Redox Signaling Mechanisms Research Articles

59 - Peroxide-Regulated Heme Transfer: A Novel Redox Signaling Mechanism

Using high-performance mass spectrometry (MS), we have uncovered an unprecedented mechanism of redox signaling by H2O2. Remarkably, many cycles of sequential binding and reduction of peroxide can occur in vitro at the heme of cytochrome c peroxidase (Ccp1) in the absence of its reducing substrate, cytochrome c. MS reveals that ~20 oxygen atoms are incorporated into Ccp1, predominantly at methionine and tryptophan residues. Additionally, extensive intramolecular dityrosine formation involving neighboring tyrosines (Y36/Y39, Y36/Y42 and Y67/Y71) was uncovered on MS sequencing of the crosslinked peptides. A total of 24 residues are oxidized by repeated hole-hopping from the heme in Ccp1 treated with a 10-fold excess of H2O2. Oxidation of the catalytic distal H52 shuts down H2O2 reduction but not until the proximal heme ligand (H175) is oxidized, which labilizes the heme. Notably, no irreversible heme modification is detected by MS, indicating that Ccp1 preserves the integrity of its heme by directing the oxidizing equivalents from H2O2 onto its polypeptide. These properties of Ccp1 in vitro are consistent with our observation that it acts as a heme chaperone for apoCta1, a heme-dependent catalase found in yeast mitochondria. When levels of H2O2 rise during the cell’s switch to respiratory metabolism, maturation of Cta1, as monitored by the rise in its catalase activity, coincides with apoCcp1 exit from mitochrondria (1). MS analysis reveals that extramitochondrial Ccp1 is ~80% oxidized at H175, which confirms that its heme has been labilized by mitochondrial H2O2 to trigger heme transfer to apoCta1. This sequence of events constitutes the first example of H2O2 regulating its own intracellular concentration by turning on catalase activity post-translationally via heme transfer. M. Kathiresan, D. Martins and A.M. English Proc. Nat. Acad. Sci., USA 2014, 111, 17468–17473.

76 - Stoichiometric Quantification of S-Glutathionylation and Total Thiol Oxidation in Mouse Macrophages

Reversible oxidative modifications of protein thiols represent a fundamental mechanism of redox signaling by modulating enzyme activities and protein functions in a variety of cellular activities, such as signaling, metabolism, gene expression, and apoptosis. The stoichiometry (or occupancy) of oxidative modifications on specific cysteine residue is important for understanding the functional role of the modification; however, this aspect of quantification has been underexplored. Herein we report the stoichiometric quantification of both S-glutathionylation (SSG) and total oxidation in macrophages at the basal physiological conditions by applying resin-assisted quantitative redox proteomics. The stoichiometry information of SSG and total oxidation on a total of ~4,100 Cys sites was quantified. The basal average oxidation level of protein thiols was ~12%, which is consistent with the overall reducing status in vivo. The average percentage of SSG in total oxidation was 32.0% at basal, supporting that SSG is one major type of thiol modifications. Interesting, we observed that the redox potential of a cellular organelle was well correlated with the level of oxidation in the organelle. For example, endoplasmic reticulum and lysosome have much higher oxidation levels than those from reducing organelles, such as nucleus and mitochondrion. In contrast, the reducing organelles have an average higher percentage of SSG in total oxidation, suggesting SSG is the dominant modification and plays an important regulatory role in these organelles. Moreover, the integration of stoichiometric and dynamic data reveals potential evidence of inhibitory and activation sites via redox modifications

64 - Low Dose Cadmium Stimulates Myofibroblast Differentiation and Lung Fibrosis by Activation of SMAD and Nuclear Thioredoxin-1

