Deep learning based prediction of species-specific protein S-glutathionylation sites

As a widespread and reversible post-translational modification of proteins, S-glutathionylation specifically generates the mixed disulfides between cysteine residues and glutathione, which regulates various biological processes including oxidative stress, nitrosative stress and signal transduction. The identification of proteins and specific sites that undergo S-glutathionylation is crucial for understanding the underlying mechanisms and regulatory effects of S-glutathionylation. Experimental identification of S-glutathionylation sites is laborious and time-consuming, whereas computational predictions are more attractive due to their high speed and convenience. Here, we developed a novel computational framework DeepGSH (http://deepgsh.cancerbio.info/) for species-specific S-glutathionylation sites prediction, based on deep learning and particle swarm optimization algorithms. 5-fold cross validation indicated that DeepGSH was able to achieve an AUC of 0.8393 and 0.8458 for Homo sapiens and Mus musculus. According to critical evaluation and comparison, DeepGSH showed excellent robustness and better performance than existing tools in both species, demonstrating DeepGSH was suitable for S-glutathionylation prediction. The prediction results of DeepGSH might provide guidance for experimental validation of S-glutathionylation sites and helpful information to understand the intrinsic mechanisms.     

Protein cysteine <Emphasis Type="Italic">S</Emphasis>-guanylation and electrophilic signal transduction by endogenous nitro-nucleotides

Nitric oxide (NO), a gaseous free radical that is synthesized in organisms by nitric oxide synthases, participates in a critical fashion in the regulation of diverse physiological functions such as vascular and neuronal signal transduction, host defense, and cell death regulation. Two major pathways of NO signaling involve production of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and posttranslational modification (PTM) of redox-sensitive cysteine thiols of proteins. We recently clarified the physiological formation of 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) as the first demonstration, since the discovery of cGMP more than 40 years ago, of a new second messenger derived from cGMP in mammals. 8-Nitro-cGMP is electrophilic and reacts efficiently with sulfhydryls of proteins to produce a novel PTM via cGMP adduction, a process that we named protein S-guanylation. 8-Nitro-cGMP may regulate electrophilic signaling on the basis of its electrophilicity through induction of S-guanylation of redox sensor proteins. Examples include S-guanylation of the redox sensor protein Kelch-like ECH-associated protein 1 (Keap1), which leads to activation of NF-E2-related factor 2 (Nrf2)-dependent expression of antioxidant and cytoprotective genes. This S-guanylation-mediated activation of an antioxidant adaptive response may play an important role in cytoprotection during bacterial infections and oxidative stress. Identification of new redox-sensitive proteins as targets for S-guanylation may help development of novel therapeutics for oxidative stress- and inflammation-related disorders and vascular diseases as well as understanding of cellular protection against oxidative stress.     

Prediction of S-Glutathionylation Sites Based on Protein Sequences

S-glutathionylation, the reversible formation of mixed disulfides between glutathione(GSH) and cysteine residues in proteins, is a specific form of post-translational modification that plays important roles in various biological processes, including signal transduction, redox homeostasis, and metabolism inside cells. Experimentally identifying S-glutathionylation sites is labor-intensive and time consuming, whereas bioinformatics methods provide an alternative way to this problem by predicting S-glutathionylation sites in silico. The bioinformatics approaches give not only candidate sites for further experimental verification but also bio-chemical insights into the mechanism of S-glutathionylation. In this paper, we firstly collect experimentally determined S-glutathionylated proteins and their corresponding modification sites from the literature, and then propose a new method for predicting S-glutathionylation sites by employing machine learning methods based on protein sequence data. Promising results are obtained by our method with an AUC (area under ROC curve) score of 0.879 in 5-fold cross-validation, which demonstrates the predictive power of our proposed method. The datasets used in this work are available at http://csb.shu.edu.cn/SGDB.     

Causes and Consequences of Cysteine S-Glutathionylation

Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress.     

GSHSite: Exploiting an Iteratively Statistical Method to Identify S-Glutathionylation Sites with Substrate Specificity

S-glutathionylation, the covalent attachment of a glutathione (GSH) to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM) that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA). TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM) is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN) and human protein tyrosine phosphatase 1b (PTP1B). Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (http://csb.cse.yzu.edu.tw/GSHSite/), for identifying uncharacterized GSH substrate sites on the protein sequences.     

Inactivation of Ca/calmodulin-dependent protein kinase I by S-glutathionylation of the active-site cysteine residue

We show that Ca²⁺/calmodulin(CaM)-dependent protein kinase I (CaMKI) is directly inhibited by its S-glutathionylation at the Cys¹⁷⁹. In vitro studies demonstrated that treatment of CaMKI with diamide and glutathione results in inactivation of the enzyme, with a concomitant S-glutathionylation of CaMKI at Cys¹⁷⁹ detected by mass spectrometry. Mutagenesis studies confirmed that S-glutathionylation of Cys¹⁷⁹ is both necessary and sufficient for the inhibition of CaMKI by diamide and glutathione. In transfected cells expressing CaMKI, treatment with diamide caused a reversible decrease in CaMKI activity. Cells expressing mutant CaMKI (179CV) proved resistant in this regard. Thus, our results indicate that the reversible regulation of CaMKI via its modification at Cys¹⁷⁹ is an important mechanism in processing calcium signal transduction in cells.     

Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu²⁺, benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.     

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H₂O₂ was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H₂O₂ to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H₂O₂ on kinase activity. Similar to the results obtained with H₂O₂-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H₂O₂ are increased. These results demonstrate that physiologically relevant concentrations of H₂O₂ can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Inhibition of tubulin polymerization by hypochlorous acid and chloramines

Protein thiol oxidation and modification by nitric oxide and glutathione are emerging as common mechanisms to regulate protein function and to modify protein structure. Also, thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. We assessed the effect of the oxidants hypochlorous acid and chloramines on the cytoskeletal protein tubulin. Total cysteine oxidation by the oxidants was monitored by labeling tubulin with the thiol-selective reagent 5-iodoacetamidofluorescein; by reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid); and by detecting interchain tubulin disulfides by Western blot under nonreducing conditions. Whereas HOCl induced both cysteine and methionine oxidation of tubulin, chloramines were predominantly cysteine oxidants. Cysteine oxidation of tubulin, rather than methionine oxidation, was associated with loss of microtubule polymerization activity, and treatment of oxidized tubulin with disulfide reducing agents restored a considerable portion of the polymerization activity that was lost after oxidation. By comparing the reactivity of hypochlorous acid and chloramines with the previously characterized oxidants, peroxynitrite and the nitroxyl donor Angeli's salt, we have identified tubulin thiol oxidation, not methionine oxidation or tyrosine nitration, as a common outcome responsible for decreased polymerization activity.     

Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity

The endo--1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.     

Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo. Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

In vivo targets of S-thiolation in Chlamydomonas reinhardtii

Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [(35)S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.     

Cysteine S-conjugate β-lyases

Summary. Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess an electron-withdrawing group attached at the sulfur. The end products of the β-lyase reaction are pyruvate, ammonium and a sulfur-containing fragment. If the sulfur-containing fragment is reactive, the parent cysteine S-conjugate may be toxic, particularly to kidney mitochondria. Halogenated alkenes are examples of electrophiles that are bioactivated (toxified) by conversion to cysteine S-conjugates. These conjugates are converted by cysteine S-conjugate β-lyases to thioacylating fragments. Several cysteine S-conjugates found in allium foods (garlic and onion) are β-lyase substrates. This finding may account in part for the chemopreventive activity of allium products. This review (1) identifies enzymes that catalyze cysteine S-conjugate β-lyase reactions, (2) suggests that toxicant channeling may contribute to halogenated cysteine S-conjugate-induced toxicity to mitochondria, and (3) proposes mechanisms that may contribute to the antiproliferative effects of sulfur-containing fragments eliminated from allium-derived cysteine S-conjugates.     

The insect repellent DEET (<Emphasis Type="Italic">N,N</Emphasis>-diethyl-3-methylbenzamide) increases the synthesis of glutathione <Emphasis Type="Italic">S</Emphasis>-transferase in cultured mosquito cells

DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET’s repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET’s toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [³⁵S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS–polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.     

Oxidative Modification of Cysteine 111 Promotes Disulfide Bond-Independent Aggregation of SOD1

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1-linked fALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanism inducing the aggregation is not understood. To study the contribution of oxidative modification of cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse model at different stages of the disease and under oxidative stress induced by H₂O₂. Our data suggest that under normal circumstance, cysteine 111 residue in SOD1 is free; however, under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S−). With the progression of the disease, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues; human mutant SOD1 could generate an upper shift band in reducing SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also detected the formation of SOD1 multimers at different stages of the disease, and found that accumulated oxidative stress facilitated the formation of aggregates, which were not mediated by disulfide bond. This oxidative modification of cysteine 111 therefore promotes the formation of disulfide bond-independent aggregation of SOD1.     

Glutathione disulfide liposomes – A research tool for the study of glutathione disulfide associated functions and dysfunctions

Glutathione disulfide (GSSG) is the oxidized form of glutathione (GSH). GSH is a tripeptide present in the biological system in mM concentration and is the major antioxidant in the body. An increase in GSSG reflects an increase in intracellular oxidative stress and is associated with disease sates. The increase has also been demonstrated to lead to an increase in protein S-glutathionylation that can affect the structure and function of proteins. Protein S-glutathionylation serves as a regulatory mechanism during cellular oxidative stress. Though GSSG is commercially available, its roles in various GSSG-associated normal/abnormal physiological functions have not been fully delineated due to the reason that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed cationic liposomes that can effectively deliver GSSG into cells. Various concentrations of GSSG liposomes can be conveniently prepared. At 1 mg/mL, the GSSG liposomes effectively increased intracellular GSSG by 27.1±6.9 folds (n=3) in 4 h and led to a significant increase in protein S-glutathionylation confirming that the increased GSSG is functionally effective. The Trypan blue assay demonstrated that GSSG liposomes were not cytotoxic; the cell viability was greater than 95% after cells were treated with the GSSG liposomes for 4 h. A stability study showed that the dry form of the GSSG liposomes were stable for at least 70 days when stored at ?80 °C. Our data demonstrate that the GSSG liposomes can be a valuable tool in studying GSSG-associated physiological/pathological functions.