-fold greater than that of Nα-acetyl-l-lysine, Nα-Boc-l-histidine and Nα-acetyl-l-arginine. Adducts formed from the reaction of 4MBQ with Nα-acetyl-l-lysine and Nα-acetyl-l-arginine were identified as quinone adducts. In contrast, those formed with Nα-acetyl-l-cysteine and Nα-Boc-l-histidine were identified as phenol adducts through UPLC-QTOF-MS/MS analysis. Irrespective of whether the molar ratio of 4MBQ to protein free thiol groups was 1:2 or 2:1, the residues of cysteine, lysine, arginine and histidine in β-lactoglobulin (β-Lg) invariably engaged in the reaction with 4MBQ. This work may provide valuable information for design polyphenol-protein covalent conjugates which can be further applied as functional additives in food industry by choosing protein with different proportion of lysine, histidine, arginine and cysteine residues.

Antioxidant activity enhancement and oxidative damage inhibition by Lagenaria breviflora fruit and Xanthosoma sagittifolium corm in hypertensive Wistar rats.

Cardiovascular diseases are the leading causes of mortality in the world today with hypertension being the major clinical presentation of these diseases. This study assessed the anti-hypertensive effects of Lagenaria breviflora whole fruit and Xanthsoma sagittifolium corms in experimentally inudced hypertensive Wistar rats. The ability of the plants to ameliorate oxidative damage accompanying hypertension was evaluated using changes in oxidative stress markers as well as monitoring of cardiovascular parameters. Hypertension was induced by intraperitoneal injection of DOCA salt twice weekly and daily inclusion of NaCl (1%) in drinking water. Methanol extracts of L.breviflora or X. sagittifolium was administered to hypertensive rats for 35 days and the outcome was compared to hypertensive rats administred with lisinopril or hydrochlorothiazide and a group of normotensive rats (control). Systolic, diastolic and mean arterial pressures were determined on day 34 and blood sample collected on day 35. The rats were thereafter humanely sacrificed, and organs were harvested. This study showed that the extracts lowered blood pressure, free protein thiols but increased toal protein, gluthathione peroxidase, reduced glutathione, glutathione S-transferase, catalase and nitric oxide in the heart, kidney and liver compared to untreated hypertensive rats. However, malondialdehyde levels and hydrogen peroxide activities were reduced. L. breviflora fruit and X. sagittifloium corm exhibited antihypertensive properties and ameliorate oxidative damage associated with hypertension by enhancing the antioxidant defense sysyem and inhibiting generation of free radicals.

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

3-Chloro-5-Substituted-1,2,4-Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers.

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8.

S-Nitrosylation of Paraxonase 1 (PON1) Elevates Its Hydrolytic and Antioxidant Activities

Covalent binding between nitric oxide (NO) and a protein’s free thiol group (SH) is termed protein S-nitrosylation. Protein S-nitrosylation is involved in cellular regulation mechanisms that underlie a wide range of critical functions, such as apoptosis, alteration of enzyme activities, and transcription-factor stability. Impaired protein S-nitrosylation is associated with a growing list of pathophysiological conditions, such as cardiovascular disease, multiple sclerosis, pulmonary hypertension, and sickle cell disease. The enzyme paraoxonase 1 (PON1) binds to high-density lipoprotein to provide many of its antiatherogenic properties. The enzyme has a strong antioxidant capacity, which protects fats, lipids, and lipoproteins from oxidation, in addition to breaking down oxidized fats. We investigated the effect of S-S transnitrosylation on PON1 activities. Incubation of recombinant PON1 (rePON1) with nitrosylated human serum albumin (HSA-NO) resulted in S-nitrosylation of about 70% of the rePON1, as measured by Q-TOF LC/MS. S-nitrosylation significantly increased rePON1 hydrolytic activities. It also increased rePON1’s ability to inhibit low-density lipoprotein oxidation induced by Cu2+. Finally, it increased the enzyme’s penetration into macrophage cells by 31%. Our findings suggest that S-nitrosylation of rePON1 improves its biological functions which may positively affect atherosclerosis disease progression.

Blood Thiol Redox State in Chronic Kidney Disease.

