Redox Targets Research Articles

Isothymusin, a Potential Inhibitor of Cancer Cell Proliferation: An In Silico and In Vitro Investigation.

Since centuries plant-based compounds are known for the treatment of cancer in both traditional and contemporary medicine. The problems like target non-specificity and toxicity are well-known regarding anticancer drugs. Therefore, target specific search of novel entities is constant. Isothymusin is a dimethoxy, trihydroxy flavone present in plants like Ocimum sanctum, and Limnophilla geoffrayi. There are limited reports available on the anticancer potential of isothymusin. The effects of isothymusin on redox status, cell cytotoxicity, and targets involved in the promotion and progression of the cancer cells have been investigated. Antiproliferative efficacy was evaluated by MTT, Neutral Red Uptake, and Sulforhodamine-B assays. The spectrophotometric methods were adopted to study the effect against selected targets. Redox activity was assessed by in vitro antioxidant assays and the interaction study, ADMET profiling, and toxicity assessments were done in silico. Isothymusin scavenges the radicals, i.e., DPPH and nitric oxide with moderate ferric reducing potential. It affected the proliferation of leukemia, colon, skin, and breast cancer cell lines by more than 50% but moderately affected prostate, kidney, lung, hepatic, and breast adenocarcinoma (up to 48%). Isothymusin inhibited the enzymes associated with the promotion stage of cancer, including cycloxygenase- 2 and lipoxygenase-5. Additionally, it also inhibited the activity of proliferation markers like cathepsin- D, dihydrofolate reductase, hyaluronidase, and ornithine-decarboxylase. Besides, in silico studies supported the in vitro enzyme inhibition assays outcome. Toxicity studies showed promising results of chemical descriptors and non-skin-irritant, moderate ocular-irritancy, and in vitro Ames test confirmed non-mutagenic nature. Isothymusin showed radical scavenging and anti-proliferative activities, which may be taken up as a phytochemical lead for the synthesis of analogues possessing enhanced anticancer potential.

Abstract P085: Microrna Profiling Of Vascular Smooth Muscle Cells In Human Hypertension Potential Regulators Of Endoplasmic Reticulum And Oxidative Stress

Numerous molecular mechanisms have been implicated in processes underlying vascular phenotypic changes and alterations in hypertension, including microRNAs (miRNAs), oxidative stress and perturbed endoplasmic reticulum (ER) function. The interplay between these elements is unclear. We assessed the VSMC miRNAs profile in hypertension focusing on oxidative and ER stress pathways. VSMCs from small arteries from normotensive (NT) and hypertensive (HT) subjects were used. miRNA profiling of 758 miRNAs was performed using TaqMan advanced miRNA assay (TaqMan Low Density Array Human microRNA). Ingenuity Pathway Analysis (IPA) was used for miRNA target prediction. Expression of vascular genes and proteins was detected by RT-PCR and immunoblotting. ROS generation (chemiluminescence) was assessed in the absence and presence of ER stress inducer tunicamycin (5μg/ml, 24h). miRNA array identified 25 miRNAs uniquely expressed in HT and 21 miRNAs uniquely expressed in NT (CT<30). Of the 332 miRNAs present in both groups, 60 miRNAs were significantly upregulated in HT (fold change >1.5), while 136 miRNAs were significantly downregulated in HT (fold change >1.5). miRNAs that were altered in hypertension, targeted genes involved in oxidative and ER stress. Pro-oxidant [Nox1 mRNA (1.71 fold), Nox4 (1.59 fold), Nox5 (2.04 fold)] and antioxidant [SOD2 mRNA (4.43 fold), GPx1 (1.97 fold)] enzymes protein levels upregulated in HT (p<0.05 vs NT). ER stress proteins, such as PERK (1.57 fold) and elF2α (2.31 fold) were also upregulated in HT (p<0.05 vs NT). IPA analysis of our miRNA library, revealed that miR-505-5p (-2.13 fold), miR-324-5p (-1.51 fold), miR-185-5p (-1.742 fold) and miR-491-5p (-1.667 fold) may regulate Nox5 levels. Moreover, miR-200b-3p (-28.57 fold) targets multiple ER stress pathways including elF2α. Treatment with tunicamycin increased ROS generation (2.29 fold) and Nox5 protein expression (1.69 fold) while downregulating SOD2 mRNA (-8.02 fold) in HT (p<0.05 vs NT). Our findings unveil the differentially expressed miRNAs and their predicted redox targets, highlighting potential interplay between VSMC ER stress, oxidative stress and miRNAs in human hypertension.

