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Although macrophages are known to be affected by their redox status, oxidation is not yet a well-recognized post-translational modification (PTM) in regulating macrophages and immune cells in general. While it has been described that the redox status of single cysteines in specific proteins is relevant for macrophage functions, global oxidation information is scarce. Hence, we globally assessed the impact of oxidation on macrophage activation using untargeted proteomics and PTM-omics. We exposed THP-1 macrophages to lipopolysaccharide (LPS) for 4 h and 24 h and applied a sequential iodoTMT labeling approach to get information on overall oxidation as well as reversible oxidation of cysteines. Thus, we identified 10452 oxidation sites, which were integratively analyzed with 5057 proteins and 7148 phosphorylation sites to investigate their co-occurance with other omics layers. Based on this integrative analysis, we found significant upregulation of several immune-related pathways, e.g. toll-like receptor 4 (TLR4) signaling, for which 19 proteins, 7 phosphorylation sites, and 39 oxidation sites were significantly affected, highlighting the relevance of oxidations in TLR4-induced macrophage activation. Co-regulation of oxidation and phosphorylation was observed, as evidenced by multiply modified proteins related to inflammatory pathways. Additionally, we observed time-dependent effects, with differences in the dynamics of oxidation sites compared to proteins and phosphorylation sites. Overall, this study highlights the importance of oxidation in regulating inflammatory processes and provides a method that can be readily applied to study the cellular redoxome globally.
Assuntos
Processamento de Proteína Pós-Traducional , Receptor 4 Toll-Like , Fosforilação , Receptor 4 Toll-Like/metabolismo , Imunidade , OxirreduçãoRESUMO
Although circadian biorhythms of mitochondria and cells are highly conserved and crucial for the well-being of complex animals, there is a paucity of studies on the reciprocal interactions between oxidative stress, redox modifications, metabolism, thermoregulation, and other major oscillatory physiological processes. To address this limitation, we hypothesize that circadian/ultradian interaction of the redoxome, bioenergetics, and temperature signaling strongly determine the differential activities of the sleep-wake cycling of mammalians and birds. Posttranslational modifications of proteins by reversible cysteine oxoforms, S-glutathionylation and S-nitrosylation are shown to play a major role in regulating mitochondrial reactive oxygen species production, protein activity, respiration, and metabolomics. Nuclear DNA repair and cellular protein synthesis are maximized during the wake phase, whereas the redoxome is restored and mitochondrial remodeling is maximized during sleep. Hence, our analysis reveals that wakefulness is more protective and restorative to the nucleus (nucleorestorative), whereas sleep is more protective and restorative to mitochondria (mitorestorative). The "redox-bioenergetics-temperature and differential mitochondrial-nuclear regulatory hypothesis" adds to the understanding of mitochondrial respiratory uncoupling, substrate cycling control and hibernation. Similarly, this hypothesis explains how the oscillatory redox-bioenergetics-temperature-regulated sleep-wake states, when perturbed by mitochondrial interactome disturbances, influence the pathogenesis of aging, cancer, spaceflight health effects, sudden infant death syndrome, and diseases of the metabolism and nervous system.
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Redox regulation plays a wide role in plant growth, development, and adaptation to stresses. Sulfenylation is one of the reversible oxidative post-transcriptional modifications. Here we performed an iodoTMT-based proteomic analysis to identify the redox sensitive proteins in vivo under freezing stress after cold acclimation in Brassica napus. Totally, we obtained 1,372 sulfenylated sites in 714 proteins. The overall sulfenylation level displayed an increased trend under freezing stress after cold acclimation. We identified 171 differentially sulfenylated proteins (DSPs) under freezing stress, which were predicted to be mainly localized in chloroplast and cytoplasm. The up-regulated DSPs were mainly enriched in photosynthesis and glycolytic processes and function of catalytic activity. Enzymes involved in various pathways such as glycolysis and Calvin-Benson-Bassham (CBB) cycle were generally sulfenylated and the metabolite levels in these pathways was significantly reduced under freezing stress after cold acclimation. Furthermore, enzyme activity assay confirmed that the activity of cytosolic pyruvate kinase and malate dehydrogenase 2 was significantly reduced under H2O2 treatment. Our study provides a landscape of redox sensitive proteins in B. napus in response to freezing stress after cold acclimation, which proposes a basis for understanding the redox regulation in plant metabolic response to freezing stress after cold acclimation.
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Despite different phenotypic manifestations, mounting evidence points to similarities in the molecular basis of major neurodegenerative diseases (ND). CNS has evolved to be robust against hazard of ROS, a common perturbation aerobic organisms are confronted with. The trade-off of robustness is system's fragility against rare and unexpected perturbations. Identifying the points of CNS fragility is key for understanding etiology of ND. We postulated that the 'primate differential redoxome' (PDR), an assembly of proteins that contain cysteine residues present only in the primate orthologues of mammals, is likely to associate with an added level of regulatory functionalities that enhanced CNS robustness against ROS and facilitated evolution. The PDR contains multiple deterministic and susceptibility factors of major ND, which cluster to form coordinated redox networks regulating various cellular processes. The PDR analysis revealed a potential CNS fragility point, which appears to associates with a non-redundant PINK1-PRKN-SQSTM1(p62) axis coordinating protein homeostasis and mitophagy.