Thiols (sulfhydryl groups) are effective antioxidants that can preserve the correct structure of proteins, and can protect cells and tissues from damage induced by oxidative stress. Abnormal levels of thiols have been measured in the blood of patients with moderate-to-severe chronic kidney disease (CKD) compared to healthy subjects, as well as in end-stage renal disease (ESRD) patients on haemodialysis or peritoneal dialysis. The levels of protein thiols (a measure of the endogenous antioxidant capacity inversely related to protein oxidation) and S-thiolated proteins (mixed disulphides of protein thiols and low molecular mass thiols), and the protein thiolation index (the molar ratio of the S-thiolated proteins to free protein thiols in plasma) have been investigated in the plasma or red blood cells of CKD and ESRD patients as possible biomarkers of oxidative stress. This type of minimally invasive analysis provides valuable information on the redox status of the less-easily accessible tissues and organs, and of the whole organism. This review provides an overview of reversible modifications in protein thiols in the setting of CKD and renal replacement therapy. The evidence suggests that protein thiols, S-thiolated proteins, and the protein thiolation index are promising biomarkers of reversible oxidative stress that could be included in the routine monitoring of CKD and ESRD patients.

Anti-inflammatory and prolonged protective effects of Artemisia herba-alba extracts via glutathione metabolism reinforcement

Artemisia herba-alba (AHA) is a medicinal plant known for its preventive capacities against chronic diseases. Inflammation is a naturally occurring cellular response to noxious stimuli, and both acute and chronic inflammation are involved in human disorders. Preventing and limiting the negative impact of inflammation by using phytocompounds has attracted a lot of attention. Here, we show that AHA extracts are potent anti-inflammatory products at low doses in white blood cells, treated with phorbol myristate acetate (PMA), a pro-inflammatory trigger. Regardless of the mode of extraction, i.e. maceration or microwave, all extracts reduced on dose-dependent manner reactive oxygen species (ROS) production induced by the pro-inflammatory compound PMA in white blood cells and leukemic Jurkat cell line. The induction of the anti-inflammatory effect was evaluated by the measurement of glutathione metabolism (glutathione reductase, transferase, and peroxidase), and protein-free thiols in Jurkat cells. Jurkat cells incubated with low to high concentrated AHA extracts, ranged from 10 to 100 g/mL, induced a fast cell response measured at one and six hours. Interestingly, extracts from the microwave showed a stronger induction, 5 to 10 fold changes at early time points, and maintained over 72 h. On the other hand, AHA extracts showed a moderate anti-proliferative effect in the leukemic Jurkat line. We concluded that AHA extracts enhance directly anti-inflammatory cell response, earlier via ROS induction. Later, however, vigorously maintained, independently from ROS, suggesting the intervention of the cellular metabolism in enhancing these effects. Considering the preventive and anti-inflammatory potential of AHA, regular consumption is advisable, and phytotherapeutic applications are encouraged to treat local inflammations.

ESI MS studies highlight the selective interaction of Auranofin with protein free thiols.

The clinically established gold drug Auranofin was reacted individually with a group of representative proteins, namely ubiquitin, ribonuclease A, carbonic anhydrase, haemoglobin and superoxide dismutase, and adduct formation was monitored in the various cases by ESI-MS analysis. We found that the reaction is highly selective for solvent exposed free cysteines that are modified through coordination of the AuPEt3+ fragment. Indeed, ESI-Q-TOF MS spectra carried out on protein samples incubated with a three fold molar excess of Auranofin allowed direct detection of the native proteins bearing bound AuPEt3+ fragments in the cases of carbonic anhydrase and haemoglobin. At variance, the two proteins that do not possess any free cysteine residue, i.e. ubiquitin and ribonuclease A, were unable to bind the gold fragment. In the case of superoxide dismutase, adduct formation is hindered by the scarce solvent accessibility of the free cysteine residue. These findings were further confirmed by a series of competition binding experiments with ebselen, a potent and selective cysteine-modifying reagent; we observed that pre-treatment with ebselen prevents the binding of the AuPEt3+ fragment to both carbonic anhydrase and haemoglobin.

Effect of negative functionalisation of gold nanorods on conformation and activity of human serum albumin

The theranostic applications of gold nanorods (AuNRs) are limited due to the presence of cytotoxic cetrimonium bromide (CTAB) stabiliser, leading to the instigation of alternate stabilisers like negatively charged polystyrene sulphonate (PSS). Despite previous reports suggesting the impact of PSS-AuNRs on cells, their effect on the most abundant protein in plasma, i.e. human serum albumin (HSA), has not been studied before. Hence, the impact of PSS-AuNRs on HSA was thoroughly examined using varied spectroscopic techniques. The absorbance and fluorescence spectroscopic findings suggested the extent of ground-state complexation and tryptophan domain disruptions of HSA for different AuNR concentrations, which were also suggested based on size measurements and activation energy calculations for complex formation. Modifications in the hydrophobic environment of HSA were evaluated using synchronous fluorescence, whereas the secondary structural damages were explained using circular dichroism (CD) and FTIR analyses. Additional studies to analyse protein denaturation, fibrillation, esterase activity, and free thiol were carried out to understand structural and functional changes. The study suggested that PSS-AuNRs showed concentration-dependent alterations in HSA structure, but the extent of protein toxicity was considerably lesser for PSS-AuNRs of similar dimensions compared to the data available for CTAB-AuNRs; thus, highlighting that PSS-AuNRs could be safer for biomedical applications.

