Beyond glucose: The crucial role of redox signaling in ß-cell metabolic adaptation.

OBJECTIVE: Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic ß-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in ß-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for ß-cell function and health. METHODS: We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic ß-cells. RESULTS: Glucose stimulation significantly increases ROS levels in ß-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. CONCLUSIONS: Proper functioning of pancreatic ß-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic ß-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in ß-cell function.

Redox proteomics of PANC-1 cells reveals the significance of HIF-1 signaling protein oxidation in pancreatic ductal adenocarcinoma pathogenesis.

BACKGROUND: Protein cysteine oxidation is substantially involved in various biological and pathogenic processes, but its implications in pancreatic cancer development remains poorly understood. METHODS AND RESULTS: In this study, we performed a global characterization of protein oxidation targets in PDAC cells through iodoTMT-based quantitative proteomics, which identified over 4300 oxidized cysteine sites in more than 2100 proteins in HPDE6c7 and PANC-1 cells. Among them, 1715 cysteine residues were shown to be differentially oxidized between HPDE6c7 and PANC-1 cells. Also, charged amino acids including aspartate, glutamate and lysine were significantly overrepresented in flanking sequences of oxidized cysteines. Differentially oxidized proteins in PANC-1 cells were enriched in multiple cancer-related biological processes and signaling pathways. Specifically, the HIF-1 signaling proteins exhibited significant oxidation alterations in PANC-1 cells, and the reduced PHD2 oxidation in human PDAC tissues was correlated with lower survival time in pancreatic cancer patients. CONCLUSION: These investigations provided new insights into protein oxidation-regulated signaling and biological processes during PDAC pathogenesis, which might be further explored for pancreatic cancer diagnosis and treatment.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

Using Redox Proteomics to Gain New Insights into Neurodegenerative Disease and Protein Modification.

Antioxidant defenses in biological systems ensure redox homeostasis, regulating baseline levels of reactive oxygen and nitrogen species (ROS and RNS). Oxidative stress (OS), characterized by a lack of antioxidant defenses or an elevation in ROS and RNS, may cause a modification of biomolecules, ROS being primarily absorbed by proteins. As a result of both genome and environment interactions, proteomics provides complete information about a cell's proteome, which changes continuously. Besides measuring protein expression levels, proteomics can also be used to identify protein modifications, localizations, the effects of added agents, and the interactions between proteins. Several oxidative processes are frequently used to modify proteins post-translationally, including carbonylation, oxidation of amino acid side chains, glycation, or lipid peroxidation, which produces highly reactive alkenals. Reactive alkenals, such as 4-hydroxy-2-nonenal, are added to cysteine (Cys), lysine (Lys), or histidine (His) residues by a Michael addition, and tyrosine (Tyr) residues are nitrated and Cys residues are nitrosylated by a Michael addition. Oxidative and nitrosative stress have been implicated in many neurodegenerative diseases as a result of oxidative damage to the brain, which may be especially vulnerable due to the large consumption of dioxygen. Therefore, the current methods applied for the detection, identification, and quantification in redox proteomics are of great interest. This review describes the main protein modifications classified as chemical reactions. Finally, we discuss the importance of redox proteomics to health and describe the analytical methods used in redox proteomics.

Redox Regulation of Salt Tolerance in Eutrema salsugineum by Proteomics.

Salt stress severely restricts plant growth and crop production, which is accompanied by accumulation of reactive oxygen species (ROS) that disturb cell redox homeostasis and oxidize redox-sensitive proteins. Eutrema salsugineum, a halophytic species closely related to Arabidopsis, shows a high level of tolerance to salinity and is increasingly used as a model plant in abiotic stress biology. To understand redox modifications and signaling pathways under salt stress, we used tandem mass tag (TMT)-based proteomics to quantify the salt-induced changes in protein redox modifications in E. salsugineum. Salt stress led to increased oxidative modification levels of 159 cysteine sites in 107 proteins, which play roles in carbohydrate and energy metabolism, transport, ROS homeostasis, cellular structure modulation, and folding and assembly. These lists of unknown redox reactive proteins in salt mustard lay the foundation for future research to understand the molecular mechanism of plant salt response. However, glutathione peroxidase (GPX) is one of the most important antioxidant enzymes in plants. Our research indicates that EsGPX may be involved in regulating ROS levels and that plants with overexpressed EsGPX have much improved salt tolerance.

