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.

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.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

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.

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.

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.

Redox proteomics analysis of hair shaft proteins upon hydrothermal and alkaline insult.

OBJECTIVE: Human hair is regularly subjected to chemical and physical insults, such as heat, UV-irradiation and alkaline hair care products. These insults result in molecular modifications at the hair protein level that underpin mechanical and sensory property changes in the fibres. These changes can manifest itself in reduced hair quality and performance attributes observable to the consumer. In this work, changes in protein modification as a result of heat and alkaline treatments are determined. METHODS: Redox proteomic profiling using high-resolution mass spectrometry was applied to map and evaluate amino acid residue modifications in human hair exposed to a combination of thermal treatments and alkali exposure with the aim to understand the underlying chemical processes. RESULTS: Our results show that an increase in redox-related modifications is associated with exposure to higher levels of hydrothermal and alkaline insult. Post-translational modification profiling at the protein primary structural level delivered some further insights into the site-specificity of these modifications, with a clear increase in the number of cysteic acid modifications noticed in samples subjected to more extreme insults. CONCLUSION: Pinpointing modification sides within proteins and the hair shaft proteome can be used as a basis for employing mitigation or repair strategies of hair protein damage caused by environmental or hair treatment-related insults.

OBJECTIF: Les cheveux humains sont sujet à de nombreuses agressions physiques et chimiques telles que la chaleur, les radiations ultra-violettes et les produits alcalins d'entretien des cheveux. Ces agressions entrainent des modifications moléculaires dans les protéines constituant les cheveux et elles conduisent aussi à des changements mécaniques et sensoriels des fibres capillaires. Les manifestations possibles de ces transformations sont une baisse, visible pour le consommateur, de la qualité et des indicateurs de performance des cheveux. Lors de cette étude, nous mettons en évidence les changements au niveau protéique liés à la chaleur et aux traitements alcalins. MÉTHODES: Les méthodes de profilage d'oxydoréduction protéomique utilisant des spectromètres de masses à haute résolution ont été utilisées afin d'évaluer les modifications des amino-acides dans les cheveux humains après exposition à plusieurs combinaisons de traitements thermiques et alcalins dans le but de comprendre les processus chimiques impliqués. RÉSULTATS: Nos résultats montrent que l'augmentation des modifications d'oxydoréduction est associée à des niveaux élevés d'exposition aux traitements thermiques et/ou alcalins. Le profilage des modifications post-translationnelles des structures primaires des protéines ont permis de mieux comprendre les spécificités de ces modifications ; notamment une augmentation nette du nombre des modifications des acides cystéiques liée aux traitements les plus agressifs. CONCLUSION: Ce travail d'identification des modifications engendrées par les agressions liées aux traitements capillaires ou environnementales peut désormais servir de base pour évaluer et mettre en place des techniques de réduction des risques, protection et de réparation des protéines des cheveux.

Characterization of cellular oxidative stress response by stoichiometric redox proteomics.

The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including S-glutathionylation (SSG), S-nitrosylation (SNO), S-sulfenylation (SOH), and S-sulfhydration (SSH). These modifications can impact various aspects of protein function such as activity, binding, conformation, localization, and interactions with other molecules. To identify novel redox proteins in signaling and regulation, it is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs. Mass spectrometry (MS)-based redox proteomics has emerged as the primary platform for broad characterization of thiol PTMs in cells and tissues. Herein, we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs at physiological or oxidative stress conditions and highlight some recent applications. Considering the relative maturity of available methods, emphasis will be on two types of modifications: 1) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and 2) S-glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives toward a better understanding of cellular redox regulatory networks in cells and tissues.

Gel-based fluorescent proteomic tools for investigating cell redox signaling. A mini-review.

The specific chemical reactivity of thiol groups makes protein cysteines susceptible to reactions with reactive oxygen species (ROS) and reactive nitrogen species (RNS) resulting in the formation of various reversible and irreversible oxidative post-translational modifications (oxPTMs). This review highlights a number of gel-based redox proteomic approaches to detect protein oxPTMs, with particular emphasis on S-nitrosylation, which we believe are currently one of the most accurate way to analyze changes in the redox status of proteins. The information collected in this review relates to the recent progress regarding methods for the enrichment and identification of redox-modified proteins, with an emphasis on fluorescent gel proteomics. Gel-based fluorescent proteomic strategies are low-cost and easy-to-use tools for investigating the thiol proteome and can provide substantial information on redox signaling.