Background Toxic effects of cadmium (Cd) from cigarette smoke and occupational sources are well studied, yet low-level Cd also occurs in human diet. Cd accumulates in vivo due to a twenty-year biological half-life. Increasing evidence suggests that exposure to dietary Cd can impact human health by potentiating inflammatory mechanism. Submicromolar Cd can alter cellular redox control mechanism, contributing to stimulation of pulmonary diseases. The pathogenesis of idiopathic pulmonary fibrosis involves excessive myofibroblast activation which leads to increased contractures in lung. Substantial evidence supports that myofibroblast differentiation is associated with disruption of redox homeostasis, yet the mechanistic effects of exogenous exposure remains unknown. Objectives To test our hypothesis that low dietary levels of Cd stimulate pulmonary fibrosis via activation of SMAD transcription factor and stimulation of myofibroblast differentiation. Results Using human fetal lung fibroblasts (HFLF), we found that two differentiation markers, α-Smooth-Muscle-actin (α-SM-actin) and Extra-Domain-A-containing fibronectin (ED-A FN), were significantly elevated in transcriptional and translational levels by low-dose Cd (0.5, 1.0 and 2.0 μM). Under the same Cd treatment, an increased formation of stress fibers was observed in HFLF. To examine the effects of low-dose Cd on SMAD activation and redox regulatory mechanism, HFLF was transfected with nuclear exporting or nuclear localized signal conjugated thioredoxin (Trx)-1 to express in cytoplasm or nucleus, respectively. The results show that Cd increased SMAD activity by 3 fold and stimulated translocation of Trx1 from cytoplasm to nucleus. Overexpression of Trx1 in cytoplasm diminished Cd induced SMAD activation, while overexpression of Trx1 in nuclei mimicked the Cd effect. This suggests that low-dose Cd stimulates differentiation of HFLF to myofibroblast by mechanism involving nuclear translocation of Trx1 followed by activation of SMAD and subsequent gene expression of α-SM-actin and ED-A FN for differentiation and fibrosis. Conclusion Low dietary exposure to Cd presents a potential risk for pulmonary fibrosis by affecting redox signaling mechanism. The improved understanding of redox regulation mechanism in differentiation and fibrosis can provide therapeutic targets to maintain pulmonary health.

54 - The Receptor for Advanced Glycation End Product (RAGE) Binding to HMGB1 and Subsequent NADPH Oxidase Activation Mediates Ectopic Intestinal Inflammation in NAFLD

Recent clinical studies found a strong association of colonic inflammation with NAFLD) yet the mechanisms remain unknown. The present study identifies HMGB1 as a key mediator of intestinal inflammation in NAFLD and outlines a detailed redox signaling mechanism for such a pathway. NAFLD mice showed liver damage and release of higher HMGB1 in systemic circulation, increased intestinal tyrosine nitration that was dependent on NADPH oxidase. Intestines from NAFLD mice showed higher TLR4 activation and proinflammatory cytokine release, an outcome strongly dependent on the existence of NAFLD pathology and NADPH oxidase. In vitro experimentation using IE6 cells showed the HMGB1 activation of Toll like receptor (TLR-4) was both NADPH oxidase and peroxynitrite dependent with the latter being formed from the activation of NADPH oxidase. Proinflammatory cytokine production was significantly blocked by the specific peroxynitrite scavenger FBA, AKT inhibitor and NADPH oxidase inhibitor Apocynin suggesting NADPH oxidase-dependent peroxynitrite as a key mediator in TLR-4 activation and cytokine release via an AKT dependent pathway. Studies to ascertain the mechanism of HMGB1-mediatied NADPH oxidase activation showed a distinct role of RAGE as the use of inhibitors targeted against RAGE prevented NADPH oxidase activation, peroxynitrite formation, TLR4 activation and finally cytokine release. Thus in conclusion, the present study identifies a novel HMGB1mediated inflammatory pathway that is RAGE and redox signaling dependent and helps promote ectopic intestinal inflammation in NAFLD. 5P30GM103336-03 and 7R00ES019875-03 awarded to Dr. Saurabh Chatterjee.