An amplified label-free electrochemical aptasensor of γ-interferon based on target-induced DNA strand transform of hairpin-to-linear conformation enabling simultaneous capture of redox probe and target

In this work, a novel and signal-amplified label-free electrochemical aptasensor was developed and enabled efficient determination of γ-interferon (IFN-γ), based on target-induced DNA strand transform of hairpin-to-linear conformation combining with simultaneous capture of redox probe and target. Gold nanoparticles (AuNPs) were electrodeposited in the matrix of poly(amidoamine) dendrimer (PAMAM), followed by drop-casting addition on MoS2 nanosheets to prepare AuNPs- PAMAM/MoS2 composites. HS-terminated hairpin-DNA aptamer of IFN-γ was conjugated with AuNPs to prepare aptamer-AuNPs-PAMAM/MoS2 onto glassy carbon electrode (GCE), by using bovine serum albumin as the cross-linker and stabilizer. Methylene blue (MB) as a redox probe was absorbed on IFN-γ aptamer. In the presence of IFN-γ, MB electrochemical signal increased gradually. The preparation processes, mechanisms and optimal experiment conditions of aptamer- AuNPs-PAMAM/MoS2/MB/GCE sensing platform were studied by electron microscope imaging technologies, spectral curves and electrochemical measurements. There is a well plotting linear relationship between the peak current intensities of MB and IFN-γ contents in the range of 0.01–1000 pg mL−1, showing a low detection limit of 2 fg mL−1. Experimental results testified that the aptasensor had highly sensitive and selective responses toward IFN-γ, over potential interferents. In real biological samples, the aptasensor of IFN-γ had superior detection recoveries, indicating its high detection performance and feasibility for practicability.

Biomarker Identification, Safety, and Efficacy of High-Dose Antioxidants for Adrenomyeloneuropathy: a Phase II Pilot Study

X-Adrenoleukodystrophy (X-ALD) and its adult-onset, most prevalent variant adrenomyeloneuropathy (AMN) are caused by mutations in the peroxisomal transporter of the very long-chain fatty acid ABCD1. AMN patients classically present spastic paraparesis that can progress over decades, and a satisfactory treatment is currently lacking. Oxidative stress is an early culprit in X-ALD pathogenesis. A combination of antioxidants halts the clinical progression and axonal damage in a murine model of AMN, providing a strong rationale for clinical translation. In this phase II pilot, open-label study, 13 subjects with AMN were administered a high dose of α-tocopherol, N-acetylcysteine, and α-lipoic acid in combination. The primary outcome was the validation of a set of biomarkers for monitoring the biological effects of this and future treatments. Functional clinical scales, the 6-minute walk test (6MWT), electrophysiological studies, and cerebral MRI served as secondary outcomes. Most biomarkers of oxidative damage and inflammation were normalized upon treatment, indicating an interlinked redox and inflammatory homeostasis. Two of the inflammatory markers, MCP1 and 15-HETE, were predictive of the response to treatment. We also observed a significant decrease in central motor conduction time, together with an improvement or stabilization of the 6MWT in 8/10 subjects. This study provides a series of biomarkers that are useful to monitor redox and pro-inflammatory target engagement in future trials, together with candidate biomarkers that may serve for patient stratification and disease progression, which merit replication in future clinical trials. Moreover, the clinical results suggest a positive signal for extending these studies to phase III randomized, placebo-controlled, longer-term trials with the actual identified dose. ClinicalTrials.gov Identifier: NCT01495260

BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4

AimNADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4.In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. ResultsTGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. Innovation & conclusionBIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.

Reactive Oxygen Species and the Redox-Regulatory Network in Cold Stress Acclimation.