Platelet and Red Blood Cell Counts, as well as the Concentrations of Uric Acid, but Not Homocysteinaemia or Oxidative Stress, Contribute Mostly to Platelet Reactivity in Older Adults.

Purpose The goal of this study was to estimate the hierarchical contribution of the most commonly recognized cardiovascular risk factors associated with atherogenesis to activation and reactivity of blood platelets in a group of men and women at ages 60-65. Methods Socioeconomic and anthropometric data were taken from questionnaires. Blood morphology and biochemistry were measured with standard diagnostic methods. Plasma serum homocysteine was measured by immunochemical method. Plasma concentrations of VCAM, ICAM, total antioxidant status, and total oxidant status were estimated with commercial ELISA kits. Markers of oxidative stress of plasma and platelet proteins (concentrations of protein free thiol and amino groups) and lipids (concentrations of lipid peroxides) and generation of superoxide anion by platelets were measured with colorimetric methods. Platelet reactivity was estimated by impedance aggregometry with arachidonate, collagen, and ADP as agonists. Expression of selectin-P and GPIIb/IIIa on blood platelets was tested by flow cytometry. Results Platelet aggregation associated significantly negatively with HGB and age and significantly positively with PLT, MPV, PCT, PDW, and P-LCR. When platelet reactivity (“cumulative platelet reactivity_aggregation”) was analyzed in a cumulated manner, the negative association with serum concentration of uric acid (Rs = −0.169, p = 0.003) was confirmed. Multivariate analysis revealed that amongst blood morphological parameters, platelet count, plateletcrit, and number of large platelets and uric acid are the most predictive variables for platelet reactivity. Conclusions The most significant contributors to platelet reactivity in older subjects are platelet morphology, plasma uricaemia, and erythrocyte morphology.

Immunoprecipitation methods to identify S-glutathionylation in target proteins

S-glutathionylation is a reversible post-translational modification of proteins that generate a mixed disulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylation has been extensively studied since it represents the cellular response to oxidative stress, in physiological as well as pathological conditions. This modification may be a protective mechanism from irreversible oxidative damage and, on the other hand, may modulate protein folding and function.Due to the importance of S-glutathionylation in cellular redox signaling, various methods have been developed to identify S-gluthationylated proteins.Herein, we describe two easy methods to recognized S-glutathionylation of a target protein after oxidative stress in cellular extracts based on different immunoprecipitation procedures. The immunoprecipitation assay allows the capture of one glutathionylated protein using a specific antibody that binds to the target protein. The presence of S-glutathionylation in the immunoprecipitated protein is identified using anti-glutathione antibody. The second type of approach is based on the detection of the glutathionylated protein with biotin/streptavidin technique. After different steps of protection of non-oxidized thiolic groups and reduction of S-glutathionylated groups, the newly-formed protein free-thiols are labeled with biotin-GSH. The modified protein can be isolate with streptavidin-beads and recognized using an antibody against target protein.•S-glutathionylation is a reversible post-translational modification of proteins that recently has been emerged as important signaling in the redox regulation of protein function.•Both methods to identify glutathionylated proteins are economic, easy and do not require particular equipment.•The setups of both methods guarantee high reproducibility.

Site-Specific Labeling of Proteins with Near-IR Heptamethine Cyanine Dyes.

Convenient labeling of proteins is important for observing its function under physiological conditions. In tissues particularly, heptamethine cyanine dyes (Cy-7) are valuable because they absorb in the near-infrared (NIR) region (750–900 nm) where light penetration is maximal. In this work, we found Cy-7 dyes with a meso-Cl functionality covalently binding to proteins with free Cys residues under physiological conditions (aqueous environments, at near neutral pH, and 37 °C). It transpired that the meso-Cl of the dye was displaced by free thiols in protein, while nucleophilic side-chains from amino acids like Tyr, Lys, and Ser did not react. This finding shows a new possibility for convenient and selective labeling of proteins with NIR fluorescent probes.