Global approaches for protein thiol redox state detection.

Due to its nucleophilicity, the thiol group of cysteine is chemically very versatile. Hence, cysteine often has important functions in a protein, be it as the active site or, in extracellular proteins, as part of a structural disulfide. Within the cytosol, cysteines are typically reduced. But the nucleophilicity of its thiol group makes it also particularly prone to post-translational oxidative modifications. These modifications often lead to an alteration of the function of the affected protein and are reversible in vivo, e.g. by the thioredoxin and glutaredoxin system. The in vivo-reversible nature of these modifications and their genesis in the presence of localized high oxidant levels led to the paradigm of thiol-based redox regulation, the adaptation, and modulation of the cellular metabolism in response to oxidative stimuli by thiol oxidation in regulative proteins. Consequently, the proteomic study of these oxidative posttranslational modifications of cysteine plays an indispensable role in redox biology.

Recent advances in mass spectrometry-based methods to investigate reversible cysteine oxidation.

The post-translational modification of cysteine to diverse oxidative states is understood as a critical cellular mechanism to combat oxidative stress. To study the role of cysteine oxidation, cysteine enrichments and subsequent analysis via mass spectrometry are necessary. As such, technologies and methods are rapidly developing for sensitive and efficient enrichments of cysteines to further explore its role in signaling pathways. In this review, we analyze recent developments in methods to miniaturize cysteine enrichments, analyze the underexplored disulfide bound redoxome, and quantify site-specific cysteine oxidation. We predict that further development of these methods will improve cysteine coverage across more diverse organisms than those previously studied and elicit novel roles cysteines play in stress response.

Ahp deficiency-induced redox imbalance leads to metabolic alterations in E.coli.

Alkyl hydroperoxide reductase (Ahp) is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli (E. coli). Ahp-deficient strains have been found to have high reactive oxygen species (ROS) levels, sufficient to cause cell damage. However, the exact role and underlying mechanisms of Ahp deficiency-induced cell damage remain largely unknown. Here, the E. coli MG1655 ΔAhp mutant strain was constructed as a model of deficiency to assess its role. The cells of the ΔAhp strain were found to be significantly longer than those of the wild strain, with elevated ROS and hydrogen peroxide (H2O2) levels. Proteome, redox proteome and metabolome analyses were performed to systematically present a global and quantitative profile and delineate the redox signaling and metabolic alterations at the proteome, metabolome, and cysteine oxidation site levels. The multiomics data revealed that Ahp deficiency disrupted the redox balance, activated the OxyR system, upregulated oxidative defense proteins and inhibited the TCA cycle to some extent. Surprisingly, the mutant strain shifted from aerobic respiration to anaerobic respiration and fermentation during the logarithmic phase in the presence of sufficient O2. The acid resistance system was activated to mitigate the effect of excessive acid produced by fermentation. Taken together, the results of this study demonstrated that Ahp deficiency triggered cellular redox imbalance and regulated metabolic pathways to confer resistance to submicromolar intracellular H2O2 levels in E. coli.

Brevetoxin induces a shift in the redox state of the proteome and unfolded protein response in human lymphoblast cells that can be alleviated with the acrolein scavenger MESNA.