Assuntos
Doenças Neurodegenerativas , Animais , Mitofagia , Doenças Neurodegenerativas/genética , Oxirredução , Primatas/metabolismo , Proteínas/metabolismoRESUMO
Vitamin C (VitC) possesses pro-oxidant properties at high pharmacologic concentrations which favor repurposing VitC as an anti-cancer therapeutic agent. However, redox-based anticancer properties of VitC are yet partially understood. We examined the difference between the reduced and oxidized forms of VitC, ascorbic acid (AA) and dehydroascorbic acid (DHA), in terms of cytotoxicity and redox mechanisms toward breast cancer cells. Our data showed that AA displayed higher cytotoxicity towards triple-negative breast cancer (TNBC) cell lines in vitro than DHA. AA exhibited a similar cytotoxicity on non-TNBC cells, while only a minor detrimental effect on noncancerous cells. Using MDA-MB-231, a representative TNBC cell line, we observed that AA- and DHA-induced cytotoxicity were linked to cellular redox-state alterations. Hydrogen peroxide (H2O2) accumulation in the extracellular medium and in different intracellular compartments, and to a lesser degree, intracellular glutathione oxidation, played a key role in AA-induced cytotoxicity. In contrast, DHA affected glutathione oxidation and had less cytotoxicity. A "redoxome" approach revealed that AA treatment altered the redox state of key antioxidants and a number of cysteine-containing proteins including many nucleic acid binding proteins and proteins involved in RNA and DNA metabolisms and in energetic processes. We showed that cell cycle arrest and translation inhibition were associated with AA-induced cytotoxicity. Finally, bioinformatics analysis and biological experiments identified that peroxiredoxin 1 (PRDX1) expression levels correlated with AA differential cytotoxicity in breast cancer cells, suggesting a potential predictive value of PRDX1. This study provides insight into the redox-based mechanisms of VitC anticancer activity, indicating that pharmacologic doses of VitC and VitC-based rational drug combinations could be novel therapeutic opportunities for triple-negative breast cancer.
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Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cisteína , Oxirredução/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Antioxidantes/química , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Biologia Computacional/métodos , Cisteína/química , Células Endoteliais/metabolismo , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas , Espécies Reativas de Oxigênio/metabolismoRESUMO
Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H2O2, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H2O2. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.
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Oxirredução , Proteoma , Proteômica , Cromatografia Líquida , Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
Fifteen years ago, in 2001, the concept of "Reactive Sulfur Species" or RSS was advocated as a working hypothesis. Since then various organic as well as inorganic RSS have attracted considerable interest and stimulated many new and often unexpected avenues in research and product development. During this time, it has become apparent that molecules with sulfur-containing functional groups are not just the passive "victims" of oxidative stress or simple conveyors of signals in cells, but can also be stressors in their own right, with pivotal roles in cellular function and homeostasis. Many "exotic" sulfur-based compounds, often of natural origin, have entered the fray in the context of nutrition, ageing, chemoprevention and therapy. In parallel, the field of inorganic RSS has come to the forefront of research, with short-lived yet metabolically important intermediates, such as various sulfur-nitrogen species and polysulfides (Sx2-), playing important roles. Between 2003 and 2005 several breath-taking discoveries emerged characterising unusual sulfur redox states in biology, and since then the truly unique role of sulfur-dependent redox systems has become apparent. Following these discoveries, over the last decade a "hunt" and, more recently, mining for such modifications has begun-and still continues-often in conjunction with new, innovative and complex labelling and analytical methods to capture the (entire) sulfur "redoxome". A key distinction for RSS is that, unlike oxygen or nitrogen, sulfur not only forms a plethora of specific reactive species, but sulfur also targets itself, as sulfur containing molecules, i.e., peptides, proteins and enzymes, preferentially react with RSS. Not surprisingly, today this sulfur-centred redox signalling and control inside the living cell is a burning issue, which has moved on from the predominantly thiol/disulfide biochemistry of the past to a complex labyrinth of interacting signalling and control pathways which involve various sulfur oxidation states, sulfur species and reactions. RSS are omnipresent and, in some instances, are even considered as the true bearers of redox control, perhaps being more important than the Reactive Oxygen Species (ROS) or Reactive Nitrogen Species (RNS) which for decades have dominated the redox field. In other(s) words, in 2017, sulfur redox is "on the rise", and the idea of RSS resonates throughout the Life Sciences. Still, the RSS story isn't over yet. Many RSS are at the heart of "mistaken identities" which urgently require clarification and may even provide the foundations for further scientific revolutions in the years to come. In light of these developments, it is therefore the perfect time to revisit the original hypotheses, to select highlights in the field and to question and eventually update our concept of "Reactive Sulfur Species".
RESUMO
Cellular redox status plays a key role in mediating various physiological and developmental processes often through modulating activities of redox-sensitive proteins. Various stresses trigger over-production of reactive oxygen/nitrogen species which lead to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. Here, we report a high-throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols. The biotin-tagged peptides are affinity purified, labeled with iTRAQ reagents, and analyzed using a paralleled HCD-CID fragmentation mode in an LTQ-Orbitrap. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. This approach allows identification of the specific redox-regulated cysteine residues, and offers an effective tool for elucidation of redox proteomes.