Formation of oxidised (OX) proteins in Entamoeba histolytica exposed to auranofin and consequences on the parasite virulence.

Metronidazole (MNZ), the first line drug for amoebiasis and auranofin (AF), an emerging antiprotozoan drug, are both inhibiting Entamoeba histolytica thioredoxin reductase. The nature of oxidised proteins (OXs) formed in AF- or MNZ-treated E. histolytica trophozoites is unknown. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the OXs formed in AF- or MNZ-treated E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry (MS). We detected 661 OXs in MNZ-treated trophozoites and 583 OXs in AF-treated trophozoites. More than 50% of these OXs were shared, and their functions include hydrolases, enzyme modulators, transferases, nucleic acid binding proteins, oxidoreductases, cytoskeletal proteins, chaperones, and ligases. Here, we report that the formation of actin filaments (F-actin) is impaired in AF-treated trophozoites. Consequently, their erythrophagocytosis, cytopathic activity, and their motility are impaired. We also observed that less than 15% of OXs present in H2 O2 -treated trophozoites are also present in AF- or MNZ-treated trophozoites. These results strongly suggest that the formation of OXs in AF- or MNZ-treated trophozoites and in H2 O2 -treated trophozoites occurred by two different mechanisms.

Differential redox proteomic profiles of serum from severe asthma patients after one month of benralizumab and mepolizumab treatment.

Mepolizumab and Benralizumab are biological drugs for severe asthma patients able to reduce moderate-to-severe exacerbation rate (peripheral eosinophilial % mepolizumab 1.6 ± 1.2; benralizumab 0; p < 0.0001), improving the quality of life and lung function parameters (FEV1%: mepolizumab 87.1 ± 21.5; benralizumab 89.7 ± 15, p < 0.04). Here we report a preliminary redox proteomic study highlighting the level of oxidative burst present in serum from patients before and after one month of both treatments. Our results highlighted apolipoprotein A1 oxidation after Mepolizumab treatment, that could be related to HDL functionality and could represent a potential biomarker for the treatment. On the other hand, after one month of Benralizumab we detected higher oxidation levels of ceruloplasmin and transthyretin, considered an important oxidative stress biomarker which action help to maintain redox homeostasis.

Bull Sperm Capacitation Is Accompanied by Redox Modifications of Proteins.

The ability to fertilise an egg is acquired by the mammalian sperm during the complex biochemical process called capacitation. Capacitation is accompanied by the production of reactive oxygen species (ROS), but the mechanism of redox regulation during capacitation has not been elucidated. This study aimed to verify whether capacitation coincides with reversible oxidative post-translational modifications of proteins (oxPTMs). Flow cytometry, fluorescence microscopy and Western blot analyses were used to verify the sperm capacitation process. A fluorescent gel-based redox proteomic approach allowed us to observe changes in the level of reversible oxPTMs manifested by the reduction or oxidation of susceptible cysteines in sperm proteins. Sperm capacitation was accompanied with redox modifications of 48 protein spots corresponding to 22 proteins involved in the production of ROS (SOD, DLD), playing a role in downstream redox signal transfer (GAPDHS and GST) related to the cAMP/PKA pathway (ROPN1L, SPA17), acrosome exocytosis (ACRB, sperm acrosome associated protein 9, IZUMO4), actin polymerisation (CAPZB) and hyperactivation (TUBB4B, TUB1A). The results demonstrated that sperm capacitation is accompanied by altered levels of oxPTMs of a group of redox responsive proteins, filling gaps in our knowledge concerning sperm capacitation.

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.

Effect of 2-Cys Peroxiredoxins Inhibition on Redox Modifications of Bull Sperm Proteins.

Sperm peroxiredoxins (PRDXs) are moonlighting proteins which, in addition to their antioxidant activity, also act as redox signal transducers through PRDX-induced oxidative post-translational modifications of proteins (oxPTMs). Despite extensive knowledge on the antioxidant activity of PRDXs, the mechanisms related to PRDX-mediated oxPTMs are poorly understood. The present study aimed to investigate the effect of bull sperm 2-Cys PRDX inhibition by Conoidin A on changes in oxPTM levels under control and oxidative stress conditions. The results showed that a group of sperm mitochondrial (LDHAL6B, CS, ACO2, SDHA, ACAPM) and actin cytoskeleton proteins (CAPZB, ALDOA, CCIN) is oxidized due to the action of 2-Cys PRDXs under control conditions. In turn, under oxidative stress conditions, 2-Cys PRDX activity seems to be focused on antioxidant function protecting glycolytic, TCA pathway, and respiratory chain enzymes; chaperones; and sperm axonemal tubulins from oxidative damage. Interestingly, the inhibition of PRDX resulted in oxidation of a group of rate-limiting glycolytic proteins, which is known to trigger the switching of glucose metabolism from glycolysis to pentose phosphate pathway (PPP). The obtained results are expected to broaden the knowledge of the potential role of bull sperm 2-Cys in both redox signal transmission and antioxidant activity.