Cold temperatures restrict plant growth, geographical extension of plant species, and agricultural practices. This review deals with cold stress above freezing temperatures often defined as chilling stress. It focuses on the redox regulatory network of the cell under cold temperature conditions. Reactive oxygen species (ROS) function as the final electron sink in this network which consists of redox input elements, transmitters, targets, and sensors. Following an introduction to the critical network components which include nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductases, thioredoxins, and peroxiredoxins, typical laboratory experiments for cold stress investigations will be described. Short term transcriptome and metabolome analyses allow for dissecting the early responses of network components and complement the vast data sets dealing with changes in the antioxidant system and ROS. This review gives examples of how such information may be integrated to advance our knowledge on the response and function of the redox regulatory network in cold stress acclimation. It will be exemplarily shown that targeting the redox network might be beneficial and supportive to improve cold stress acclimation and plant yield in cold climate.

The Plasma Membrane: A Platform for Intra- and Intercellular Redox Signaling.

Membranes are of outmost importance to allow for specific signal transduction due to their ability to localize, amplify, and direct signals. However, due to the double-edged nature of reactive oxygen species (ROS)—toxic at high concentrations but essential signal molecules—subcellular localization of ROS-producing systems to the plasma membrane has been traditionally regarded as a protective strategy to defend cells from unwanted side-effects. Nevertheless, specialized regions, such as lipid rafts and caveolae, house and regulate the activated/inhibited states of important ROS-producing systems and concentrate redox targets, demonstrating that plasma membrane functions may go beyond acting as a securing lipid barrier. This is nicely evinced by nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases (NOX), enzymes whose primary function is to generate ROS and which have been shown to reside in specific lipid compartments. In addition, membrane-inserted bidirectional H2O2-transporters modulate their conductance precisely during the passage of the molecules through the lipid bilayer, ensuring time-scaled delivery of the signal. This review aims to summarize current evidence supporting the role of the plasma membrane as an organizing center that serves as a platform for redox signal transmission, particularly NOX-driven, providing specificity at the same time that limits undesirable oxidative damage in case of malfunction. As an example of malfunction, we explore several pathological situations in which an inflammatory component is present, such as inflammatory bowel disease and neurodegenerative disorders, to illustrate how dysregulation of plasma-membrane-localized redox signaling impacts normal cell physiology.

Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

Ruthenium and manganese metalloporphyrins modified screen-printed electrodes for bio-relevant electroactive targets

Ruthenium(II) 5-(4[Formula: see text]-sulfanylmethylphenyl)-10,15,20-triphenylporphyrin (Ru-TPP-SH) and manganese(III) 5-(4[Formula: see text]-sulfanylmethylphenyl)-10,15,20-triphenylporphyrin (Mn-TPP-SH) were synthesized, spectroscopically characterized and drop casted to modify screen-printed electrodes (SPEs). The modified SPEs were then tested against the redox target [Fe(CN)6][Formula: see text] in comparison with the bare SPE and SPE modified with the free porphyrin. The best performing one, [Formula: see text]. Mn-TPP-SH was used for the electrochemical detection of 1,4–benzoquinone, serotonin, caffeic and ascorbic acids, the latter also in association with uric acid, showing good electrocatalytic properties. The tunability of the metal-TPP-SH through the choice of the coordinating metal, the drop casting conditions and possible further functionalization make this type of porphyrin a good candidate for further developments of porphyrin-modified SPEs.