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.

Copper Increases Brain Oxidative Stress and Enhances the Ability of 6-Hydroxydopamine to Cause Dopaminergic Degeneration in a Rat Model of Parkinson's Disease.

Redox properties enable copper to perform its essential role in many biological processes, but they can also convert it into a potentially hazardous element. Its dyshomeostasis may have serious neurological consequences, and its possible involvement in Parkinson's disease and other neurodegenerative disorders has been suggested. The in vitro and ex vivo ability of copper to increase oxidative stress has already been demonstrated, and the aim of the present study was to assess in vivo the capacity of copper to cause brain oxidative damage and its ability to increase the dopaminergic degeneration induced by 6-hydroxydopamine. We found that chronic copper administration (10mgCu2+/kg/day, IP) causes its accumulation in different brain areas (cortex, striatum, nigra) and was accompanied by an increase in TBARS levels and a decrease in protein free-thiol content in the cortex. A decrease in catalase activity and an increase in glutathione peroxidase activity were also observed in the cortex. The intrastriatal administration of Cu2+ caused an increase in some indices of oxidative stress (TBARS and protein free-thiol content) in striatum and nigra, but was unable to induce dopaminergic degeneration. However, when copper was intrastriatally coadministered with 6-hydroxydopamine, it increased dopaminergic degeneration, a fact that was also accompanied by an increase in the assayed indices of oxidative stress, a decrease in catalase activity, and an augmentation in glutathione activity. Evidently, copper cannot cause neurodegeneration per se, but may potentiate the action of other factors involved in the pathogenesis of Parkinson's disease through oxidative stress.

The exceptional sensitivity of brain mitochondria to copper

Wilson disease (WD) is characterized by a disrupted copper homeostasis resulting in dramatically increased copper levels, mainly in liver and brain. While copper damage to mitochondria is an established feature in WD livers, much less is known about such detrimental copper effects in other organs. We therefore assessed the mitochondrial sensitivity to copper in a tissue specific manner, namely of isolated rat liver, kidney, heart, and brain mitochondria. Brain mitochondria presented with exceptional copper sensitivity, as evidenced by a comparatively early membrane potential loss, profound structural changes already at low copper dose, and a dose-dependent reduced capacity to produce ATP. This sensitivity was likely due to a copper-dependent attack on free protein thiols and due to a decreased copper reactive defense system, as further evidenced in neuroblastoma SHSY5Y cells. In contrast, an increased production of reactive oxygen species was found to be a late-stage event, only occurring in destroyed mitochondria. We therefore propose mitochondrial protein thiols as major targets of mitochondrial copper toxicity.

Mitochondrial protein sulfenation during aging in the rat brain

There is accumulating evidence that cysteine sulfenation (cys-SOH) in proteins plays an important role in cellular response to oxidative stress. The purpose of the present study was to identify mitochondrial proteins that undergo changes in cys-SOH during aging. Studies were conducted in rats when they were 5 or 30 months of age. Following blocking of free protein thiols with N-ethylmaleimide, protein sulfenic acids were reduced by arsenite to free thiol groups that were subsequently labeled with biotin-maleimide. Samples were then comparatively analyzed by two-dimensional Western blots, and proteins showing changes in sulfenation were selectively identified by mass spectrometry peptide sequencing. As a result, five proteins were identified. Proteins showing an age-related decrease in sulfenation include pyruvate carboxylase and pyruvate dehydrogenase; while those showing an age-related increase in sulfenation include aconitase, mitofilin, and tubulin (α-1). Results of the present study provide a general picture of mitochondrial protein sulfenation in brain oxidative stress and implicate the involvement of protein sulfenation in overall decline of mitochondrial function during brain aging.

Systems Biology Approaches to Redox Metabolism in Stress and Disease States.

All cellular metabolic processes are tied to the cellular redox environment. Therefore, maintaining redox homeostasis is critically important for normal cell function. Indeed, redox stress contributes to the pathobiology of many human diseases. The cellular redox response system is composed of numerous interconnected components, including free radicals, redox couples, protein thiols, enzymes, metabolites, and transcription factors. Moreover, interactions between and among these factors are regulated in time and space. Owing to their complexity, systems biology approaches to the characterization of the cellular redox response system may provide insights into novel homeostatic mechanisms and methods of therapeutic reprogramming. Recent Advances: The emergence and development of systems biology has brought forth a set of innovative technologies that provide new avenues for studying redox metabolism. This article will review these systems biology approaches and their potential application to the study of redox metabolism in stress and disease states. Clarifying the scope of biological intermediaries affected by dysregulated redox metabolism requires methods that are suitable for analyzing big datasets as classical methods that do not account for multiple interactions are unlikely to portray the totality of perturbed metabolic systems. Given the diverse redox microenvironments within cells, it will be important to improve the spatial resolution of omic approaches. Futures studies on the integration of multiple systems-based methods and heterogeneous omics data for redox metabolism are required to accelerate the development of the field of redox systems biology. Antioxid. Redox Signal. 29, 953-972.