Thymoquinone, a biologically active component of Nigella sativa, induces mitochondrial production of reactive oxygen species and programmed death of tumor cells

Mechanisms of tumor-cell responses to 2-isopropyl-5-methyl-1,4-benzoquinone (thymoquinone) and 1,4-benzoquinone were studied using fluorescence and the inhibition assay. It was shown that quinones enhanced the intracellular production of reactive oxygen species, reduced the mitochondrial membrane potential, and induced tumor-cell death through different pathways. It was found that thymoquinone, which induced lower production of reactive oxygen species than 1,4-benzoquinone, was more toxic to tumor cells. It was established that reactive oxygen species produced due to exposure to thymoquinone are involved in redox signaling processes that lead to the formation of mitochondrial permeability transition pores and activation of programmed cell death. These results suggest that the functioning of the established redox signaling mechanism is enabled by the colocalization of mitochondrial oxidoreductases involved in the production of reactive oxygen species and of protein targets of reactive oxygen species involved in the activation of apoptosis.

Application of Mitochondria-Targeted Pharmaceuticals for the Treatment of Heart Disease.

Mitochondria fulfill the massive energy demands of the human heart through oxidative phosphorylation (OXPHOS) which couples nutrient oxidation and the reduction of molecular oxygen (O2) to the phosphorylation of ADP. Reactive oxygen species (ROS) are also generated during OXPHOS which can be damaging at high levels but serve as secondary messengers when produced in a controlled manner. Here, I review how disruption of control over mitochondrial ROS production can lead to the pathogenesis of a range of cardiovascular diseases (CVD) including decompensated left ventricular hypertrophy, alcoholic and diabetic hypertrophy, myocardial infarction (MI), ischemic-reperfusion injury (IR), and heart failure. In particular I focus on the function of protein S-glutathionylation (PGlu) reactions, a rapid and reversible redox signaling mechanism that involves the conjugation and removal of glutathione from cysteine switches, in the modulation of ROS production in myocardial mitochondria and how these reactions become deregulated in heart disease. I also discuss the use of mitochondria penetrating antioxidants in the treatment of heart disease. I propose that heart disease related to deregulated PGlu reactions can be treated with a novel and hypothetical class of mitochondria penetrating reduced glutathione (GSH) molecules called MitoGSH. This synthetic form of GSH can be tagged with either SS peptides or triphenylphosphonium ions to ensure accumulation in mitochondria which could restore glutathione levels and preserve redox buffering networks. Mitochondria penetrating antioxidants have been shown to be efficient at restoring mitochondrial antioxidant defense in CVD. However, CVD and various other disorders are associated with a depletion of GSH pools. Use of mitochondria-targeted GSH analogs could serve as a more efficient means of treating heart disease since it would allow for the direct restoration of GSH levels and preserve mitochondrial redox buffering and signaling capacity.

A simple and efficient method for generating high-quality recombinant Mical enzyme for in vitro assays

We have recently identified a new family of multidomain oxidoreductase (redox) enzymes, the MICALs, that directly regulate the actin cytoskeletal elements necessary for the morphology, motility, and trajectory of cells. Our genetic assays reveal that Mical is both necessary and sufficient for actin organization and cellular effects in vivo and our biochemical assays with purified Mical protein reveal that Mical utilizes its redox activity to directly disassemble actin filaments. These results identify Mical proteins as novel actin disassembly factors and uncover a redox signaling mechanism that directly regulates the actin cytoskeleton. These results have also set the stage for in-depth characterization of the Mical enzyme. However, it has been difficult to obtain sufficient amounts of highly-pure Mical protein to conduct further biochemical, structural, imaging, catalytic, and other high-precision studies. Herein, we describe a means for expressing high levels of soluble recombinant Mical protein in bacteria. Likewise, we have designed a new purification strategy that enables the rapid and efficient purification of milligram quantities of highly-pure and >99% active Mical protein. This new strategy for generating large amounts of highly-pure and active Mical protein will aid research objectives designed to characterize the biochemical, enzymology, and structural biology of Mical and its effects on actin filament dynamics.