CARDIOVASCULAR RISK STRATIFICATION IN YOUNG POPULATION

Objective: Cardiovascular (CV) diseases are the leading cause of death in developed countries and present high prevalence in developing countries. Nevertheless, there are no biomarkers available to detect the causal physiopathological process of the clinical illness, implying a high socioeconomic impact. Nowadays, CV risk estimators focus on age as the most important risk factor in CV events and mortality. The current study is focused on CV risk stratification attending to oxidative stress for the discovery of new markers for early detection of the physiopathological process. Design and method: 24 blood samples from a cohort of patients in average age of life (30–50 years old) classified in 3 groups of study according to their CV risk (healthy controls, patients with CV risk factors and patients with reported CV event in the last 3 years) were analyzed using FASILOX, a novel strategy for analysis of the dynamic thiol redox proteome. To our knowledge, this study constitutes the first description of reversible Cys oxidation in human plasma according to CV risk stratification. Results: We identified that reversible oxidations are decreased in CV events individuals compared with healthy subjects. Only proteins involved in coagulation cascade have reversible oxidations increased in CV events, biologic function directly linked with CV pathology, whose alteration could result in a CV event.We have also showed down-regulation of proteins involved in antioxidant respond, which is in line with the loss of redox homeostasis in CV events. Conclusions: CV events present a severe oxidative stress, exceeding antioxidant mechanisms as reversible oxidations of Cys, generating irreversible oxidations that together with the down-regulation of redoxins, generate an increase of molecular damage.The present draft of redox targets together with the quantification of protein and oxidative changes may help to better

Multifaceted Roles of Glutathione and Glutathione-Based Systems in Carcinogenesis and Anticancer Drug Resistance.

Glutathione is the most abundant antioxidant molecule in living organisms and has multiple functions. Intracellular glutathione homeostasis, through its synthesis, consumption, and degradation, is an intricately balanced process. Glutathione levels are often high in tumor cells before treatment, and there is a strong correlation between elevated levels of intracellular glutathione/sustained glutathione-mediated redox activity and resistance to pro-oxidant anticancer therapy. Recent Advances: Ample evidence demonstrates that glutathione and glutathione-based systems are particularly relevant in cancer initiation, progression, and the development of anticancer drug resistance. This review highlights the multifaceted roles of glutathione and glutathione-based systems in carcinogenesis, anticancer drug resistance, and clinical applications. The evidence summarized here underscores the important role played by glutathione and the glutathione-based systems in carcinogenesis and anticancer drug resistance. Future studies should address mechanistic questions regarding the distinct roles of glutathione in different stages of cancer development and cancer cell death. It will be important to study how metabolic alterations in cancer cells can influence glutathione homeostasis. Sensitive approaches to monitor glutathione dynamics in subcellular compartments will be an indispensible step. Therapeutic perspectives should focus on mechanism-based rational drug combinations that are directed against multiple redox targets using effective, specific, and clinically safe inhibitors. This new strategy is expected to produce a synergistic effect, prevent drug resistance, and diminish doses of single drugs. Antioxid. Redox Signal. 27, 1217-1234.

P 041 - Redox control of renal metabolism and transport function by the NADPH oxidase Nox4

The NADPH oxidase isoform Nox4 produces H2O2, a potent modulator of cellular metabolism. It oxidizes iron and cysteines and is thus able to directly influence the activity of metabolic enzymes or the dynamics of metabolites. Nox4 is highly expressed in the renal cortex and shows a protective role in chronic renal disease whereas its expression is lost in renal pathology. However, its physiological function in the kidney is still largely unknown. In situ hybridization (RNAscope) showed an exclusive expression of Nox4 in proximal tubular epithelial cells, responsible for reabsorption and secretion of NaCl, metabolites and NaHCO3. To investigate a redox-depend tubular function and metabolism, we performed metabolite profiling by 1H-NMR and semi-quantitative LC/MS combined with redox proteomics (BIAM assay for oxidized cysteines) in different models of gain/loss of function of Nox4. A unique decrease of α-ketoglutarate (KG) in plasma and urine in contrast to an increased concentration in renal tissue of Nox4-/- was identified as compared to WT mice. As other TCA cycle metabolites were not altered, this highly suggests a redox-dependent transport of KG. Moreover, a sodium-bicarbonate co-transporter (Slc4A7) which contributes to pHi, was identified as redox target in HEK cells overexpressing Nox4. Redox proteomics of renal cells isolated from Nox4-/- mice showed glutamine synthetase as an oxidation target of Nox4. Accordingly, glutamine concentration is increased in HEK cells overexpressing Nox4. Our results using metabolomics and redox proteomics add insights on the redox regulation of physiological renal function by Nox4.