Human lymphoblast cells were treated with the marine algal toxin, brevetoxin-2 (PbTx-2), and its effects on the proteome were assessed by redox proteomics using cysteine reactive tandem mass tags (TMT). Additionally, cells were simultaneously treated with PbTx-2 and the antioxidant and acrolein scavenger sodium 2-mercaptoethylsulfonate (MESNA) to determine if MESNA could prevent the proteomic effects of brevetoxin-2. A massive shift in the redox state of the proteome of brevetoxin-2 treated cells was observed. The main pathway affected was genetic information processing. Significantly oxidized proteins included Trx-1, peroxyredoxins (Prxs), ribosomal proteins, and the eukaryotic initiation factor 2 ß subunit (eIF2ß). Proteins that were overexpressed in brevetoxin-treated cells included four folding chaperones. These effects were diminished in the presence of MESNA indicating that MESNA may act through its antioxidant properties or as a brevetoxin scavenger. These studies provide novel insights into new prophylactics for brevetoxicosis in humans and wildlife.

Thiol redox proteomics: Characterization of thiol-based post-translational modifications.

Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various biological processes. Redox proteomics approaches aim to characterize the landscape of redox PTMs at the systems level. These approaches facilitate studies of condition-specific, dynamic processes implicating redox PTMs and have furthered our understanding of redox signaling and regulation. Mass spectrometry (MS) is a powerful tool for such analyses which has been demonstrated by significant advances in redox proteomics during the last decade. A group of well-established approaches involves the initial blocking of free thiols followed by selective reduction of oxidized PTMs and subsequent enrichment for downstream detection. Alternatively, novel chemoselective probe-based approaches have been developed for various redox PTMs. Direct detection of redox PTMs without any enrichment has also been demonstrated given the sensitivity of contemporary MS instruments. This review discusses the general principles behind different analytical strategies and covers recent advances in redox proteomics. Several applications of redox proteomics are also highlighted to illustrate how large-scale redox proteomics data can lead to novel biological insights.

Proteome-wide quantitative analysis of redox cysteine availability in the Drosophila melanogaster eye reveals oxidation of phototransduction machinery during blue light exposure and age.

The retina is one of the highest oxygen-consuming tissues because visual transduction and light signaling processes require large amounts of ATP. Thus, because of the high energy demand, oxygen-rich environment, and tissue transparency, the eye is susceptible to excess production of reactive oxygen species (ROS) resulting in oxidative stress. Oxidative stress in the eye is associated with the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy. ROS can modify and damage cellular proteins, but can also be involved in redox signaling. In particular, the thiol groups of cysteines can undergo reversible or irreversible oxidative post-translational modifications (PTMs). Identifying the redox-sensitive cysteines on a proteome-wide scale provides insight into those proteins that act as redox sensors or become irreversibly damaged upon exposure to oxidative stress. In this study, we profiled the redox proteome of the Drosophila eye under prolonged, high intensity blue light exposure and age using iodoacetamide isobaric label sixplex reagents (iodo-TMT) to identify changes in cysteine availability. Although redox metabolite analysis of the major antioxidant, glutathione, revealed similar ratios of its oxidized and reduced form in aged or light-stressed eyes, we observed different changes in the redox proteome under these conditions. Both conditions resulted in significant oxidation of proteins involved in phototransduction and photoreceptor maintenance but affected distinct targets and cysteine residues. Moreover, redox changes induced by blue light exposure were accompanied by a large reduction in light sensitivity that did not arise from a reduction in the photopigment level, suggesting that the redox-sensitive cysteines we identified in the phototransduction machinery might contribute to light adaptation. Our data provide a comprehensive description of the redox proteome of Drosophila eye tissue under light stress and aging and suggest how redox signaling might contribute to light adaptation in response to acute light stress.

IodoTMT-labeled redox proteomics reveals the involvement of oxidative post-translational modification in response to para-hydroxybenzoic acid and hydrogen peroxide stresses in poplar.