Administering CO2 Anesthesia to Male Sprague‐Dawley Rats Prior to Euthanasia for Postmortem Brain Slicing Alters Cardiorespiration (In Vivo) and O2‐/CO2‐sensitive Redox Signaling in Dorsocaudal Medullary Neurons in Brain Slices

Inducing CO2 anesthesia during euthanasia is anticipated to produce CO2 narcosis in parallel with escalating levels of cellular hypercapnia, acidosis, and hypoxia during the 3–5min preceding euthanasia. The goal of this study was to identify cardiorespiratory responses activated in CO2 anesthetized rats used to harvest brain slices for studies of redox and nitrosative signaling in neurons in a cardiorespiratory control center (caudal solitary complex, cSC). We hypothesized that medullary tissue slices harvested from CO2 anesthetized rats would exhibit differences in production of reactive O2 and N2 species (RONS) during normocapnic hyperoxia and hypercapnic hyperoxia as compared to brain slices harvested without CO2 anesthesia prior to euthanasia. In vivo: Telemetric measurements of cortical EEG, respiratory muscle EMG, ECG, and body temperature (TB) were made in adult male rats implanted with a DSI 4‐ET radio‐transmitter. Gradual replacement of air with CO2 (0.03–100%) over ~3–5min caused sequential changes in breathing pattern that correlated with changes in the cortical EEG. Onset of CO2 breathing increased ventilation without measureable changes in EEG, decreased heart rate (HR), and induced exploring behavior. At ~20%CO2 respiratory frequency (fRESP) decreased while tidal volume (VT) was unchanged or increased further. HR either remained unchanged or decreased further. The EEG decreased at ~25–30%CO2 and exploring behavior ceased with no further movements other than occasional blinking. At ~45%CO2 inspiratory duration increased for several breaths with visible gasping efforts while HR increased transiently followed by gradual slowing. At ~60%CO2 there was a transient burst in the EEG lasting ~1.4min during which fRESP increased but the rat remained prone and inactive. Cortical activity completely ceased following the EEG burst and breathing slowly decreased until the last breath ~1min later at ~95%CO2. HR continued on average for another 5.3min. TB decreased on average by 0.3°C as CO2 increased from 0.03 to 100%. In vitro: Transverse brain slices (400μm, 34–36°C) harvested from CO2 anesthetized rats exhibited increased superoxide production (DHE) during normocapnic hyperoxia (40% to 95%O2) compared to slices harvested without using CO2 anesthesia. CO2 anesthesia also decreased the stimulatory effects of hypercapnic acidosis (5% to 10%CO2) on aggregate RONS production (DHR123) during hyperoxia in brain slices. By contrast, nitric oxide production (DAF‐FM DA) during normocapnic hyperoxia was unchanged in brain slices harvested from rats ±CO2 anesthesia. We postulate that CO2 anesthesia activates multiple respiratory patterns and affects HR leading to long‐lasting changes in redox signaling mechanisms in cSC neurons studied under in vitro conditions. Specifically, we hypothesize that CO2 anesthesia decreases superoxide dismutase activity in brain slices causing increased superoxide accumulation while also shifting downstream RONS production during hypercapnic hyperoxia from the H+‐Fenton reaction to the CO2‐peroxynitrite reaction.Support or Funding InformationONR Undersea Medicine Program

Redox Regulation of the M1/M2 Shift in Microglia: Programming the Deleterious Phenotype

Microglia, the resident innate immune cells in the brain, are implicated in the progressive nature of diverse neurodegenerative diseases associated with aging, including Parkinson's disease. Our recent findings point to the loss of NF‐κB p50 function due to reactive oxygen species as a key mechanism driving microglial failure to resolve the M1 (pro‐inflammatory) response and impaired M2 (alternative) activation, where loss of NF‐κB p50 function leads to a CNS‐specific vulnerability to chronic inflammation. Moreover, our current work indicates that aging synergistically augments the brain M1 activation response in NF‐κB p50−/− mice, implicating a role for loss of NF‐κB p50 function in age‐associated microglial priming (heightened sensitivity to pro‐inflammatory stimuli) and dopaminergic neurotoxicity. Together, these findings implicate NF‐κB p50 as pivotal redox signaling mechanism regulating the M1/M2 activation balance in microglia, where loss of function tips this balance to favor a M1 polarized and neurotoxic phenotype. These findings support that loss of NF‐κB p50 function and aging may interact to excessively augment microglial priming and point to a novel pro‐inflammatory signaling mechanism independent the NF‐κB p50/p65 transcription factor in this process.Support or Funding InformationNIEHS/NIH ONES Award R01ES016951; DOD GW120068

Abstract B21: The effect of mutant Kras associated changes in redox signaling in pancreatic neoplasia.