P 093 - Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling

Hydrogen peroxide's (H2O2) best characterized signaling actions in mammalian cells involve the oxidation of thiols in cytoplasmic targets within minutes of stimulation. However, these redox targets are orders of magnitude less H2O2-reactive and abundant than cytoplasmic peroxiredoxins (Prx). How can they be oxidized in minutes? Our computational results show that at H2O2 supply rates commensurate with mitogenic signaling a H2O2 concentration gradient of a few tenths of µm is established. Even near supply sites H2O2 concentrations are too low to oxidize typical targets in minutes. Further, any Prx inhibition or increase in H2O2 supply able to strongly increase the local H2O2 concentration would collapse the gradient and/or extensively oxidize PrxI/II, inconsistent with observations. In turn, the local concentrations of Prx sulfenate and disulfide forms strongly exceed those of H2O2. Redox targets reacting with these forms at rate constants much lower than that for thioredoxin could be oxidized in

Cysteines as Redox Molecular Switches and Targets of Disease.

Thiol groups can undergo numerous modifications, making cysteine a unique molecular switch. Cysteine plays structural and regulatory roles as part of proteins or glutathione, contributing to maintain redox homeostasis and regulate signaling within and amongst cells. Not surprisingly therefore, cysteines are associated with many hereditary and acquired diseases. Mutations in the primary protein sequence (gain or loss of a cysteine) are most frequent in membrane and secretory proteins, correlating with the key roles of disulfide bonds. On the contrary, in the cytosol and nucleus, aberrant post-translational oxidative modifications of thiol groups, reflecting redox changes in the surrounding environment, are a more frequent cause of dysregulation of protein function. This essay highlights the regulatory functions performed by protein cysteine residues and provides a framework for understanding how mutation and/or (in)activation of this key amino acid can cause disease.

Rab7-a novel redox target that modulates inflammatory pain processing.

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.

The role of micrornas in regulating redox modulation in bipolar disorder

IntroductionAlterations in redox modulation are consistently reported in bipolar disorder (BD). MicroRNAs are targeted regulators of gene expression.Objectives and aimsWe aimed to examine if microRNAs that target redox modulators can discriminate between BD and healthy controls.MethodsData from brains of individuals with and without BD were obtained from Array Collection datasets. MicroRNAs targeting redox modulators were assessed for their ability to discriminate BD from the control group using machine-learning algorithms. Methylation of microRNAs, expression of their transcription factors and redox targets were assessed with ANCOVA with FDR correction. For validation, we acquired plasma samples belonging to 2 families of individuals with and without BD (n = 9). Plasma microRNAs were sequenced using the Ion Total RNA Sequencing Kit (Thermo Fisher Scientific), and microRNAs identified from the in silico analysis were examined in the validation dataset.ResultsWe identified 5 miRNAs (hsa-miR-299, hsa-miR-125a, hsa-miR-145, hsa-miR-30b, hsa-miR424) that were common in two of the four in silico datasets. Target genes glutathione peroxidase 4, ATP5A1, ATP5G1, NDUFS1, NDUFC2, and catalase were expressed at different levels between BD and the control group. Furthermore, our results showed that transcription factors CTCF and USF1 might control the expression of hsa-miR-145, while methylation differences were not found. Finally, hsa-miR-30b was significantly increased in the plasma of patients with BD compared to controls in the validation experiment.ConclusionsOur study demonstrates that microRNAs may have an important role in the initiation of redox changes in BD.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Rational Design of a Stimuli-Responsive Polymer Electrode Interface Coupled with in Vivo Microdialysis for Measurement of Sialic Acid in Live Mouse Brain in Alzheimer's Disease.