Poplar is widely planted as an economic and ecological tree species. However, accumulation of the phenolic acid allelochemical para-hydroxybenzoic acid (pHBA) in soil is a severe threat to the growth and productivity of poplar. pHBA stress leads to excessive production of reactive oxygen species (ROS). However, it is unclear which redox-sensitive proteins are involved in the pHBA-induced cellular homeostasis regulatory mechanism. We here identified reversible redox-modified proteins and modified cysteine (Cys) sites in exogenous pHBA- and hydrogen peroxide (H2O2)-treated poplar seedling leaves by using the iodoacetyl tandem mass tag-labeled redox proteomics method. In total, 4786 redox modification sites were identified in 3176 proteins, with 104 and 91 proteins being differentially modified at 118 and 101 Cys sites in response to pHBA and H2O2 stresses, respectively. The differentially modified proteins (DMPs) were predicted to be mainly localized in the chloroplast and cytoplasm, with most proteins being enzymes with catalytic activities. The KEGG enrichment analysis of these DMPs revealed that proteins related to the MAPK signaling pathway, soluble sugar metabolism, amino acid metabolism, photosynthesis, and phagosome pathways were extensively regulated by redox modifications. Moreover, combined with our previous quantitative proteomics data, 8 proteins were upregulated and oxidized under both pHBA and H2O2 stresses. Reversible oxidation of Cys sites in these proteins might be actively responsible for the regulation of tolerance to pHBA-induced oxidative stress. Based on the aforementioned results, a redox regulatory model activated by pHBA- and H2O2-induced oxidative stress was proposed. This study conducts the first redox proteomics analysis of poplar in response to pHBA stress and provides a new insight into the mechanistic framework of reversible oxidative post-translational modifications to gain a better understanding of pHBA-induced chemosensory effects on poplar.

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures.

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (∼96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general.

Radiotherapy Side Effects: Comprehensive Proteomic Study Unraveled Neural Stem Cell Degenerative Differentiation upon Ionizing Radiation.

Cranial radiation therapy is one of the most effective treatments for childhood brain cancers. Despite the ameliorated survival rate of juvenile patients, radiation exposure-induced brain neurogenic region injury could markedly impair patients' cognitive functions and even their quality of life. Determining the mechanism underlying neural stem cells (NSCs) response to irradiation stress is a crucial therapeutic strategy for cognitive impairment. The present study demonstrated that X-ray irradiation arrested NSCs' cell cycle and impacted cell differentiation. To further characterize irradiation-induced molecular alterations in NSCs, two-dimensional high-resolution mass spectrometry-based quantitative proteomics analyses were conducted to explore the mechanism underlying ionizing radiation's influence on stem cell differentiation. We observed that ionizing radiation suppressed intracellular protein transport, neuron projection development, etc., particularly in differentiated cells. Redox proteomics was performed for the quantification of cysteine thiol modifications in order to profile the oxidation-reduction status of proteins in stem cells that underwent ionizing radiation treatment. Via conjoint screening of protein expression abundance and redox status datasets, several significantly expressed and oxidized proteins were identified in differentiating NSCs subjected to X-ray irradiation. Among these proteins, succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial (sdha) and the acyl carrier protein, mitochondrial (Ndufab1) were highly related to neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, illustrating the dual-character of NSCs in cell differentiation: following exposure to ionizing radiation, the normal differentiation of NSCs was compromised, and the upregulated oxidized proteins implied a degenerative differentiation trajectory. These findings could be integrated into research on neurodegenerative diseases and future preventive strategies.

Defining the S-Glutathionylation Proteome by Biochemical and Mass Spectrometric Approaches.

Protein S-glutathionylation (SSG) is a reversible post-translational modification (PTM) featuring the conjugation of glutathione to a protein cysteine thiol. SSG can alter protein structure, activity, subcellular localization, and interaction with small molecules and other proteins. Thus, it plays a critical role in redox signaling and regulation in various physiological activities and pathological events. In this review, we summarize current biochemical and analytical approaches for characterizing SSG at both the proteome level and at individual protein levels. To illustrate the mechanism underlying SSG-mediated redox regulation, we highlight recent examples of functional and structural consequences of SSG modifications. Finally, we discuss the analytical challenges in characterizing SSG and the thiol PTM landscape, future directions for understanding of the role of SSG in redox signaling and regulation and its interplay with other PTMs, and the potential role of computational approaches to accelerate functional discovery.

A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model.

Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In-depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5,608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where a ∼2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.