Abstract Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer (PaCa) tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), that are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. We are therefore investigating the role of Prdx-1 and its oxidation states in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data suggests that throughout tumor progression, Prdx-1 is differentially regulated and that its localization changes. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total prdx-1 expression and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by NOX inhibitors and fatty acids that control pancreatic neoplasia. In addition, AKT, ERK , and FOXO3a phosphorylation were also differntially modified by DPI treatment and fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. We therefore conclude that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia. Citation Format: Michelle A. Schultz, Rital Shah, Brian DeCant, Windel E. Mascarinas, David Bentrem, Paul Grippo. The effect of mutant Kras associated changes in redox signaling in pancreatic neoplasia.. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B21.

Titanium Dioxide Nanoparticles Trigger Loss of Function and Perturbation of Mitochondrial Dynamics in Primary Hepatocytes.

Titanium dioxide (TiO2) nanoparticles are one of the most highly manufactured and employed nanomaterials in the world with applications in copious industrial and consumer products. The liver is a major accumulation site for many nanoparticles, including TiO2, directly through intentional exposure or indirectly through unintentional ingestion via water, food or animals and increased environmental contamination. Growing concerns over the current usage of TiO2 coupled with the lack of mechanistic understanding of its potential health risk is the motivation for this study. Here we determined the toxic effect of three different TiO2 nanoparticles (commercially available rutile, anatase and P25) on primary rat hepatocytes. Specifically, we evaluated events related to hepatocyte functions and mitochondrial dynamics: (1) urea and albumin synthesis using colorimetric and ELISA assays, respectively; (2) redox signaling mechanisms by measuring reactive oxygen species (ROS) production, manganese superoxide dismutase (MnSOD) activity and mitochondrial membrane potential (MMP); (3) OPA1 and Mfn-1 expression that mediates the mitochondrial dynamics by PCR; and (4) mitochondrial morphology by MitoTracker Green FM staining. All three TiO2 nanoparticles induced a significant loss (p < 0.05) in hepatocyte functions even at concentrations as low as 50 ppm with commercially used P25 causing maximum damage. TiO2 nanoparticles induced a strong oxidative stress in primary hepatocytes. TiO2 nanoparticles exposure also resulted in morphological changes in mitochondria and substantial loss in the fusion process, thus impairing the mitochondrial dynamics. Although this study demonstrated that TiO2 nanoparticles exposure resulted in substantial damage to primary hepatocytes, more in vitro and in vivo studies are required to determine the complete toxicological mechanism in primary hepatocytes and subsequently liver function.

Abstract 2132: Mutant Kras associated protein oxidation in pancreatic neoplasia

Abstract Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer (PaCa) tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), thioredoxin (Txn), and sulfiredoxin (Srxn), which are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. Txn and Srxn prevent Prdx-1 overoxidation. We are therefore investigating the role of Prdx-1 oxidation and Txn and Srxn regulation in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data suggest that throughout tumor progression, Prdx-1 expression and localization are differentially regulated. These changes also correlated with reduced Txn and Srxn expression in lesions. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total prdx-1 expression and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by NOX inhibitors and omega-3 fatty acids that control pancreatic neoplasia. ERK inhibitors also differentially affected Prdx1 expression in pancreatic cells in vitro. In addition, AKT, ERK, and FOXO3a phosphorylation were also modified by DPI treatment (NOX inhibitor) and omega-3 fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. We therefore conclude that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras-mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia. Citation Format: Michelle Schultz, Brian DeCant, Andrew Diaz, Sharon Solman, Rital Shah, David Bentrem, Paul J. Grippo. Mutant Kras associated protein oxidation in pancreatic neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2132. doi:10.1158/1538-7445.AM2015-2132