126 - Identification of Covalent Adducts Between Docosahexaenoic Acid and Its Oxidation Products with Thiols: The Potencial Significance of Thiol-Ene Reaction for Redox Signalling Processes

Covalent coupling between unsaturated bonds in fatty acids and thiol groups in peptides and proteins can occur through a free radical (thiol-ene) and Michael addition reactions. Recently, we have observed that fatty acid induced-SOD1 aggregation in vitro depends on protein free thiol groups and the presence of least one unsaturated bond in the fatty acid structure. Docosahexaenoic acid (DHA; C22:6n-3) is highly enriched in the brain and its oxidation generates products bearing hydroperoxides, hydroxides and ketone moieties. The later, is known to form covalent adducts with thiol groups via Michael addition while unsaturated fatty acid can undergo a thiol-ene addition reaction. To better understand how DHA and its oxidation products could lead to thiol modifications we have investigated their reaction with thiols in a tri-peptide (glutathione, GSH) and a protein (Keap1) in vitro by LC-MS/MS and biotin-PEAC5-maleimide (BPM) assay. A new covalent adduct between DHA and glutathione (GS-DHA adduct, m/z 634.3168) was detected by LC-MS/MS. However, this adduct was only detected when incubated with AAPH, a free radical-generating azo compound, which suggeststhat GS-DHA is generated in presence of thiylradical. On the other hand, DHA derived hydroperoxides and ketones reacted with GSH generating a GS-oxo-DHA adduct (m/z 648.2960) while the hydroxides did not. To evaluate the reactivity of DHA and its oxidation products under more physiological conditions we evaluated their reactivity towards a thiol-rich protein, Keap1, the negative regulator of Nrf2. Incubations of DHA with Keap1 showed protein thiolate consumption as well as aggregation. Studies is now being conducted to characterize the presence of covalent DHA-thiol adducts in the protein and also to compare the reactivity between DHA and its oxidized derivatives. Overall, our data show that both non-oxidized and oxidized DHA derivatives can modify thiol groups in peptides and proteins, which may potentially affect redox signaling pathways.

Protein glutathionylation protects wheat (Triticum aestivum Var. Sonalika) against Fusarium induced oxidative stress

Fusarium induced oxidative stress could be recovered by reversible protein oxidative modification through the process of glutathionylation in co-stressed (low-dose (50 μM) Cd2+ pre-treatment followed by Fusarium inoculation) wheat seedlings. Co-stressed seedlings showed low disease severity index as compared to Fusarium infected seedlings. A reduced level of hydrogen peroxide (H2O2) and carbonyl contents due to irreversible protein oxidation were observed in co-stressed seedlings as compared to Fusarium infected seedlings. Further, a comparative biochemical assay showed an enhanced glutathione content in co-stressed tissues as compared to Fusarium infected tissues. In an investigation, reduced glutathione pre-coated agarose gel beads were used to pull down proteins having affinity with GSH. Fructose-1, 6-bisphosphate aldolase and 3-Phosphoglycerate kinase were observed to be co-existed in co-stressed seedlings when analysed by LC-MS/MS after being processed through protein-pull assay. Co-stressed tissues showed an enhanced free protein thiol content as compared to Fusarium infected tissues. The ratio of free thiol to thiol disulfides was also observed to be increased in co-stressed tissues as compared to Fusarium infected tissues. In contrast, the quantitative assay by Ellman's reagent and qualitative analysis by diagonal gel electrophoresis showed enhanced protein thiol disulfides in Fusarium infected tissues as compared to co-stressed tissues. Further, glutaredoxin, responsible for the reverse reduction of proteins was observed to be enhanced in co-stressed tissues as compared to Fusarium infected tissues. Thus, a low dose Cd2+ triggered glutathionylation is suggestive of offering tolerance against Fusarium induced oxidative stress and protects target proteins from irreversible modification and permanent damage in wheat.