Regulation of hyperoxia-induced neonatal lung injury via post-translational cysteine redox modifications.

Preterm infants and patients with lung disease often have excess fluid in the lungs and are frequently treated with oxygen, however long-term exposure to hyperoxia results in irreversible lung injury. Although the adverse effects of hyperoxia are mediated by reactive oxygen species, the full extent of the impact of hyperoxia on redox-dependent regulation in the lung is unclear. In this study, neonatal mice overexpressing the beta-subunit of the epithelial sodium channel (ß-ENaC) encoded by Scnn1b and their wild type (WT; C57Bl6) littermates were utilized to study the pathogenesis of high fraction inspired oxygen (FiO2)-induced lung injury. Results showed that O2-induced lung injury in transgenic Scnn1b mice is attenuated following chronic O2 exposure. To test the hypothesis that reversible cysteine-redox-modifications of proteins play an important role in O2-induced lung injury, we performed proteome-wide profiling of protein S-glutathionylation (SSG) in both WT and Scnn1b overexpressing mice maintained at 21% O2 (normoxia) or FiO2 85% (hyperoxia) from birth to 11-15 days postnatal. Over 7700 unique Cys sites with SSG modifications were identified and quantified, covering more than 3000 proteins in the lung. In both mouse models, hyperoxia resulted in a significant alteration of the SSG levels of Cys sites belonging to a diverse range of proteins. In addition, substantial SSG changes were observed in the Scnn1b overexpressing mice exposed to hyperoxia, suggesting that ENaC plays a critically important role in cellular regulation. Hyperoxia-induced SSG changes were further supported by the results observed for thiol total oxidation, the overall level of reversible oxidation on protein cysteine residues. Differential analyses reveal that Scnn1b overexpression may protect against hyperoxia-induced lung injury via modulation of specific processes such as cell adhesion, blood coagulation, and proteolysis. This study provides a landscape view of protein oxidation in the lung and highlights the importance of redox regulation in O2-induced lung injury.

Abscisic acid-controlled redox proteome of Arabidopsis and its regulation by heterotrimeric Gß protein.

The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G-proteins are key mediators of ABA responses. Both ABA and G-proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G-protein signaling remains uncharacterized. To probe the role of reversible protein oxidation in plant stress response and its dependence on G-proteins, we determined the ABA-dependent reversible redoxome of wild-type and Gß-protein null mutant agb1 of Arabidopsis. We quantified 6891 uniquely oxidized cysteine-containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G-proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA- and G-protein-dependent redox changes, many of which occurred on proteins not previously linked to them. We report the most comprehensive ABA-dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G-proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G-proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses.

Quantitative analysis of redox proteome reveals oxidation-sensitive protein thiols acting in fundamental processes of developmental hematopoiesis.

Fetal and adult hematopoietic stem and progenitor cells (HSPCs) are characterized by distinct redox homeostasis that may influence their differential cellular behavior in normal and malignant hematopoiesis. In this work, we have applied a quantitative mass spectrometry-based redox proteomic approach to comprehensively describe reversible cysteine modifications in primary mouse fetal and adult HSPCs. We defined the redox state of 4,438 cysteines in fetal and adult HSPCs and demonstrated a higher susceptibility to oxidation of protein thiols in fetal HSPCs. Our data identified ontogenic changes to oxidation state of thiols in proteins with a pronounced role in metabolism and protein homeostasis. Additional redox proteomic analysis identified oxidation changes to thiols acting in mitochondrial respiration as well as protein homeostasis to be triggered during onset of MLL-ENL leukemogenesis in fetal HSPCs. Our data has demonstrated that redox signaling contributes to the regulation of fundamental processes of developmental hematopoiesis and has pinpointed potential targetable redox-sensitive proteins in in utero-initiated MLL-rearranged leukemia.

Redox proteome analysis of auranofin exposed ovarian cancer cells (A2780).

The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, metabolic changes associated with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and the mitochondrial functions and results into severe ER stress. Results are discussed in the context of the current mechanistic knowledge existing on AF.