Abstract A16: Mutant Kras associated protein oxidation in pancreatic neoplasia

Abstract Recent studies have suggested that mutant Kras associated activation of the Nrf2 pathway may have a significant role in pancreatic cancer tumorigenesis. Nrf2 regulates a battery of antioxidant genes, including peroxiredoxin-1 (Prdx-1), that are responsible for maintaining redox homeostasis in the cell. While ROS detoxification may have significant roles in cancer, several non-classical mechanisms of redox signaling associated with protein oxidation and phosphorylation are also important in tumorigenesis. Prdx-1 can regulate transcription as a cofactor in multiple oxidation states, represses tumorigenesis in its reduced state, and can act as a nuclear chaperone in elevated states of oxidation and when phosphorylated. We are therefore investigating the role of Prdx-1 and its oxidation states in oncogenic signaling pathways in mutant Kras models of pancreatic tumorigenesis and neoplasia. Our data demonstrate that Prdx1 is localized to the nucleus in normal c57-BL6 mice and exclusively located in the cytoplasm in normal pancreatic cells in the EL-Kras model of pancreatic neoplasia. Moreover, in acinar to ductal metaplatic pancreatic cells in EL-Kras mice, there is an increase in cytoplasmic Prdx1 expression and evidence of nuclear staining. Papillary cells in lesions of EL-Kras mice have apical Prdx-1 expression and ductal cells lining these lesions have higher cytoplasmic Prdx-1 expression and some nuclear Prdx-1 localization. This suggests that throughout tumor progression, Prdx-1 is differentially regulated. In vitro, neoplastic and tumorigenic pancreatic cell lines have more total Prdx-1 and oxidized Prdx-1 expression than non-tumorigenic cell lines. Moreover, cells that have mutant Kras were associated with higher levels of protein oxidation than those with wildtype Kras expression. Interestingly, mutant Kras was also associated with differences in pERK interaction with Prdx-1, which was also differentially regulated by fatty acids that control pancreatic neoplasia. In addition, ERK phosphorylation was also modified by fatty acid treatment in wildtype and mutant Kras normal, neoplastic, and tumorigenic cell lines. Our data therefore suggests that mutant Kras is associated with elevated levels of Prdx-1 oxidation and that Kras mediated activation of ERK could be associated with changes in Prdx-1 signaling in pancreatic neoplasia. Citation Format: Michelle A. Schultz, Rital Shah, Brian DeCant, David Bentrem, Paul Grippo. Mutant Kras associated protein oxidation in pancreatic neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A16.

The basic chemistry of exercise-induced DNA oxidation: oxidative damage, redox signaling, and their interplay.

Acute exercise increases reactive oxygen and nitrogen species generation. This phenomenon is associated with two major outcomes: (1) redox signaling and (2) macromolecule damage. Mechanistic knowledge of how exercise-induced redox signaling and macromolecule damage are interlinked is limited. This review focuses on the interplay between exercise-induced redox signaling and DNA damage, using hydroxyl radical (·OH) and hydrogen peroxide (H2O2) as exemplars. It is postulated that the biological fate of H2O2 links the two processes and thus represents a bifurcation point between redox signaling and damage. Indeed, H2O2 can participate in two electron signaling reactions but its diffusion and chemical properties permit DNA oxidation following reaction with transition metals and ·OH generation. It is also considered that the sensing of DNA oxidation by repair proteins constitutes a non-canonical redox signaling mechanism. Further layers of interaction are provided by the redox regulation of DNA repair proteins and their capacity to modulate intracellular H2O2 levels. Overall, exercise-induced redox signaling and DNA damage may be interlinked to a greater extent than was previously thought but this requires further investigation.