Sensitive and selective monitoring of sialic acid (SA) in cerebral nervous system is of great importance for studying the role that SA plays in the pathological process of Alzheimer's disease (AD). In this work, we first reported an electrochemical biosensor based on a novel stimuli-responsive copolymer for selective and sensitive detection of SA in mouse brain. Notably, through synergetic hydrogen-bonding interactions, the copolymer could translate the recognition of SA into their conformational transition and wettability switch, which facilitated the access and enrichment of redox labels and targets to the electrode surface, thus significantly improving the detection sensitivity with the detection limit down to 0.4 pM. Besides amplified sensing signals, the proposed method exhibited good selectivity toward SA in comparison to potential interference molecules coexisting in the complex brain system due to the combination of high affinity between phenylboronic acid (PBA) and SA and the directional hydrogen-bonding interactions in the copolymer. The electrochemical biosensor with remarkable analytical performance was successfully applied to evaluate the dynamic change of SA level in live mouse brain with AD combined with in vivo midrodialysis. The accurate concentration of SA in different brain regions of live mouse with AD has been reported for the first time, which is beneficial for progressing our understanding of the role that SA plays in physiological and pathological events in the brain.

Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling

Hydrogen peroxide (H2O2) is a key signaling agent. Its best characterized signaling actions in mammalian cells involve the early oxidation of thiols in cytoplasmic phosphatases, kinases and transcription factors. However, these redox targets are orders of magnitude less H2O2-reactive and abundant than cytoplasmic peroxiredoxins. How can they be oxidized in a signaling time frame? Here we investigate this question using computational reaction-diffusion models of H2O2 signaling. The results show that at H2O2 supply rates commensurate with mitogenic signaling a H2O2 concentration gradient with a length scale of a few tenths of μm is established. Even near the supply sites H2O2 concentrations are far too low to oxidize typical targets in an early mitogenic signaling time frame. Furthermore, any inhibition of the peroxiredoxin or increase in H2O2 supply able to drastically increase the local H2O2 concentration would collapse the concentration gradient and/or cause an extensive oxidation of the peroxiredoxins I and II, inconsistent with experimental observations. In turn, the local concentrations of peroxiredoxin sulfenate and disulfide forms exceed those of H2O2 by several orders of magnitude. Redox targets reacting with these forms at rate constants much lower than that for, say, thioredoxin could be oxidized within seconds. Moreover, the spatial distribution of the concentrations of these peroxiredoxin forms allows them to reach targets within 1 μm from the H2O2 sites while maintaining signaling localized. The recruitment of peroxiredoxins to specific sites such as caveolae can dramatically increase the local concentrations of the sulfenic and disulfide forms, thus further helping these species to outcompete H2O2 for the oxidation of redox targets. Altogether, these results suggest that H2O2 signaling is mediated by localized redox relays whereby peroxiredoxins are oxidized to sulfenate and disulfide forms at H2O2 supply sites and these forms in turn oxidize the redox targets near these sites.

Differential proteomic and oxidative profiles unveil dysfunctional protein import to adipocyte mitochondria in obesity-associated aging and diabetes

Human age-related diseases, including obesity and type 2 diabetes (T2DM), have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS) subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly) pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM.

SRB1 as a new redox target of cigarette smoke in human sebocytes

For its critical location, the skin represents the major interface between the body and the environment, therefore is one of the major biological barriers against the outdoor environmental stressors. Among the several oxidative environmental stressors, cigarette smoke (CS) has been associated with the development and worsening of many skin pathologies such as acne, dermatitis, delayed wound healing, aging and skin cancer. In our previous work we have demonstrated that CS is able to affect genes involved in skin cholesterol trafficking, among which SRB1, a receptor involved in the uptake of cholesterol from HDL, seems to be very susceptible to the oxidative stress induced by CS.In the present work we wanted to investigate the presence of SRB1 in human sebocytes and whether CS can affect cholesterol cellular uptake via the redox modulation of SRB1.By using a co-culture system of keratinocytes/sebocytes, we found that CS exposure induced a SRB1 protein loss without affecting sebocytes viability. The decrease of SRB1 levels was a consequence of SRB1/HNE adducts formation that leads to SRB1 ubiquitination and degradation. Moreover, the CS-induced loss of SRB1 induced an alteration of sebocytes lipid content, also demonstrated by cholesterol quantification in SRB1 siRNA experiments.In conclusion, exposure to CS, induced SRB1 post-translational modifications in sebocytes and this might affect sebocytes/skin functionality.