Redox stress and signaling during vertebrate embryonic development: Regulation and responses.

Vertebrate embryonic development requires specific signaling events that regulate cell proliferation and differentiation to occur at the correct place and the correct time in order to build a healthy embryo. Signaling pathways are sensitive to perturbations of the endogenous redox state, and are also susceptible to modulation by reactive species and antioxidant defenses, contributing to a spectrum of passive vs. active effects that can affect redox signaling and redox stress. Here we take a multi-level, integrative approach to discuss the importance of redox status for vertebrate developmental signaling pathways and cell fate decisions, with a focus on glutathione/glutathione disulfide, thioredoxin, and cysteine/cystine redox potentials and the implications for protein function in development. We present a tissue-specific example of the important role that reactive species play in pancreatic development and metabolic regulation. We discuss NFE2L2 (also known as NRF2) and related proteins, their roles in redox signaling, and their regulation of glutathione during development. Finally, we provide examples of xenobiotic compounds that disrupt redox signaling in the context of vertebrate embryonic development. Collectively, this review provides a systems-level perspective on the innate and inducible antioxidant defenses, as well as their roles in maintaining redox balance during chemical exposures that occur in critical windows of development.

Redox Proteomes in Human Physiology and Disease Mechanisms.

Redox proteomics is a field of proteomics that is concerned with the characterization of the oxidation state of proteins to gain information about their modulated structure, function, activity, and involvement in different physiological pathways. Oxidative modifications of proteins have been shown to be implicated in normal physiological processes of cells as well as in pathomechanisms leading to the development of cancer, diabetes, neurodegenerative diseases, and some rare hereditary metabolic diseases, like classic galactosemia. Reactive oxygen species generate a variety of reversible and irreversible modifications in amino acid residue side chains and within the protein backbone. These oxidative post-translational modifications (Ox-PTMs) can participate in the activation of signal transduction pathways and mediate the toxicity of harmful oxidants. Thus the application of advanced redox proteomics technologies is important for gaining insights into molecular mechanisms of diseases. Mass-spectrometry-based proteomics is one of the most powerful methods that can be used to give detailed qualitative and quantitative information on protein modifications and allows us to characterize redox proteomes associated with diseases. This Review illustrates the role and biological consequences of Ox-PTMs under basal and oxidative stress conditions by focusing on protein carbonylation and S-glutathionylation, two abundant modifications with an impact on cellular pathways that have been intensively studied during the past decade.

The Proteomic Landscape of Cysteine Oxidation That Underpins Retinoic Acid-Induced Neuronal Differentiation.

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.

S-Nitroso-Proteome Revealed in Stomatal Guard Cell Response to Flg22.

Nitric oxide (NO) plays an important role in stomata closure induced by environmental stimuli including pathogens. During pathogen challenge, nitric oxide (NO) acts as a second messenger in guard cell signaling networks to activate downstream responses leading to stomata closure. One means by which NO's action is achieved is through the posttranslational modification of cysteine residue(s) of target proteins. Although the roles of NO have been well studied in plant tissues and seedlings, far less is known about NO signaling and, more specifically, protein S-nitrosylation (SNO) in stomatal guard cells. In this study, using iodoTMTRAQ quantitative proteomics technology, we analyzed changes in protein SNO modification in guard cells of reference plant Arabidopsis thaliana in response to flg22, an elicitor-active peptide derived from bacterial flagellin. A total of 41 SNO-modified peptides corresponding to 35 proteins were identified. The proteins cover a wide range of functions, including energy metabolism, transport, stress response, photosynthesis, and cell-cell communication. This study creates the first inventory of previously unknown NO responsive proteins in guard cell immune responses and establishes a foundation for future research toward understanding the molecular mechanisms and regulatory roles of SNO in stomata immunity against bacterial pathogens.

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

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