Molecular dynamics simulations of the transport of reactive oxygen species by mammalian and plant aquaporins

BackgroundAquaporins are responsible for water transport across lipid membranes. They are also able to transport reactive oxygen species, playing an important role in redox signaling. Certain plant aquaporins have even the ability to be regulated by oxidative stress. However, the underlying mechanisms are still not fully understood. MethodsHere, molecular dynamics simulations were employed to determine the activation free energies related to the transport of reactive oxygen species through both mammalian and plant aquaporin models. Results and conclusionsBoth aquaporins may transport hydrogen peroxide (H2O2) and the protonated form of superoxide radicals (HO2). The solution-to-pore transfer free energies were low for small oxy-radicals, suggesting that even highly reactive hydroxyl radicals (HO) might have access to the pore interior and oxidize amino acids responsible for channel selectivity. In the plant aquaporin, no significant change in water permeability was observed upon oxidation of the solvent-exposed disulfide bonds at the extracellular region. During the simulated time scale, the existence of a direct oxidative gating mechanism involving these disulfide bonds could not be demonstrated. General significanceSimulation results may improve the understanding of redox signaling mechanisms and help in the interpretation of protein oxidative labeling experiments.

The Protective Role of Glutaredoxin‐1 in Endothelial Barrier Regulation and Atherosclerosis

Vascular oxidative stress is widely invoked as an important pathogenic mechanism for atherosclerosis (AS). Protein S‐glutathionylation (P‐SSG), the prevalent post‐translational oxidative modification, is emerging as a critical redox signaling mechanism in cardiovascular diseases. However, its role in AS is not clear yet. P‐SSG can be removed by glutaredoxin‐1 (Glrx), a thiol‐transferase which plays a pivotal role in redox signaling. This study is aimed to investigate the role of P‐SSG in high fat‐induced endothelial barrier dysfunction and AS through genetic manipulation of Glrx in human aortic endothelial cell (HAEC) and mice. Overexpression of Glrx attenuates high palmitate high glucose (HPHG)‐induced reversible oxidation of proteins and hyper‐permeability of HAEC monolayer to dextran. Consistently, high fat diet‐induced aortic vessel leakage indexed by dye extravasation is markedly decreased in Glrx transgenic mice when compared with WT mice. Overexpression of hGlrx‐1 in HAECs stimulates cortical actin filament formation under both basal and HPHG challenge conditions. Employing biotin‐switch assays, we showed that Glrx up‐regulation diminished HPHG‐induced oxidative modification of actin and the RhoGTPases, Rac‐1. More importantly, Glrx‐1 corrected HPHG‐induced Rac1 inactivation, a key mechanism of impaired endothelial barrier integrity. Oxidative stress‐induced endothelial barrier dysfunction is an initial step of AS, we thus assessed the high fat diet‐induced AS lesions by en face Oil red O staining in Glrx‐1 transgenic mice with ApoE deficiency background, and found that Glrx‐1 up‐regulation significantly attenuates AS lesion formation. These results suggest a causal role of P‐SSG in the pathogenesis of AS and that Glrx may have therapeutic potential for AS.

Cytotoxin-induced NADPH oxides activation: roles in regulation of cell death.

Numerous studies have shown that a variety of cytotoxic agents can activate the NADPH oxidase system and induce redox-dependent regulation of cellular functions. Cytotoxin-induced NADPH oxidase activation may either exert cytoprotective actions (e.g., survival, proliferation, and stress tolerance) or cause cell death. Here we summarize the experimental evidence showing the context-dependent dichotomous effects of NADPH oxidase on cell fate under cytotoxic stress conditions and the potential redox signaling mechanisms underlying this phenomenon. Clearly, it is difficult to create a unified paradigm on the toxicological implications of NADPH oxidase activation in response to cytotoxic stimuli. We suggest that interventional strategies targeting the NADPH oxidase system to prevent the adverse impacts of cytotoxins need to be contemplated in a stimuli- and cell type-specific manner.

TGFβ1 rapidly activates Src through a non-canonical redox signaling mechanism

Transforming growth factor-β1 (TGF-β) is involved in multiple cellular processes through Src activation. In the canonical pathway, Src activation is initiated by pTyr530 dephosphorylation followed by a conformational change allowing Tyr419 auto-phosphorylation. A non-canonical pathway in which oxidation of cysteine allows bypassing of pTyr530 dephosphorylation has been reported. Here, we examined how TGF-β activates Src in H358 cells, a small cell lung carcinoma cell line. TGF-β increased Src Tyr419 phosphorylation, but surprisingly, Tyr530 phosphorylation was increased rather than decreased. Vanadate, a protein tyrosine phosphatase inhibitor, stimulated Src activation itself, but rather than inhibiting Src activation by TGF-β, activation by vanadate was additive with TGF-β showing that pTyr530 dephosphorylation was not required. Thus, the involvement of the non-canonical oxidative activation was suspected. TGF-β increased extracellular H2O2 transiently while GSH-ester and catalase abrogated Src activation by TGF-β. Apocynin, a NADPH oxidase inhibitor, inhibited TGF-β-stimulated H2O2 production. Furthermore, mutation of cysteines to alanine, 248C/A, 277C/A, or 501C/A abrogated, while 490C/A significantly reduced, TGF-β-mediated Src activation. Taken together, the results indicate that TGF-β-mediated Src activation operates largely through a redox dependent mechanism, resulting from enhanced H2O2 production through an NADPH oxidase and that cysteines 248, 277, 490, and 501 are critical for this activation.

Mitochondrial dysfunction and loss of glutamate uptake in primary astrocytes exposed to titanium dioxide nanoparticles.

Titanium dioxide (TiO2) nanoparticles are currently the second most produced engineered nanomaterial in the world with vast usage in consumer products leading to recurrent human exposure. Animal studies indicate significant nanoparticle accumulation in the brain while cellular toxicity studies demonstrate negative effects on neuronal cell viability and function. However, the toxicological effects of nanoparticles on astrocytes, the most abundant cells in the brain, have not been extensively investigated. Therefore, we determined the sub-toxic effect of three different TiO2 nanoparticles (rutile, anatase and commercially available P25 TiO2 nanoparticles) on primary rat cortical astrocytes. We evaluated some events related to astrocyte functions and mitochondrial dysregulation: (1) glutamate uptake; (2) redox signaling mechanisms by measuring ROS production; (3) the expression patterns of dynamin-related proteins (DRPs) and mitofusins 1 and 2, whose expression is central to mitochondrial dynamics; and (4) mitochondrial morphology by MitoTracker® Red CMXRos staining. Anatase, rutile and P25 were found to have LC50 values of 88.22 ± 10.56 ppm, 136.0 ± 31.73 ppm and 62.37 ± 9.06 ppm respectively indicating nanoparticle specific toxicity. All three TiO2 nanoparticles induced a significant loss in glutamate uptake indicative of a loss in vital astrocyte function. TiO2 nanoparticles also induced an increase in reactive oxygen species generation, and a decrease in mitochondrial membrane potential, suggesting mitochondrial damage. TiO2 nanoparticle exposure altered expression patterns of DRPs at low concentrations (25 ppm) and apoptotic fission at high concentrations (100 ppm). TiO2 nanoparticle exposure also resulted in changes to mitochondrial morphology confirmed by mitochondrial staining. Collectively, our data provide compelling evidence that TiO2 nanoparticle exposure has potential implications in astrocyte-mediated neurological dysfunction.

New Insight into the Role of Reactive Oxygen Species (ROS) in Cellular Signal-Transduction Processes.

Reactive oxygen species (ROS) were once considered to be deleterious agents, contributing to a vast range of pathologies. But, now their protective effects are being appreciated. Both their damaging and beneficial effects are initiated when they target distinct molecules and consequently begin functioning as part of complex signal-transduction pathways. The recognition of ROS as signaling mediators has driven a wealth of research into their roles in both normal and pathophysiological states. The present review assesses the relevant recent literature to outline the current perspectives on redox-signaling mechanisms, physiological implications, and therapeutic strategies. This study highlights that a more fundamental knowledge about many aspects of redox signaling will allow better targeting of ROS, which would in turn improve prophylactic and pharmacotherapy for redox-associated diseases.